related peptide (CGRP) is known to exist in the neuroendocrine cells and in ... CGRP immunoreactivity was present in granules of serous cells throughout the ...
Calcitonin Gene-related Peptide in Secretory Granules of Serous Cells in the Rat Tracheal Epithelium Peter Baluk, Jay A. Nadel, and Donald M. McDonald Cardiovascular Research Institute and Departments of Medicine, Physiology, and Anatomy, University of California, San Francisco, California
The tracheal epithelium of pathogen-free rats consists mainly of serous-type secretory cells, ciliated cells, basal cells, and a few neuroendocrine cells. Mucus-containing goblet cells are rare. Calcitonin generelated peptide (CGRP) is known to exist in the neuroendocrine cells and in sensory nerves of the tracheal mucosa and is released into the airway lumen by sensory nerve stimulation. In this study, we determined whether epithelial serous cells are another source of CGRP. Tracheas of adult male specific pathogen-free F344 rats were immunostained by an avidin-biotin technique either as whole mounts or as cryostat sections using two different polyclonal primary antibodies to rat CGRP. Some specimens were stained for CGRP-like immunofluorescence and examined with a confocal microscope. CGRP immunoreactivity was present in granules of serous cells throughout the trachea. In whole mounts, the stained cells were most abundant between the cartilaginous rings, especially in the rostral trachea, where they constituted 56% of the epithelial cells in contact with the tracheal lumen. Serous cells were easily distinguished from neuroendocrine cells and nerve fibers with CGRP immunoreactivity. In evidence that the CGRP immunoreactivity was specific, the staining of serous cells was abolished by omitting the primary antibody and by absorption with 10 j.tg/ml CGRP. Antibodies to substance P, vasoactive intestinal polypeptide, and tyrosine hydroxylase did not stain epithelial serous cells. An antibody to protein gene product 9.5 labeled neuroendocrine cells, but not serous cells. Injection of capsaicin (150 j.tg/kg intravenously), a substance known to degranulate epithelial serous cells, reduced the staining ofthe serous cells for CGRP. CGRP-like immunoreactivity thus appears to be a useful marker of epithelial serous cells. We conclude that CGRP is present in the granules of epithelial serous cells in the rat trachea and is secreted into the tracheal lumen by stimuli that degranulate these cells.
Calcitonin gene-related peptide (CGRP) has been demonstrated by radioimmunoassay and by immunohistochemistry in the respiratory tract of several species, including humans. There is general agreement that some unmyelinated sensory nerve fibers of the respiratory tract are CGRP-immunoreactive and that in some axons CGRP is co-localized with substance P (SP) and other tachykinins (1, 2). It has been suggested that CGRP appearing in the tracheal lumen after nerve stimulation originates from sensory nerves (3, 4), although CGRP is also present in "neuroendocrine" cells of the airway epithelium (1, 5-7, 44). However, it is not known whether CGRP is contained.in any other cells in the epithelium in the respiratory tract. (Received in original form August 12, 1992 and in revised form December 7, 1992)
In this study, we sought to demonstrate whether CGRP is present in granules of secretory cells of the rat tracheal epithelium. We also determined whether CGRP could be released into the tracheal lumen by stimuli that degranulate these cells. We used a sensitive immunohistochemical staining method to localize CGRP and we used whole mount preparations of the trachea of specific pathogen-free rats so that all cells of the tracheal epithelium could be examined. In these animals, the tracheal secretory cells consist mainly of serous cells; few goblet cells are present (8). We also determined whether SP, protein gene product 9.5 (PGP 9.5), vasoactive intestinal polypeptide (VIP), and tyrosine hydroxylase (TH) , all of which are known to be present in some tracheal nerve fibers, are also present in the secretory cells of the airway epithelium. Some of these results were reported previously in an abstract (9).
Address correspondence to: Dr. Peter Baluk, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0130.
