Division of Pathology, Departments of 1 Pediatric Laboratory Medicine and Pathobiology and 2 Physiology and Cell Biology, Research Institute and Hospital for Sick Children and University of Toronto, Toronto, Ontario, Canada M5G 1X8
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ABSTRACT |
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The pulmonary neuroendocrine
cell system comprises solitary neuroendocrine cells and clusters of
innervated cells or neuroepithelial bodies (NEBs). NEBs figure
prominently during the perinatal period when they are postulated to be
involved in physiological adaptation to air breathing. Previous studies
have documented hyperplasia of NEBs in cystic fibrosis (CF) lungs and
increased neuropeptide (bombesin) content produced by these cells,
possibly secondary to chronic hypoxia related to CF lung disease.
However, little is known about the role of NEBs in the pathogenesis of
CF lung disease. In the present study, using a panel of cystic fibrosis transmembrane conductance regulator (CFTR)-specific antibodies and
confocal microscopy in combination with RT-PCR, we demonstrate expression of CFTR message and protein in NEB cells of rabbit neonatal
lungs. NEB cells expressed CFTR along with neuroendocrine markers.
Confocal microscopy established apical membrane localization of the
CFTR protein in NEB cells. Cl conductances corresponding
to functional CFTR were demonstrated in NEB cells in a fresh lung slice
preparation. Our findings suggest that NEBs, and related neuroendocrine
mechanisms, likely play a role in the pathogenesis of CF lung disease,
including the early stages before establishment of chronic infection
and chronic lung disease.
cystic fibrosis transmembrane conductance regulator; expression in neuroepithelial bodies; chloride
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INTRODUCTION |
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THE PULMONARY NEUROENDOCRINE cell (PNEC) system, dispersed throughout the mucosa of the airways, is composed of single cells and innervated clusters, termed neuroepithelial bodies (NEBs; see Refs. 12 and 60). In animals and humans, NEBs express a variety of neuropeptides and a bioactive amine, serotonin (5-HT; see Refs. 10, 11, 67, 72). The expression of bombesin (BN) or gastrin-releasing peptide (GRP) in mammals is of relevance to lung development since GRP receptors have been found in the surrounding mesenchyme and submucosal glands that produce mucus secretions (19, 64). BN/GRP has mitogenic effects (34, 69), and its levels were found to be increased under a variety of pathological conditions (1). The mechanism by which expression is regulated in NEBs is not well known, but BN/GRP and amine (5-HT; residing in dense core vesicles) are released under conditions of hypoxia (13). Evidence for an O2 sensor in NEBs has come from studies in fetal/neonatal rabbits where it has been shown that NEBs possess a membrane-localized O2 sensor complex consisting of a NADPH oxidase coupled to an H2O2-sensitive K+ channel (65). This membrane-bound sensor channel complex initiates hypoxia-induced signal transduction via activation of Ca2+ channels and exocytosis of amine/peptide neurotransmitters (65, 73). In the case of innervated NEBs, the hypoxia signal is transmitted to the brain stem via vagal afferents to affect control of breathing (61). In addition, local paracrine release of mediators could affect other airway functions (47).
The status of PNECs in cystic fibrosis (CF) lung disease has been examined in a few studies. Johnson et al. (30), in a series of CF cases ranging in age from 3 days to 55 yr, found up to a sixfold increase in BN/GRP-immunoreactive cells in bronchioles compared with that in normal controls and cases with prolonged mechanical ventilation. Linear hyperplasia of BN/GRP/calcitonin-positive cells was noted in a 4 yr old with CF lung disease and focal hyperplasia in a 5 yr old with CF (70).
Although cystic fibrosis transmembrane conductance regulator (CFTR)
expression has been well documented in lung epithelial cells
(44), there is no information about CFTR expression in PNECs, including NEBs. We present here for the first time evidence in a
rabbit neonatal model that CFTR is expressed at both mRNA and protein
levels in NEB cells, together with the demonstration of
Cl conductances corresponding to functional CFTR in these
cells. The occurrence of CFTR in the pulmonary "neuroendocrine"
cell type is not surprising given that these cells are an integral component of airway epithelium, and CFTR expression has been recently demonstrated in neural cells (68). The highly specialized
nature of NEB cells, including secretion of a variety of bioactive
molecules, may impact on several aspects of the pathogenesis of CF lung
disease, particularly during the early preinfectious stage.
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METHODS |
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Lung tissue removal and preparation. New Zealand rabbits and newborns on day 2 were killed by lethal injection in accordance with Canadian Council on Animal Care guidelines, and the lungs were dissected. For whole mount staining, excised lungs were immersed in CO2-independent medium (GIBCO BRL, Burlington, Ontario, Canada), and the lung parenchyma was removed under a dissecting microscope to leave behind intact bronchial trees. To study NEBs in situ, fresh and formaldehyde-fixed lung slices (200-400 µm) were obtained with a Leica vibratome (model 1000s).
Cell culture. T84 colon carcinoma cells, which served as a positive control for CFTR, were grown in RPMI medium supplemented with 10% FBS.
Immunocytochemistry and confocal microscopy.
Immunocytochemistry was performed on vibratome sections and dissected
bronchial trees using modifications of procedures previously published
(65). The CFTR-specific antibodies used in this study are
described in Table 1 and include the
well-characterized CFTR antibody (M24-1; Genzyme), the recently
introduced TAM18 monoclonal antibody against the COOH-terminal amino
acid residues 1468-1480 (Neomarkers; see Ref. 9), and
those provided by Transgene (MATG1061 and MATG1031; see Ref.
