Factors influencing fetal macrophage development: III. Immunocytochemical localization of cytokines and time-resolved expression of differentiation markers in organ-cultured rat lungs.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Effects of hydrogen and bicarbonate ions on endocrine cells in fetal rat lung organ cultures.

Concentrations of H+ and HCO3- rise in fluid lining hypercapnic airways. Effects of these ions on pulmonary endocrine cells were studied in 119 fetal rat lung organ cultures by semiquantitative staining for calcitonin gene-related peptide (CGRP)-like immunoreactive material. Intracellular CGRP was determined in cultures under "no-release" baseline conditions and after incubation in control or test media. After exposure to HCO3(-)-free medium at pH 7.4 (incubation control), CGRP fell moderately from no-release levels. Bombesin (1 ng/ml) promoted further significant loss of peptide, which was dependent on extracellular Ca2+ and inhibited by somatostatin and [D-Arg(1),D-Pro(2),D-Trp(7,9),Leu(11)]substance P, a bombesin receptor antagonist. CGRP staining of explants incubated with 24 mM HCO3- maintained no-release levels at and above pH 7.1 but decreased significantly at pH 6.8. The drop was blocked by somatostatin or exclusion of HCO3- and was not augmented by bombesin or 48 mM HCO3-. Results suggest that pulmonary endocrine cells may respond to hypercapnia by releasing bioactive peptides like CGRP, thus stimulating afferent nerves and altering patterns of ventilation and perfusion.

Factors influencing fetal macrophage development: I. Reactions of the tumor necrosis factor-alpha cascade and their inhibitors.

BACKGROUND: When fetal rat lungs are explanted to organ culture, precursor angular cells soon convert to nascent macrophages that multiply rapidly as they mature into efficient phagocytes. The present study examines the influence of proinflammatory early cytokines of the tumor necrosis factor-alpha (TNF alpha) cascade on this initial expression of the macrophage phenotype. METHODS: Fourteen- and 15-day fetal rat lungs were grown for varying periods on an agar-solidified medium with and without test factors added singly or in combination. Growth of the macrophage population was followed daily by light microscopy and quantified by measuring the area of coronas formed as cells emerged from explants. RESULTS: TNF alpha interleukin-1 beta (IL-1 beta) stimulated growth of the macrophage population, as had macrophage- and granulocyte-macrophage colony-stimulating factors (M- and GM-CSFs) in prior studies. Inhibition was obtained by exposure to IL-1 receptor antagonist and antibodies neutralizing the CSFs. Only the effects of TNF alpha were sufficiently delayed to discount possible influence on conversion and growth of nascent macrophages. Two transcription blockers, dexamethasone and pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor NF-kappa B, both profoundly suppressed macrophage growth without preventing conversion of precursors. Effects of dexamethasone were significantly ameliorated by IL-1 beta alone and combined with GM-CSF; those of PDTC were mitigated by M-CSF and a combination of IL-1 beta and TNF alpha but not by GM-CSF. CONCLUSIONS: IL-1 beta, M-CSF, and GM-CSF all promote growth of the young macrophage population. TNF alpha is effective only later on, likely because early-stage cells lack its receptors which normally use intracellular signalling pathways similar to those for IL-1. The severity of PDTC inhibition to population growth indicates that NF-kappa B is important for transmitting proliferative signals in these cells.

Factors influencing fetal macrophage development: II. Effects of the PDGF subfamily of protein-tyrosine kinase receptor ligands as studied in organ-cultured rat lungs.