Materials and Methods
Abbreviations: avidin-biotin-peroxidase complex, ABC; calcitonin generelated peptide, CGRP; phosphate-buffered saline, PBS; protein gene product 9.5, PGP 9.5; substance P, SP; tyrosine hydroxylase, TH; vasoactive intestinal peptide, VIP. Am. J. Respir. Cell Mol. BioI. Vol. 8. pp. 446-453, 1993
Animals Pathogen-free, male F344 rats (weight, 240 to 270 g; age, 12 wk) were purchased from Simonsen Inc. (Gilroy, CA). They were housed under barrier conditions to prevent re-
Baluk, Nadel, and McDonald: CORP in Rat Tracheal Epithelial Cells
spiratory infections, because infections can alter the types and proportions of epithelial cells present in the airways (8). The rats were anesthetized with sodium methohexital (70 mg/kg intraperitoneally; Brevital; Eli Lilly & Co., Indianapolis, IN). Immunohistochemistry Specimens of the rat trachea were prepared as whole mounts or as 8-p,m cryostat sections. For whole mount preparations, the trachea was cut open along the ventral midline and pinned flat on pieces of Sylgard (Dow Corning Corp., Midland, MI). Specimens were fixed overnight at 4°C in picric acid-paraformaldehyde fixative, washed thoroughly with distilled water, and then made permeable to antibodies by dehydration in ethanol, clearing in xylene, and rehydration in ethanol, as described previously (10). Whole mounts and cryostat sections of rat trachea were stained immunohistochemically for the presence of CORP, SP, PGP 9.5, VIP, and TH. Whole mounts were incubated in phosphatebuffered saline (PBS) containing 5 % normal goat serum (Tago Inc., Burlingame, CA) to block nonspecific binding of the antibodies and then incubated for 24 to 48 h in primary rabbit polyclonal antibody to CORP diluted 1:1,000 in PBS containing 1% normal goat serum. The characteristics of the primary antibodies used are described in Table 1. Primary antibodies were diluted in PBS containing 0.3 % Triton X-I00 (Sigma Chemical Co., St. Louis, MO) and 0.01% thimerosal (Sigma) as an antibacterial agent. The binding of the primary antibody was visualized with an avidin-biotinperoxidase complex (ABC) method according to the manufacturer's recommended dilutions (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) using diaminobenzidine as the chromogen. Cryostat sections were incubated in diluted antibody solutions for 1 h, and whole mount preparations were incubated for 24 h. The stained whole mounts were washed in distilled water, dehydrated with ethanol, cleared with toluene, and mounted with the epithelial surface up on glass slides in Permount (Fisher Scientific, San Francisco, CA). All incubations were done at room temperature on a rotator. The numerical densities of immunoreactive cells observed in whole mount preparations were determined with a computer-assisted morphometry system described previously (10). In making these measurements, we examined a total epithelial area of 100,000 p,m2 in the rostral trachea (rings 6 to 10) and caudal trachea (rings 20 to 24) in each of 5 rats. To test the specificity of the staining for CGRP, the primary antibody, diluted 1:1,000, was incubated overnight at
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4°C with synthetic rat CGRP (1 to 10 p,g/ml; Peninsula Laboratories, Belmont, CA). This CGRP antibody (RPN 1842) has also previously been shown not to cross-react with VIP, neuropeptide Y, SP, galanin, leu-enkephalin, gastrinreleasing peptide, calcitonin, or bombesin (6). In other experiments, the primary CGRP antibody was omitted to test for nonspecific binding of the secondary antibody. To confirm the identity of structures that were stained in whole mounts of the trachea, some specimens were frozen, cut as 8-p,m cryostat sections, and immunostained. Other specimens were fixed and embedded in epoxy resin as described previously (8). Sections 0.5 to 1 p,m in thickness were stained with toluidine blue or PAStAlcian Blue and observed with brightfield or Nomarski optics. In other experiments, optical sections of serous cells and nerves in whole mount preparations, in which the primary antibody to CGRP was visualized by a fluorescein-labeled secondary goat anti-rabbit antibody (Tago Inc.) diluted 1:50, were examined with a Nikon x60 oil-immersion objective lens and a BioRad MRC 600 confocal microscope (BioRad Microsciences Division, Cambridge, MA). In 5 rats, secretion was induced in serous cells of the trachea by injecting capsaicin (150 p,g/kg intravenously) 5 min before killing the rats, as described previously (8). Statistical Analysis All values are expressed as mean ± SEM. Differences between means were considered statistically significant when P values < 0.05, as determined by Student's unpaired t test or by ANOVA (11).
