ARTICLE |
Correspondence to: Herman Yeger, Dept. of Paediatric Laboratory Medicine, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. E-mail: hermie@sickkids.on.ca
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Summary |
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The FORSE-1 (forebrain-surface-embryonic) monoclonal antibody (MAb) recognizes a carbohydrate cell surface epitope related to the Lewis-X (LeX) and stage-specific embryonic antigens (SSEAs). In the developing CNS, the FORSE-1 epitope is believed to serve as a marker of progenitor cells. We studied the expression of the FORSE-1 epitope in pulmonary neuroendocrine cells (PNECs) and related neuroepithelial bodies (NEBs), cell types implicated in paracrine regulation of lung development. We used dual immunolabeling to identify PNECs/NEBs in tissue sections from developing rabbit fetal lungs and corresponding primary lung cell cultures. During the early stage (E16), the FORSE-1 MAb labeled primitive airway epithelium, whereas serotonin (5HT) immunoreactivity, a marker of PNEC/NEB differentiation, was negative. After E18, FORSE-1 labeling became restricted to PNECs and NEBs, identified by co-expression with 5HT, then decreased coincident with an increase in 5HT. Expression of the FORSE-1 epitope correlated inversely with 5HT expression in PNEC/NEB cells. FORSE-1 immunoreactivity correlated with cell proliferation assessed by BrdU labeling. Downregulation of the FORSE-1 epitope correlated with maturation of PNECs/NEBs. The presence of few FORSE-1/5HT-positive cells in postnatal lung suggests retention of progenitors. The FORSE-1 epitope was associated with a high molecular weight (286 kD) glycoprotein that decreased with increasing gestational age, as demonstrated by immunoblotting. Overall expression of SSEA-1, -3, and -4 antigens was similar to FORSE-1/5HT, although the former was preferentially localized to neurite-like processes. Because the role of the FORSE-1 epitope in the CNS probably involves cell adhesion and differentiation, we propose a similar function in developing lung. The demonstration of LeX/SSEA antigen expression in the PNEC/NEB cell lineage underscores the importance of these cells in developing lung. Furthermore, the FORSE-1 antigen may identify committed progenitors of the PNEC/NEB cell system. (J Histochem Cytochem 50:15671578, 2002)
Key Words: FORSE-1, stage-specific embryonic, antigens, Lewis antigens, pulmonary neuroendocrine, cells, neuroepithelial bodies, neuroendocrine differentiation, lung development
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Introduction |
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PULMONARY NEUROENDOCRINE CELLS (PNECs) are distributed throughout the tracheobronchial mucosa as solitary cells and innervated clusters, neuroepithelial bodies (NEBs) located mostly in intrapulmonary airways (
The FORSE-1 (forebrain-surface-embryonic) MAb raised against a surface antigen expressed by retinoic acid-induced human embryocarcinoma (NT2D1) was shown to specifically recognize a cell surface antigen related to the Lewis-X (Le-X) carbohydrate epitope expressed during neuronal development (
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Materials and Methods |
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Animals
New Zealand White does at 16, 18, 21, 24, 26, and 28 days' gestation (E16E28) and postnatal days 1, 3, and 7 (P1P7) litters were sacrificed by lethal injection. The fetuses were removed and the entire lungs aseptically dissected. For primary culture, excised lungs were kept immersed in cold CO2 Independent Medium (Gibco BRL; Burlington, Ontario, Canada). The parenchyma and vasculature were teased away from airways under a dissecting microscope, leaving tracheal/bronchial trees largely intact. To perform immunochemical labeling, lung slices were fixed with 4% paraformaldehyde and tissue slices (200400 µm) were prepared using a Leica Vibratome 1000S.
Cell Culture
Primary cultures of fetal rabbit lung cells were established by enzymatic dissociation of whole lungs (E16E21 gestation) and dissected bronchial trees (E24P7). On average, 2 million cells/well were maintained in -MEM supplemented with 10% FBS and cultured on Lab-Tek slides at 37C in 5% CO2. To label proliferating cells, cultures were incubated in medium containing 10 µM 5-bromo-2'-deoxyuridine (BrdU) for 3 hr and then fixed in 3.8% paraformaldehyde after 3, 5, 7, or 10 days in culture.
