ARTICLE |
Correspondence to: Laura Guembe, Dept. of Histology and Pathology, University of Navarra, 31080 Pamplona, Spain. E-mail: lguembe@unav.es
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Summary |
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Endothelins (ET) are a family of regulatory peptides displaying, among other abilities, potent constrictor actions. We studied the perinatal time course expression and distribution of ET in the mouse airway epithelium. In fetal mouse, ET-immunoreactivity (IR) appeared earlier (gestational Day 18) in the epithelium of upper (bronchi and large bronchioles) than in lower airways, being scarce and mainly located in the apical cytoplasm. As the lung developed, ET-IR became gradually stronger and extended throughout the cell in both bronchi and bronchioles. ET-IR was found in most airway epithelial cells. Clara cells were positive for ET, whereas ciliated and endocrine cells were not. In adult lungs, part of the myocytes and parenchymal cells also showed ET-IR. In both developing and adult mouse lungs, the cell distribution of ET-IR in the epithelium is compatible with apical and/or basal secretion. The presence of ET in mouse airway epithelium during the perinatal period may indicate a role for ET as a growth factor in lung development and its involvement in control of lung ventilation at birth. (J Histochem Cytochem 49:13011309, 2001)
Key Words: mouse lung, development, endothelin, Clara cells, smooth muscle, parenchymal cells
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Introduction |
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ENDOTHELIN (ET), a 21 amino-acid peptide isolated from culture medium of porcine endothelial cells (
In the perinatal lung, a role for ET in the control of pulmonary vascular tone has been suggested because both vasoconstrictor (
The present study was carried out in the mouse, a species in which only postnatal studies concerning the presence of ET in lung have been made, rendering contradictory results. Immunocytochemical techniques were applied in a systematic manner to developing (fetal and early postnatal) and adult lungs to determine the time of appearance of ET-IR, not reported to date, as well as the further time course pattern along pulmonary development.
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Materials and Methods |
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Pathogen-free Swiss mice bred at the Centro de Investigación en Farmacología Aplicada (CIFA; University of Navarra, Spain) were used in this study. Lungs of 36 mice were studied: eight adult lungs (six of them pregnant females: P-ad) and 28 subjects at various stages of development [gestational Days (E) 17, 18, and 19 and postnatal days (P) 0, 1, 2 and 6], the age at which mouse lung is considered to be mature (Four subjects of each age).
The adults were anesthetized with 12.5% urethane (1 ml/100 g body wt). The abdomen of pregnant females was opened and the fetuses were rapidly removed from the uterus and chilled in ice. The newborn mice were anesthetized by hypothermia (
Immunocytochemistry
Paraffin sections (4 µm thick) were mounted on glass slides and immunocytochemical staining was performed using the avidinbiotin complex (ABC) method (
Controls
Several controls were performed: (a) omission of the primary antibody or substitution by a non-immune serum; (b) positive control with mammalian tissues (rat stomach); and (c) absorption tests with the corresponding antigens. Antisera were preincubated for 12 hr at 4C with their respective synthetic antigens at a concentration of 0.110 nmol per ml of optimally diluted primary antiserum before application to tissue sections.
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Results |
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In the present study, using various antisera raised against ET-1, IR was found in airway epithelial cells in both developing and adult mouse lung.
In developing lungs (Fig 1 and Fig 2) ET-1-IR was detected for the first time at E-18 (Fig 1A and Fig 1B) in upper airways (bronchi and large bronchioles) and gradually extended towards the rest of the bronchiolar tree (Fig 2). Early ET-1-IR was scarce and was mainly located in the apical cytoplasm of epithelial cells (Fig 1B). While development continued (end of gestation and first days of postnatal life), the immunostained area gradually increased, extending towards the base of the cells (Fig 1C and Fig 1D). From approximately P-6 onwards, the entire cytoplasm of epithelial cells was immunoreactive (Fig 1E and Fig 1F). In bronchiolar epithelium (Fig 2), ET-1-IR, although appearing later, showed a similar cytological pattern, sparse and mainly apical at the beginning (Fig 2B) and then extending throughout the cell (Fig 2C and Fig 2D). The application of antiserum against CGRP and ET-1 to serial reversed-face sections showed that fetal CGRP-positive endocrine cells (Fig 2E) did not stain with the antiserum against ET (Fig 2F).
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The time of appearance of ET-1-IR depended not only on the size of airways but also on the antiserum used. In bronchi, the earliest IR (E-18) was obtained with the 2020 antiserum (Fig 1A and Fig 1B). Conversely, 2092 antiserum gave IR for the first time in postnatal lungs (P-1; data not shown). Nevertheless, the evolution pattern of ET-1-IR (apical to the entire cytoplasm) was similar with all the antisera.