Results In whole mounts of the rat trachea, CORP immunoreactivity was found in numerous epithelial cells and in nerve fibers (Figure 1). Two easily distinguished types of epithelial cells had CGRP immunoreactivity: serous-type secretory cells and neuroendocrine cells. When examined from the luminal surface, the serous cells were round or oval in shape and had a diameter of 5.8 ± 0.2 p,m(mean ± SEM; n = 50; Figures 2 and 6). By focusing on different levels of the whole mount preparations, it was clear that most of the CGRP immunoreactivity was present in .the apical portions of the serous cells, and some staining was clearly localized in granules (Figure 2). Granules were particularly well resolved in optical sections of the serous cells obtained by confocal microscopy (Figure 3). CORP-immunoreactive serous cells were most abundant between the cartilaginous rings of the rostral trachea (Figure 1). In these regions, 10,070 ± 970 stained cells were present per mm' of epithelial surface (n = 5
TABLE 1
Primary antibodies used Antigen
Calcitonin gene-related peptide Calcitonin gene-related peptide Substance P Protein gene product 9.5 Vasoactive intestinal peptide Tyrosine hydroxylase
Code No.
Dilution
Source
Reference
RPN. 1842 RIO 20064 13C4 MaVIP TE 101
1:1,000 1:1,000 1:1,000 1: 1,000 1: 1,000 1:1,000
Amersham Corp., Arlington Heights, IL Dr. Catia Sternini, CURE, UCLA Incstar Corp., Stillwater, MN Ultraclone Ltd., Isle of Wight, UK East Acres Biologicals, Southbridge, MA Eugene Tech Inc., Allendale, NY
6 23 10 41 42 43
Figure 1. A low-magnification view of the rat tracheal mucosa in a whole mount preparation viewed from the luminal side. CGRP immunoreactivity is present in many epithelial cells (small dark spots) and nerve fibers (arrows). Stained cells are more abundant in the epithelium over the intercartilaginous spaces than over the cartilages (asterisks). Bar = 100 pm. Figure 2. A view obtained by conventional brightfield microscopy of a whole mount of the rat trachea stained for CGRP immunoreactivity. The plane of focus is near the apical surface of the epithelial cells. Many cells are so intensely stained that granules are clearly visible only in a few of them. Bar = 10 JJ-m. Figure 3. An optical section about 0.5 JJ-m in thickness obtained by confocal microscopy of a whole mount of the rat trachea stained for CGRP immunofluorescence. The plane of focus is near the apical surface of the epithelial cells. Granules are clearly visible in some of the cells. Bar as in Figure 2.