Immunocytochemistry, Antibodies, Signal Amplification, and Confocal Microscopy
Specific antibodies recognizing variant epitopes on LeX, FORSE-1, and SSEA-1, SSEA-3, and SSEA-4 were obtained from the Developmental Studies Hybridoma Bank (University of Iowa; Iowa City, IA). All other antibodies were purchased from commercial sources and applied as previously described (
In triple-labeling experiments after incubation with both FORSE-1 and anti-5HT antibodies, donkey anti-mouse IgMHRP conjugate and donkey anti-goat IgGAMCA conjugate antibodies were applied to lung slices and cultured cells. The TRITCtyramine CARD system was also used for signal amplification of FORSE-1 (
Fluorescent images (dual labeling, FITC/TRITC, or triple staining, FITC/TRITC/AMCA) of FORSE-1, 5HT, and/or BrdU in cultured cells and paraffin section were obtained using an Olympus fluorescent microscope with a cooled 3CCD color camera. A Leica confocal laser scanning microscope with SCANWARE software was used to visualize FITC/TRITC dual signal in tissue slices. Separate color images of FORSE-1, 5HT, and BrdU from either immunofluorescence microscope or confocal scanner were merged to form a composite FORSE-1/PNEC/NEB marker image. Image processing was done using Adobe Photoshop 4.0 software.
Quantitation of Labeled Cells
FORSE-1- and 5HT-positive cells/clusters and co-labeled cells/clusters were counted in the entire areas of 22 x 22 mm Lab-Tek chambers, using a grid scale and in triplicate, in the developmental series, E16 to P7 and in cultures maintained for 3, 5, 7, and 10 days. To determine PNEC/NEB proliferation, BrdU-positive cells were counted in association with either 5HT, FORSE-1, or co-labeled cell populations. These data were normalized for the total number of keratin-positive epithelial cells. Cell numbers of different mixed cell populations in culture, were compared to epithelial cells expressing cytokeratin and calculated as follows:
Normalized cell no. = cell number x K
The data were calculated as mean ± SD using Prism GraphPad statistical software.
Immunoblotting
Lungs from different developmental stages (embryonic and newborn rabbits) were freshly dissected and homogenized in a homogenization buffer plus protease inhibitors as described by
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Results |
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Frequency and Distribution of FORSE-1 Immunoreactivity in Developing Lung
In tissue slice preparations and paraffin sections of gestational stage E16 rabbit fetal lungs, the FORSE-1 MAb strongly labeled airway epithelium in both large and small airways but not in the surrounding mesenchyme (Fig 1A and Fig 1B). At this early stage, no 5HT-immunoreactive cells were identified within the primitive epithelium. At E18, while FORSE-1 epitope expression was still prominent on airway epithelial cells, co-localization of FORSE-1 antigen and positive 5HT immunoreactivity became apparent in some cells (Fig 1C and Fig 1C'). Whereas FORSE-1 antigen was localized mostly on the apical surface of epithelial cells, 5HT expression was cytoplasmic (Fig 1C and Fig 1C'). Between E21 and E28, FORSE-1 immunoreactivity became restricted to NEB cells, as identified by co-expression with 5HT (Fig 1D1G). The FORSE-1 labeling exhibited a mostly apical and/or basolateral cell membrane localization. At E28 a low level of FORSE-1 epitope expression was detectable on most NEB cells and on surrounding cells in the vicinity of NEBs. However, a small number of cells within NEBs retained strong membrane FORSE-1 immunoreactivity. By comparison, 5HT immunoreactivity appeared diffusely distributed throughout the cytoplasm of NEB cells (Fig 1G and Fig 1G'). During later gestational stages only trace amounts of FORSE-1 antigen were detected in some NEBs (not shown). Therefore, FORSE-1 epitope expression became gradually restricted to maturing NEBs, reinforcing the notion of developmentally regulated expression.