BACKGROUND: Macrophage precursors in pseudoglandular rat lungs rapidly differentiate into phagocytes in organ culture, although this occurs only gradually in vivo. Macrophage colony-stimulating factor is vital for the process, but the possible importance of other ligands in the platelet-derived growth factor (PDGF) subfamily is scarcely appreciated. METHODS: Macrophage development was compared in 15-day fetal rat lungs cultured on solid, serum-containing media with and without added stem cell factor (SCF) (100 ng/mL) or antibodies to PDGF-AA and -BB (10-15 micrograms/mL each). In addition, organ cultures and intact lungs were immunostained for PDGF-AA and -BB to confirm their presence in the tissues. Macrophage population growth was measured by coronal area assay. RESULTS: SCF initially stimulated macrophage production. Thereafter, results varied depending on baseline production by control cultures: where this was vigorous, SCF-exposed explants performed similarly; where this was moderate, the SCF explants outperformed them 1.5-2.6 times over (P < 0.01-0.001). Inhibition of macrophage production by pyrrolidine dithiocarbamate (100 microM) was not significantly diminished in the presence of SCF (10 ng/mL). Immunoreactivity for PDGF-AA and -BB was prevalent in cells of the airway epithelium and stroma during the period macrophage precursors were converting, and both isoforms were detected in differentiating macrophages as early as 2 days in vitro. Nonetheless, exposure of cultures to anti-PDGFs had no significant effect on macrophage population growth. CONCLUSIONS: Ligands of the PDGF subfamily differ greatly in their influence over development of fetal macrophages. Whereas the PDGFs are ineffective, SCF stimulates growth of macrophage precursors and early differentiating forms and enhances survival of older cells. It appears to act mainly in synergy with other growth factors present in fetal lungs. Furthermore, in the hierarchy of hematopoietic progenitors, the macrophage precursors may be ranked on a par with burst-forming units in the red cell lineage.

Exogenous cytokines enhance survival of macrophages from organ cultured embryonic rat tissues.

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.

Precursors of macrophages in embryonic rat lungs fail to exhibit granulocyte-forming potential.

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.

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 B4 (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 approximately 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.

Neuroepithelial bodies and growth of the airway epithelium in developing hamster lung.

Clusters of small-granule endocrine cells, neuroepithelial bodies (NEBs), appear in the airway lining of pseudoglandular lungs, but their prenatal function has remained obscure. Transplacental labeling of S-phase cells in Syrian golden hamsters has allowed us to relate NEBs to patterns of replication in the surrounding endoderm. Two methods were used: 1) continuous exposure to 3H-thymidine for the last 25% (4 days) of gestation, and 2) 2-hr exposure to 5-bromo-2'-deoxyuridine (BrdU) on fetal day 15. 3H-thymidine incorporation was assessed in autoradiographs of neonatal lung by grain counting from 923 nonendocrine and 251 endocrine cells in 28 airway epithelial terrains, each centered on a NEB: 12 in the perihilar, 8 in the middle, and 8 in the distal third of the left axial bronchus. Grain densities for 10-25 nonendocrine cells on either side of the NEB were plotted vs. position relative to the endocrine cell cluster and analyzed by rank-order correlation and linear regression. Label was highest in cells closest to NEBs in all 12 terrains (P < 0.05-0.001) in the perihilar airway, in 3 of 8 terrains (P < 0.025-0.001) in the middle third of the bronchus, and in respective, pooled populations (P < 0.001). The effect was not demonstrable in the distal third of the airway. In the 15-day fetus 243 mm of airway perimeter were measured and 3,218 BrdU-labeled epithelial cells counted from sections through the entire length of the left axial airway and the lobar bronchus, intermediate, and terminal bronchioles of the infracardiac (IC) lobe.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium and ionophore A23187 lower calcitonin gene-related peptide-like immunoreactivity in endocrine cells of organ cultured fetal rat lungs.

Small-granule endocrine cells differentiate in airway epithelium of intact and cultured fetal rat lungs. We noted that the cells store calcitonin gene-related peptide (CGRP) in vitro as well as in vivo and used the ionophore A23187 to test the effects of calcium on peptide secretion in this system. Lungs of 14-day and 15-day fetal rats, organ cultured for 6-9 days, were divided into groups of 5 explants each and incubated for 15 min at 37 degrees C in the basic medium containing 0 mM, 1 mM, or 10 mM CaCl2, with or without 8 microM A23187, or 10 mM EGTA. Intracellular CGRP in these explants was quantified by supraoptimal dilution peroxidase immunocytochemistry (Springall et al.: J. Pathol. 155:259-267, 1988): counts were made of endocrine cells stained with a 1/60,000 dilution of anti-CGRP and repeated on the same sections after restaining with antibody diluted at 1/1,000. Results, analyzed by Chi-square test, were expressed as % cells stained with antibody at 1/60,000 vs. those stained at 1/1,000. Immunoreactivity for CGRP was significantly reduced by A23187 in the presence of high extracellular Ca2+ (10 mM), the inference being that these cells secrete peptide hormones in response to Ca2+ influx across the plasma membrane. The organ cultures evidently can be used to assess certain physiological responses of lung endocrine cells in an accessible, relatively organotypical setting.