Big-ET-1-IR showed in the epithelium a similar developmental pattern to that of ET-1: first in upper airways, first in the apical cytoplasm, and extending afterwards towards the rest of the cell. However, big-ET-1 was detected later in development (P-2 lungs) than ET-1 (data not shown).
In adult lung (Fig 3), IR for ET-1 and big-ET-1 was obtained in most epithelial cells of both bronchi and bronchioles (Fig 3A, Fig 3C, and Fig 3D). Among the cell types present in airway mouse epithelium, Clara cells were clearly identified as immunoreactive for ET-1 (Fig 3C). Conversely, ciliated cells, when their identification was certain, were negative (Fig 3C). CGRP-positive endocrine cells (Fig 3E) were also negative for ET-1 (Fig 3F). The specificity of IR was tested by absorption controls (Fig 3G and Fig 3H).
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In adult lungs, in addition to epithelial cells, ET-1-IR and big-ET-1-IR were obtained in part of the smooth muscle population of airway walls. Positive myocytes were especially numerous in bronchi (Fig 4A4C). In blood vessels, some smooth muscle cells also exhibited immunostaining (Fig 4D and Fig 4E), although usually with weaker intensity than in airways. Parenchymal cells of adult lungs also showed IR (Fig 4F). Comparing IR for ET and the marker of Type II pneumocytes (SP-C), a similar general distribution pattern was observed (Fig 4F and Fig 4G), although no clear cellcell correspondence was found. No IR in endothelial cells was found in any age with any of the antisera used.
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Discussion |
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Little is known concerning the appearance and evolution of ET in mammalian lung development. In the present work, IR for ET-1 and big-ET-1 was detected in the airway epithelium of both developing and adult mouse lungs. IR was found in most epithelial cells (Clara cells). Conversely, IR in airway and vascular muscle and parenchymal cells was observed only in adults.
The perinatal role of ET is now being studied. ET could have both a constrictor or a dilator effect on pulmonary smooth muscle and appears to play a role in the maintenance of the bronchial and vasomotor tone both in gestation (
In relation to the appearance and evolution of ET in mammalian lung there is no concordance in the data shown by different authors both in the same or in different species. In our study, ET-1-IR was demonstrated in most developing mouse airway epithelial cells, as reported in fetal rat (
The presence in mouse lung of IR for big-ET-1, the precursor of ET, was an expected result. Nevertheless, big-ET-1-IR was detected later (newborns) than ET-1-IR (fetuses). Apart from the possible lower sensitivity of the antiserum against big-ET-1 used, this unexpected result could be due to differences in the activity of the ET-1 converting enzyme compared to the rate of big-ET-1 translation in different stages of development. In fact, in pig lung a greater activity for the converting enzyme in the early postnatal period has been suggested (
In the present work we found ET-1-IR in most airway epithelial cells of developing and adult mouse, confirming the data obtained by part of the authors in several mammalian species. Immunoreactive cells found in the present study have been clearly identified as Clara cells, as reported in mice and rats (
The finding that in mouse ET-IR was located in the apical region of airway epithelial cells suggests the secretion of ET towards the airway lumen in both developing and adult lungs. In fact, apical ET-IR is the only one present in the epithelium at the beginning, before extending towards the base. Apical secretion of ET would explain the presence of ET in the bronchoalveolar lavage (
The presence of ET-1-IR in airway smooth muscle cells reported in the present work has been previously described in several mammalian species. Nevertheless, in mouse lung only some of the previous studies had reported ET-IR of smooth muscle (
As indicated, ET-IR was found in cells of pulmonary parenchyma and the distribution of these cells was similar to that of Type II pneumocytes. In agreement with this possibility, there are some reports on the expression of ET in this cell type (
In summary, the appearance and gradual increase of IR for ET in mouse lung at late gestation and during the neonatal period in most airway epithelial cells supports the importance of ET in the perinatal period. According to our results, not only basal but also apical secretion of ET could take place both in development and in adulthood. Apical secretion could be present from the beginning, whereas basal secretion would appear later and appears to enhance along development. In addition to the reported control of ventilation in both development and adulthood, ET could also act as a growth factor in developing lung.
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Acknowledgments |
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Supported by the Spanish Ministry of Education and Science (DGCYT project no. PB93-0711) and the University of Navarra (PIUNA), and by a grant from the Departamento de Educación y Cultura del Gobierno de Navarra, Spain (LG).
We would like to thank Prof J.M. Polak for the antisera against ET and CGRP and for critically reading the manuscript. We thank Dr Whitsett for the antiserum against SP-C. We thank I. Ordoqui and A. Urbiola for their technical assistance.
Received for publication November 8, 2000; accepted April 18, 2001.
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