Baluk, Nadel, and McDonald: CORP in Rat Tracheal Epithelial Cells
tracheas). This number constituted 56% of the epithelial cells in contact with the tracheal lumen. CORP-immunoreactive serous cells were also present in the epithelium overlying the trachealis muscle. They were significantly less frequent in the caudal trachea, constituting only 3,000 ± 560 cells/rum' or approximately 18% of the epithelial cells. The immunoreactive serous cells were less numerous in the mainstem bronchi and were absent from the smaller airways. The same staining pattern was obtained with both of the antibodies to CORP used in this study. Immunostaining of sections of CORP confirmed the impressions gained from whole mount preparations. CORP immunoreactivity was present in nerves and in the apical portions of serous cells (Figure 4), often protruding into the airway lumen. Ciliated cells, basal cells, globule leukocytes, and submucous gland cells were unstained. When cryostat sections (Figure 5) or whole mount specimens were incubated with antibody to CORP which had previously been absorbed with 1 to 10 j.tg/ml of CORP, no staining was observed. Likewise, there was no staining when the primary antibody was omitted. Toluidine blue-stained sections of the trachea showed that the secretory cells that corresponded to the CORP-immunoreactive cells had a columnar shape and contained granules, mainly in the apical portions (Figure 6). The granules were strongly stained by toluidine blue but only faintly stained by PASIAlcian Blue. Mucous goblet cells were absent. The globule leukocytes within the tracheal epithelium stained more intensely with toluidine blue and had granules that were larger and more variable in their shape than those in secretory cells. Neuroendocrine cells of tracheal epithelium were also CORP-immunoreactive (Figures 4 and 9). These cells were easily distinguished from serous cells on the basis of appearance, size, number, and distribution. In neuroendocrine cells, the staining for CORP was stronger and did not appear granular. These cells were rare in cryostat sections, but when present, they were located near the epithelial basal lamina (Figure 4). In whole mounts, their cell bodies (8.5 ± 0.2 j.tm in diameter; n = 50) had broad processes that extended between other epithelial cells for an average distance of 19.9 ± 0.7 um, i.e., across a width corresponding to several epithelial cells (Figure 9). By focusing on different levels of the whole mounts, we confirmed that these cells were located near the base of the epithelium close to the plexus of CORP-immunoreactive nerve fibers. Though sparse, neuroendocrine cells were relatively uniformly distributed in the trachea: there were 45.8 ± 13.1 cells/mm' in the rostral trachea and 46.0 ± 9.7 cells/mm' in the caudal trachea. Neuroendocrine cells constituted only 0.5 to 1.4% of the CORP-containing epithelial cells in the rostral and caudal trachea, respectively. In the trachea, neuroendocrine cells invariably occurred as single cells, but clusters of up to 20 of the cells were frequently located at branch points of small bronchi. In the trachea of rats that received an intravenous injection of capsaicin, the number of serous cells with detectable immunoreactivity (6,010 ± 490 cells/rum' in the rostral trachea) was about 60% of the control value (P < 0.05; Figures 7 and 8) and the staining intensity of the remaining
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cells was reduced. In contrast, the number of neuroendocrine cells (33.4 ± 6.1 cells/nun') was not statistically different from the control and the intensity of staining for CORP appeared unaltered. The nerve plexus also appeared unchanged by this dose of capsaicin. None of the antibodies other than anti-CORP used in this study stained the serous epithelial cells. The antibody to POP 9.5 stained the network of nerves in and near the basal region of the epithelium and also stained the neuroendocrine cells (Figure 10). These POP 9.5-immunoreactive neuroendocrine cells appeared to correspond in their shape and distribution to those stained by the antibodies to CORP. The antibody to SP stained the network of nerves in and near the basal region of the epithelium but did not stain any epithelial cells or neuroendocrine cells (Figure 11). The antibody to VIP stained only a few epithelial nerve fibers, and the antibody to TH stained nerve fibers around blood vessels.
Discussion The novel and unexpected finding of this study was the presence of CORP immunoreactivity in granules of serous-type secretory cells. Intraepithelial nerves and neuroendocrine cells had CORP immunoreactivity, as has been described previously (1, 5-7, 12, 13,44). Therefore, in the rat trachea, CORP immunoreactivity is present in three separate cell populations: serous-type secretory cells, neuroendocrine cells, and nerve fibers. There are several reasons why we detected CORP immunoreactivity in rat tracheal epithelial cells while previous studies have not. First, we studied pathogen-free rats, where it is known that serous-type epithelial cells are abundant (14-16). It will be of interest to see if CORP immunoreactivity is found in epithelial cells of other species where serous cells are not abundant. Second, we used whole mount preparations to view all of the epithelial cells rather than histological sections. Third, we used a sensitive avidin-biotin-peroxidase complex (ABC) method, rather than fluorescently labeled secondary antibodies. Both of these technical factors improve the sensitivity of the immunohistochemical staining. In fact, our finding is not altogether unprecedented: Domeij and associates (17) reported that the apical portions of some epithelial cells in/the rat larynx had CORP immunoreactivity. However, they interpreted these cells as neuroendocrine cells. We feel justified in identifying the CORP-immunoreactive cells observed in this study as serous cells because they correspond in shape, distribution, and presence of granules. Serous cells are also easily distinguished from CORP-containing neuroendocrine cells, which are less numerous and have a different shape and distribution (6, 7). Ciliated cells are the only other cell type present in large numbers on the luminal surface of the tracheal epithelium of pathogen-free rats (8, 14); goblet cells are rare (18). Several observations suggest that the staining we observed was specific for CORP. First, the staining pattern was the same for both of the anti-CORP antibodies we used. Second, staining was abolished by preincubation with exogenous CORP. Third, none of the other antibodies to neuropeptides or other molecules in autonomic or sensory nerves stained the secretory granules in epithelial cells.