CFU-rAM, the origin of lung macrophages, and the macrophage lineage.

Macrophage precursors and their progeny have been identified in early rat embryos with the use of a peroxidase-coupled marker, isolectin B4 of Griffonia simplicifolia (GSA I-B4). The macrophage lineage can be traced back to actively dividing GSA-positive angular cells present in the mesenchyme of late neurulas. These increase in number and establish residence successively in rudiments of the central nervous system, liver, and lungs. In organ-cultured fetal lungs they transform directly into a self-replicating population of macrophages responsive to colony-stimulating factors. The angular cell therefore can be seen as the source of lung macrophages during prenatal life. The extent to which this manner of production continues in postnatal life is unclear, but it appears that central hematopoietic tissues (bone marrow, spleen) may generate macrophages by a direct pathway from early-committed progenitors as well as indirectly through a series of intermediate stem cells. Considering the wealth of new information available from diverse studies in specialized culture environments and to a lesser extent from studies in vivo, it is time to integrate these findings into a more comprehensive theory of macrophage origin and fate than we have at present.

Macrophage development: IV. Effects of blood factors on macrophages from prenatal rat lung cultures.

Effects of colony-stimulating factors M-CSF, GM-CSF, G-CSF, and IL-3 were assessed on cells of macrophage lineage present in organ cultured 14-day prenatal rat lungs. Treatment groups were compared between one another and against control lungs grown on standard medium containing 40% fetal bovine serum without added factors, where a monoculture of macrophages rapidly develops from precursors present at explantation, leading to appearance of a large mature population on the pleural surface outside the lungs. Studies were carried out in living cultures and by light and electron microscopy using peroxidase-coupled isolectin B4 of Griffonia simplicifolia to identify macrophages and their precursors. In the first experiment, 14-day prenatal lung explants (14 + 0 days) containing macrophage precursors but not matured cells were exposed to individual CSFs for 7 days in an attempt to determine whether precursors are committed irrevocably to the macrophage line or can be altered by exposure to factors promoting significant granulocyte development. In succeeding experiments, 4- and 7-day-old cultures (14 + 4, 14 + 7 days) containing matured macrophages were targeted to see whether macrophage survival can be extended beyond expectations in controls and whether mitotic activity is stimulated. Recombinant CSFs were used at dosage levels known to promote colony formation in vitro (200-1,000 CFU/ml). Cultures exposed from prenatal day 14 to M-, GM-, G-CSF, or IL-3 yielded a monoculture of macrophages without exception. Populations developed in the presence of M- or GM-CSF were much larger than in controls or cultures grown with the other blood factors. GM-CSF-exposed cultures produced by far the largest macrophages, among them many multinucleate giant cells. Macrophages developed in the presence of G-CSF were also significantly larger than controls. Growth of the mature macrophage population was greatly stimulated by exposure to M-CSF or GM-CSF but not by IL-3 or G-CSF. Mitotic figures were noted in the coronas of emerged cells surrounding stimulated cultures, compared to none in the controls. Ultrastructurally, macrophages stimulated by M-CSF retained a mature appearance like macrophages in control, IL-3, and G-CSF treatment groups, whereas many in the GM-CSF group became less differentiated. As to long-term survival, a single 14-day explant was grown for 8 days on standard medium (the equivalent date for birth), then placed in a soft agar medium containing M-CSF.(ABSTRACT TRUNCATED AT 400 WORDS)

Macrophage development: II. Early ontogeny of macrophage populations in brain, liver, and lungs of rat embryos as revealed by a lectin marker.

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.

Macrophage development: III. Transformation of pulmonary macrophages from precursors in fetal lungs and their later maturation in organ culture.

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.

Neuroepithelial bodies stimulate proliferation of airway epithelium in fetal hamster lung.