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Primary cultures established from E16 lungs were maintained in vitro for up to 3 days to identify co-expression of the FORSE-1 epitope and 5HT. These cultures were also preincubated with BrdU to assess the proliferative capacity of the cells expressing the FORSE-1 epitope and 5HT immunoreactivity. The results of these experiments recapitulate the developmental patterns observed in situ. Most primitive epithelial cells were FORSE-1-immunopositive at E16, with evidence of rare cell clusters exhibiting coordinate expression with 5HT (Fig 2 and inset). The number of cells showing concomitant expression of FORSE-1 epitope and 5HT (Fig 2 and inset) increased between E18 and E21. However, this ratio then shifted; most cells expressed 5HT only by E28 (Fig 2). The number of NEB clusters increased significantly between E18 and E28. At E28, large NEBs contained cells with strong immunoreactivity for both the FORSE-1 epitope and 5HT. In cultures of lung on postnatal days 1(P1), P3, and P7, cells of large NEBs were predominantly 5HT-positive. Occasional FORSE-1-immunoreactive cells were observed. Cultures of cells from lungs at E16 and E24 maintained for up to 3, 5, 7, and 10 days in vitro and labeled with BrdU revealed that the number of BrdU-positive cells within NEBs and/or cells expressing FORSE-1 and 5HT also gradually decreased with gestational age (Fig 3A). Furthermore, in day 3 cultures of E24 lung the cells exhibiting concomitant expression of FORSE-1 and 5HT were BrdU-positive (Fig 3B), as were those cells expressing 5HT only. The fact that the BrdU-labeled 5HT-positive cells showed restricted growth, i.e., did not form colonies, suggests that this cell population has a long doubling time, as previously reported using [3H]-thymidine labeling (
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Quantitation of FORSE-1- and 5HT-expressing Cell Populations
The numbers of single cells and cell clusters (four to ten cells and >10 cells) staining for FORSE-1, 5HT, and FORSE-1/5HT were counted both in short-term (4-day) cultures established from the gestational series lungs (Fig 4 and Fig 5) and E16 lung maintained for up to 10 days in vitro (Fig 6 and Fig 7). The numbers of FORSE-1-positive single cells declined dramatically with increasing gestational age or with time in culture, whereas the number of FORSE-1 positive cell clusters varied little. In contrast, there was a dramatic increase in 5HT expression in both single cells and NEB cell clusters. BrdU labeling of short-term cultures established from the gestational series of lungs showed a significant decrease in BrdU incorporation in single cells and cells within clusters co-expressing FORSE-1 and 5HT and a minimal decrease in cells or clusters expressing only FORSE-1 or 5HT (Fig 5). Therefore, quantitation of labeled cells revealed at least two subpopulations of cells, suggesting that co-expression of FORSE-1 and 5HT was associated with developmental maturation.
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This quantitative pattern of FORSE-1/5HT expression and correlation with BrdU labeling was essentially recapitulated in cultures of E16 lungs maintained for up to 10 days in culture (Fig 6). In this case, single cells, small clusters, and large clusters were scored for expression of FORSE-1, 5HT, and for incorporation of BrdU. Because FORSE-1 epitope expression diminished linearly with time in culture, 5HT expression increased and peaked in the day 57 culture period for single cells, in the 710 day period for small (410 cells) clusters, and by 10 days for large (>10 cells) clusters (Fig 6D). In contrast, cells co-expressing FORSE-1/5HT cells decreased gradually with time in culture, coordinate with decrease in the number of FORSE-1 only expressing cells and a significant increase in 5HT only expressing cells. Cells expressing 5HT appeared to cease proliferation only with increased time in culture, as observed in cultures labeled with BrdU (Fig 7). Overall, our data demonstrate a developmental sequence of coordinated expression of FORSE-1 and 5HT coinciding with phenotypic maturation of NEBs.