Figure 4. A cryostat section of the rat trachea stained for CORP immunoreactivity. Staining is present in the apical portions of serous cells, in a neuroendocrine cell located at the base of the epithelium (arrow), and in nerve fibers (arrowheads), but not in ciliated cells.
Bar = 10 JLm. Figure 5. A cryostat section of the rat trachea similar to that in Figure 4 but incubated with CORP antiserum that had been pre-absorbed
with 10 JLg/ml CORP. All specific staining is abolished. Bar as in Figure 4. Figure 6. A toluidine blue-stained section of a pathogen-free rat trachea embedded in epoxy resin. Two types of epithelial cells reach
the lumen: ciliated cells and-serous secretory cells. The apical portions of serous cells contain granules and project slightly into the lumen (arrowheads). A globule leukocyte with large granules is present within the epithelium (arrow). A profile of part of a submucous gland contains a few ciliated cells and a globule leukocyte (arrow). The other nonciliated epithelial cells do not have clearly visible granules. Bar = 10 JLm.
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Figure 7. A view of the tracheal mucosa from a control rat with the focus on the apical surface of the epithelial cells. Many epithelial cells have strong CGRP immunoreactivity, which in some cases is clearly localized to granules. Bar = 10 pm. Figure 8. A region of the tracheal mucosa similar to that in Figure 7 but from a rat treated with capsaicin (150 p.g/kg intravenously) 5 min earlier. The number and intensity of staining of CGRP-immunoreactive cells are reduced. Bar as in Figure 7. Figure 9. Tracheal mucosa incubated for CGRP immunoreactivity with the focus on the basal surface of the epithelial cells. Nerve fibers and three neuroendocrine cells (arrows) are stained. Bar as in Figure 7. Figure 10. A whole mount preparation of the rat trachea similar to that in Figure 8 but incubated with an antibody to protein gene product 9.5. The focus is on the basal surface of the epithelium. Nerve fibers and a few neuroendocrine cells (arrows) are stained. Bar as in Figure 7. Figure 11. A whole mount preparation of the rat trachea similar to that in Figure 8 but incubated with an antibody to substance P. The focus is on the basal surface of the epithelium. Nerve fibers are stained in a pattern similar to that for CGRP, but neuroendocrine cells are not stained. Bar as in Figure 7.