Autoradiographs were prepared from lungs of a newborn Syrian golden hamster exposed continuously to [3H]thymidine throughout the final 4.5 days of gestation. Silver grains were counted over nuclei of 1,145 nonendocrine airway epithelial cells adjacent to 28 mature neuroepithelial bodies (NEBs). Generally, accumulated label was greater in cells nearer a NEB than in those further away. Diminution of label with increasing distance from the closet NEB was confirmed statistically. In 24 of 28 instances, both rank-order correlation and linear regression were significant (P less than 0.001-0.05); in two others, only one test was significant; in another two, neither test was significant. The pattern was consistent and widespread. Rank-order correlations and linear regressions were significant (P less than 0.001) in populations pooled separately from left lung, right upper, and right lower lobes, and the three regression lines were superimposable. Confirmation was obtained in another animal by labeling S-phase cells with a 2-h transplacental pulse of bromodeoxyuridine (BrdU) on fetal day 15. Of 322 BrdU-positive cells counted in 270,204 microns 2 of bronchial epithelium, 174 (54%) lay within 20 microns of a neuroepithelial body. This concentration of dividing cells was significant by chi-square test: chi 2[1] = 101.62; P less than 0.001. We conclude that established NEBs promote growth of the developing airway by stimulating proliferation of local endoderm. A few daughter cells may enter NEBs; most move away to join the expanding nonendocrine airway lining.

Dynamics of neuroepithelial body (NEB) formation in developing hamster lung: light microscopic autoradiography after 3H-thymidine labeling in vivo.

Autoradiographs were prepared from lungs of a newborn Syrian golden hamster exposed continuously to 3H-thymidine for the final 4.5 days of a normal 16 day gestation. Silver grains were counted over nuclei of 1,298 small-granule endocrine cells in 165 neuroepithelial bodies (NEBs) in the right upper lobe and along the left axial bronchus, where nodal NEBs occurred at branch points and internodal NEBs in the airway between them. Nuclei of 1,005 nonendocrine airway epithelial cells were counted next to the NEBs. Label was distributed differently in the two populations: All nonendocrine cells were labeled, whereas many endocrine cells were not. In NEBs of the right upper lobe, total label (net grains/nuclear profile) averaged only 23% of that in nonendocrine cells. Along the left axial bronchus, mean label in nonendocrine cells and internodal NEBs rose 10-fold between the hilum and the periphery. Increases for both populations were linear and parallel, but total label in the NEBs was consistently lower than that in the surrounding epithelium by 15 grains/nuclear profile. Nodal NEBs were more lightly labeled than those of the internodes, consistent with their earlier formation. A few very heavily labeled small-granule cells (0.9%) occurred singly in the periphery of large, otherwise lightly labeled NEBs. Statistically these belonged to the labeling distribution of nearby nonendocrine cells. In contrast to NEBs, neurons in 10 bronchial ganglia of the right lung were virtually unlabeled. These arise from vagal neural crest and seem to comprise an entirely distinct population. We conclude that NEBs belong intrinsically to pulmonary endoderm, not neural crest. During fetal life each develops from a cell or cells programmed to stop dividing well ahead of other elements in the epithelium. Their formation is linked closely to early proliferation of the bronchial tree and is an integral part of growth and differentiation of the airway lining. After a wave of initial formation has passed down the airway, small-granule cells are added slowly to mature NEBs, probably through differentiation from adjoining airway epithelial cells--a potential mechanism for cell replacement in adult life.

Nonimmune-mediated phagocytosis by "premedullary" lung macrophages: effects of concanavalin A, tuftsin, and macrophage-inhibitory peptide.

Free cells arising in organ-cultured embryonic rat and hamster lungs share ultrastructural, lysosomal enzyme, and cell membrane properties with typical alveolar macrophages, expressing the developmental potential of the earliest-macrophage precursors resident in the lungs. In the lung culture environment cell proliferation is supported and macrophage attributes are developed despite absence of lymphocytes from the system. We have shown previously that among these attributes, the cells respond with increased phagocytosis of erythrocytes if these are opsonized with immunoglobulin G. Attention has now been turned to the question of nonimmune-mediated phagocytosis by the same population. Living macrophages that emerged from lung cultures bound rhodamine-coupled soybean and wheat germ agglutinins to a greater degree than concanavalin A (Con A), which nevertheless promoted lateral translocation of occupied receptors in the cell membrane. Emerged cells also phagocytosed living bacteria and native yeast cells (Y). The percentage of macrophages ingesting 3 or more yeast cells increased 400 (hamsters) to 500% (rats) when yeast was preincubated with Con A (200 micrograms/ml). Pretreatment of macrophages with Tuftsin (100 microM) enhanced uptake of Y by 100 (hamster) to 200% (rat). Pretreatment of macrophages with macrophage-inhibitory peptide (500 microM) appeared to inhibit phagocytosis of Y by 60% in hamsters but had no significant effect on cells from rat lung cultures.