FORSE-1 Epitope Is Associated with a 286-kD Carrier Glycoprotein
Extracts of lungs from E16 to E28 and postnatal days 1, 3, and 7 were resolved by SDS-PAGE under non-reducing conditions and FORSE-1 epitope-containing proteins were immunoblotted with the FORSE-1 MAb and detected by the chemiluminescence method. The FORSE-1 epitope was associated with a major 286-kD protein band (Fig 2). This protein probably represents a glycoprotein and/or proteoglycan, based on polysaccharide composition of the FORSE-1 epitope previously established in brain studies (
FORSE-1 Is Co-expressed with Stage-specific Embryonic Antigens (SSEAs)
The FORSE-1 epitope is a component of the LeX antigen, as demonstrated in studies on brain (
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Discussion |
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Current thinking in the PNEC/NEB field supports the notion that the primitive undifferentiated pulmonary epithelium expresses a pluripotent phenotype before further commitment and differentiation into the respective specialized cell subtypes of the lung (
Here we show that FORSE-1-positive cells acquire expression of 5HT in a temporal manner during gestation corresponding to the development of the airways (Fig 4 and Fig 5). Cells co-expressing the FORSE-1 epitope and 5HT were replaced by mainly 5HT-expressing cells, signifying PNEC/NEB differentiation. Residual expression of a small number of the FORSE-1-positive cells was detectable in maturing NEBs. Recent studies have implicated NEB loci as the site for retention of a cell population with regenerative potential in the distal airways (
Studies on developing hamster lung have provided some additional insights into factors involved in the control of neuroendocrine differentiation and the role of NEBs in pulmonary epithelial proliferation (
The observation that the FORSE-1 epitope, equivalent to LeX, is gradually lost from PNECs/NEBs coincident with maturation of PNEC/NEB cells and acquisition of 5HT, suggests a potential role for FORSE-1 in this developmental programming. Co-labeling with antibodies to SSEA antigens supports the embryonic phenotype of the neuroendocrine-programmed cells that ultimately lose SSEA antigens as neuroendocrine maturation progresses towards term. Therefore, we found co-expression and patterns suggesting a sequential progression with NEB maturation. Earlier studies of SSEA-1-related antigens in developing human lung found that LeY, LeX, and sialyl LeX-i were strongly expressed in cells in lung buds, bronchial buds, and terminal buds, coincident with formation of the corresponding lung structures (
Glycosylation patterns of proteins and lipids residing on the cell surface have been extensively investigated as markers of embryogenesis and fetal development and as determinants of tumor-associated antigens and tumor properties, particularly metastasis (
Interestingly, our immunolabeling results suggest that the FORSE-1 epitope may be expressed exclusive of SSEA antigens or may precede SSEA antigen expression (Fig 6). Because FORSE-1 is essentially equivalent to LeX and the globo-series SSEA antigens must become fucosylated to become the lactoseries antigens as defined by Lewis X (LeX) and variants of LeX, such as sialylLeX, which represent terminal carbohydrate structures, the latter possibility is less likely. Therefore, FORSE-1 epitope expression may still constitute a unique antigen within the Lewis antigen group.
The Lewis antigens are identifiable with specific monoclonal antibodies and are strongly associated with a number of different tumors (
Confocal microscopy revealed localization of the FORSE-1 epitope to the plasma membrane of this progenitor subpopulation of PNECs/NEBs, suggesting probable involvement in cellcell interactions and/or cellmatrix interactions. Western blotting analysis demonstrated the FORSE-1 epitope on a 286-kD carrier protein which decreased significantly by postnatal day 7.
Among the molecules that are involved in cellcell interactions, we previously identified NCAM (neural cell adhesion molecule) on PNECs/NEBs using the MOC-1 monoclonal antibody (
Taken together, these earlier studies and our present data favor the idea of a potential progenitor cell type that is capable of multipotent differentiation depending on requirements of the pulmonary epithelium for repair after injury or in response to environmental influences (e.g., hypoxia). Retention of FORSE-1-expressing cells residing within mature NEBs suggests that this may be the cell type that is programmed for regeneration of pulmonary epithelium after toxic injury (
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Acknowledgments |
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Supported by funding from Canadian Institutes of Health Research (CIHR), operating and Group Grant on Lung Development (EC and HY, MOP12742 and MGP15270).
We thank Ms Elizabeth Bienkowski and Ms Veronica Wong for technical assistance. The FORSE-1 monoclonal antibody developed by Dr P.H. Patterson was obtained from the Developmental Studies Hybridoma Bank developed under auspices of the NICHD and maintained by the University of Iowa (Department of Biological Sciences; Iowa City, IA).
Received for publication April 3, 2002; accepted July 19, 2002.
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