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CORP is known to be released into the lumen of the trachea by a capsaicin-sensitive mechanism (2). In a recent study, it was shown that electrical stimulation of the sensory nerves of the guinea pig trachea results in release of CORP into the lumen (3). Likewise, it was shown that capsaicin and high concentrations of potassium cause the release of CORP, neurokinin A, and SP into the lumen of the rat trachea (4). In both of these studies, the CORP was assumed to come directly from the sensory nerves. The latter investigators also noted that CORP was released into the lumen in much larger amounts than neurokinin A and SP. However, for two reasons, it is difficult to imagine that all of the CGRP found in the lumen comes from the nerve fibers. First, most of the nerve fibers are present at the base of the epithelium and relatively few reach the luminal surface (10, 14, 19). Second, the CORP presumed to be released from nerves would have to penetrate the barrier of tight junctions that links the apical surfaces of the epithelial cells. The present study suggests that serous cells may be a more likely source of CORP because secretory cells are clearly polarized to release their granules into the lumen, and capsaicin stimulates sensory nerves and degranulates serous cells in the doses that we used (8). Furthermore, our histological evidence suggests that serous cell granules contain -a large proportion of the CORP in the trachea. Additional work is needed to determine what fraction of the total CORP pool exists in serous cell granules, in neuroendocrine cells, and in nerves. Other questions worth investigating are whether serous cells synthesize CORP or whether they have a mechanism for taking up exogenous CGRP released from sensory nerves. The presence of CGRP in cells of non-neuronal origin is well documented in several other organs: CORP is present in thyroid parafollicular cells (20, 21), skin Merkel cells (22), some cells in the islets of Langerhans in the pancreas (23), and some chromaffin cells of adrenal glands (24). The CORP in secretory granules of tracheal epithelial cells may have effects on other epithelial cells. Indeed, CORP receptors have been described in the epithelium (25). Furthermore, CORP increases the beat frequency of cilia of dog epithelial cells grown in culture (26). In addition, CORP may have a trophic effect on the replication or growth of tracheal epithelial cells; CORP has been reported to stimulate the proliferation of endothelial cells (27). Other airway cell types may also be targets for CORP released into the lumen. Tracheal smooth muscle cells contract and blood vessels vasodilate in response to CORP (2, 28). However, the indirect routes that CORP from serous cells would have to take to reach these targets makes these effects unlikely. Sensory nerves are another possible target. One of the roles of unmyelinated sensory nerves in the respiratory tract is to release SP and other tachykinins that mediate "neurogenic inflammation" (19). Furthermore, CORP coexists with tachykinins in these sensory nerves (2). Recently, it has been reported that CORP inhibits plasma extravasation caused by some inflammatory mediators (29). Therefore, tachykinins from nerves and CGRP from nerves and epithelial cells may have opposing pro- and anti-inflammatory actions which serve to limit the extent of "neurogenic inflammation." A further role for CORP in the airways may be to attract inflammatory cells to sites of inflammation. It
has been shown that CGRP and peptide fragments resulting from its degradation by endopeptidases are chemotactic for eosinophils (30-32). In addition to the physiological implications of the presence of CGRP immunoreactivity in serous cells, the neuropeptide may be a useful marker for serous-type secretory cells. Reliable markers exist for rat airway ciliated cells, basal cells, Clara cells (33,34), and globule leukocytes (35). However, serous secretory cells stain poorly with PAS/AIcian Blue (18) and previously have been identified with certainty only with electron microscopy (14-16). The immunohistochemical markers for rat airway secretory cells which are currently available stain both mucous and serous cells or only subpopulations of them (34). Therefore, the availability of a convenient marker may permit studies of changes of epithelial cell populations in pathologic conditions in which mucous-type epithelial cells proliferate. Examples of such conditions are chronic exposure to sulfur dioxide (36) or tobacco smoke (37, 38) or respiratory tract infections (8). It has only recently been realized that the secretory cells constitute a major population of progenitor cells that can differentiate into mucous cells (39, 40). Future studies may show how CGRP immunoreactivity in the serous cells changes when the rats become infected and the serous cells are transformed into mucous cells. In conclusion, CGRP is present in the granules of tracheal epithelial serous cells of pathogen-free rats and is secreted into the tracheal lumen by stimuli that degranulate these cells. Acknowledgments: This work was supported in part by a University of California Tobacco-Related Disease Research Program Grant and by NIH Program Project Grant HL 24136. Simona Ikeda gave excellent technical assistance. We thank Jim Hayden and Steve Pfeiffer of BioRad Microsciences Division (Cambridge, MA) for access to a BioRad MRC 600 confocal microscope. We also thank Dr. Catia Sternini (Center for Ulcer Research and Education, University of California, Los Angeles) for a gift of her antibody to CGRP.
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