Population dynamics of adult-formed granule neurons of the rat olfactory bulb.

The population dynamics of internal granule cells in the rat olfactory bulb during adult life were analyzed in histological sections and in autoradiograms with (1) counts of granule cells, (2) counts of labeled granule cells 1 month after injection of 3H-thymidine at various ages, and (3) counts of labeled granule cells at varying survival times (up to 18 months) after injection at 3 months and 24 months. The total number of granule cells increases linearly throughout life, approximately doubling between 3 and 31 months. Autoradiographic studies show that the rate of production of new granule cells decreases from 3 to 12 months and then is approximately constant during the rest of the life span. The number of labeled cells found 6 months after injection at 3 and 24 months is about one-fourth and one-half, respectively, that of the number at a 1-month survival, suggesting that many of the cells produced to do not survive. However, at least some granule cells labeled at 3 months survive for 18 months. A model is suggested in which granule cells are produced continuously throughout life and control of the total number of granule cells is effected chiefly through the rate of cell death.

Aging in the rat olfactory system: relative stability of piriform cortex contrasts with changes in olfactory bulb and olfactory epithelium.

Previous studies have quantified growth and atrophy of the olfactory bulb and olfactory epithelium of the Sprague-Dawley rat from maturity to senescence. Major events occurring in these structures include changes in the volume of mitral cells and changes in the number of septal olfactory receptors. These effects are large, consist of a growth phase followed by atrophy, and are temporally related in that events in the olfactory epithelium precede those in the mitral cells. A hypothesis of aging based on transneuronal effects would predict that these changes would be similarly transmitted to the next synaptic station in the olfactory pathway. Therefore, cells and synapses of the piriform cortex were studied in rats 3, 12, 18, 24, 27, 30, and 33 months of age. Alternate Vibratome sections through brains perfused with mixed aldehydes were processed for light and electron microscopy. No significant age effects were found for the volumes of cortical laminae Ia and Ib. Both numerical and surface density of synaptic apposition zones in layer Ia, formed primarily by mitral cell axons, were stable with age. A modest (18%) but significant decline in the proportion of layer Ia occupied by dendrites and spines was mirrored by an increase in the proportion of glial processes; no change in the proportion of axons and terminals was observed. Neither nuclear volume, nor soma volume, nor numerical density of layer II neurons changed with age. Thus, contacts made in the piriform cortex by mitral cell axons remain relatively stable in senescence, despite the marked volumetric changes in the mitral cell somata, changes which were confirmed again in this study. Age-related dendritic regression in layer II neurons may be attributable to functional deafferentation subsequent to reduced receptor input to mitral cells.

Intranuclear inclusions in rat piriform cortex: increase with age and preferential location within superficial layer II.

Intranuclear inclusions have been observed in layer II neurons of rat piriform cortex. These inclusions have the form of a filamentous lattice and resemble those described by others previously. The frequency of lattice-containing nuclei shows a significant fourfold increase over a period of 3-33 months of age, with the largest increase occurring after 18 months. The incidence of these inclusions is highest in the superficial third of layer II and is significantly greater than what would be expected from the distribution of all neuronal nuclei in layer II. The presence of intranuclear lattices may be related to the high level of electrical activity in piriform cortex, and their increase with age may reflect a long-term cumulative effect of this activity.

An autoradiographic study of the mouse olfactory epithelium: evidence for long-lived receptors.

In order to try to determine whether differentiated olfactory receptors turn over (die and are replaced by newly differentiated cells) during adult life, mice were injected with a single dose of 3H-thymidine at either 2 or 4 months of age and allowed to survive for up to 12 months; they were caged in a laminar flow unit to prevent rhinitis. Counts of labeled receptor cells detected autoradiographically after injection at 2 months of age revealed that, following an initial decrease from 1 to 3 months of survival, numbers of labeled cells remained approximately constant, at least up to 12 months of survival. Cells still labeled at 12 months of survival were confirmed as receptor cells by electron microscopic examination of reembedded sections. The hypothesis is suggested that in the absence of disease-related destruction of the olfactory epithelium, most or all receptor cell turnover represents newly formed cells that fail to establish synapses with the olfactory bulb; fully differentiated receptor cells may be quite long-lived.