45). The specificity of these antibodies was verified by
Western blot on CFTR-positive human Caco-2 cell line extracts using a
method published previously (41). Specificity for rabbit
CFTR was verified in whole rabbit lung extracts as previously described
(40, 75). Neuroendocrine markers were demonstrated with
antibodies against 5-HT (DiaSorin, Stillwater, MN) and neural cell
adhesion molecule (NCAM) epitopes (MOC-1 and a rat monoclonal) obtained
from Euro-Diagnostica (Arnhem, The Netherlands) and Immunotech
(Marseille, France), respectively. The specificity of these
latter antibodies has been described previously (13, 50).
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Isolation of NEBs for RT-PCR. A procedure to remove individual NEBs from vibratome slices used siliconized micropipettes (similar to patch-clamp pipettes). Micropipettes were autoclaved, and the pipette buffer (PBS) was treated with diethyl pyrocarbonate to achieve a RNase-free condition. NEBs were visualized by staining with neutral red (74) and were aspirated in the pipettes mounted in a micromanipulator. Aspirated NEBs were ejected into a RNA microfuge tube, and RNA was extracted from NEBs and control cell lines and tissues using a NucleoSpin kit (Clontech).
Primers selected for amplification of CFTR were previously published (41) and corresponded to the exon 13-exon 14 junction, nuclear transcripts (nt) 2481-2512 (5'-TCACCGAAAGACAACAGCATCCACACGAAAAG-3'), and nt 2747-2777 (5'-CACAGCACAACCAAAGAAGCAGCCACCTC-3') of human CFTR cDNA. For normalization of total RNA, a primer pair representing humanElectrophysiology. For electrophysiological studies, neonatal New Zealand rabbits of both sexes were used between 1 and 5 days of age. The lungs were perfused with Krebs solution and then inflated with 2% agarose (FMC Bioproducts, Rockland, ME). Transverse lung slices (150-300 µm) were cut with a Vibratome (Ted Pella, Redding, CA). Sectioning was performed with tissue immersed in ice-cold Krebs solution that had the following composition (in mM): 140 NaCl, 3 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, and 5 glucose at pH 7.3 adjusted with HCl. To identify NEB cells in fresh lung tissue, the slices were incubated with vital dye neutral red (0.02 mg/ml) for 15 min at 37°C as previously described (20, 74). For electrophysiological recordings, the lung slices were transferred to a recording chamber mounted on the stage of a Nikon microscope (Optiphot-2UD; Nikon, Tokyo, Japan). For the demonstration of voltage-activated, hypoxia-sensitive K+ current, a feature characteristic of NEB cells, the perfusing Krebs solution for the K+ current recording had the following composition (in mM): 130 NaCl, 3 KCl, 2.5 CaCl2, 1 MgCl2, 10 NaHCO3, 10 HEPES, and 10 glucose at pH 7.35. When K+ currents were recorded, the pipette solution contained (in mM) 30 KCl, 100 potassium gluconate, 1 MgCl2, 4 MgATP, 5 EGTA, and 10 HEPES (72). Whole cell patch recordings were performed as described by Hamill et al. (23). Hypoxia in the bath solution was achieved by bubbling the reservoir that fed the perfusion chamber with 95% N2 and connecting the reservoir to the chamber with low-gas permeability tubing. The PO2 level in the perfusion medium was 15-20 mmHg (20).
For assessment of Cl ![]() |
RESULTS |
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CFTR localization in lung cells as determined by confocal
microscopy.
Confocal microscopy on lung slices was employed to discriminate double
labeling of NEBs with combinations of antibodies. Antibodies to NCAM
epitopes, both MOC-1 and the rat anti-mouse NCAM antibody, labeled the
plasma membrane surrounding individual neuroendocrine cells within the
NEBs (Fig. 1, A and
B). We used an anti-laminin antibody to outline the
bronchiolar basement membrane and showed that the NEB surface is
exposed to the airway lumen (Fig. 1A). The surrounding
mucociliary epithelium did not stain with NCAM-specific antibodies.
NEBs were also identified by their content of 5-HT, as
demonstrated with a specific antibody to 5-HT (Fig.
1C).
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Expression of mRNAs for tryptophan hydroxylase and CFTR in NEBs
using RT-PCR.
To confirm that CFTR is expressed in NEB cells, we applied RT-PCR to
NEB clusters that were removed using patch-clamp pipettes (Fig. 3,
A-C). T84 cells served as a positive control
for CFTR message expression, and rabbit cortical brain served as a
positive control for expression of tryptophan hydroxylase (TH), a
marker of NEBs, since it is the rate-limiting enzyme for synthesis of 5-HT.
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Electrophysiology.
We first established that the neutral red-positive NEBs in lung slices
exhibited voltage-gated K+ currents, as reported previously
(74). Depolarizing steps from a holding potential of 60
to +30 mV evoked outward K+ currents in the majority of the
cells tested (95%; Fig.
5A). Again, as previously reported, the exposure to a hypoxic stimulus resulted in a rapid and reversible reduction in amplitude and time
course of the outward K+ currents in NEB cells. Current
amplitudes at +30-mV test potential were reduced by 34% after hypoxic
exposure (Figs. 5B). Washout of the hypoxic solution led to
recovery of the outward K+ current (Fig. 5C).
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DISCUSSION |
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Our studies demonstrate for the first time that CFTR is expressed
in an airway cell type that has not been previously considered as a
potential contributing component in the pathobiology of CF lung
disease. Anti-CFTR antibodies have been used extensively in a variety
of studies to demonstrate expression of CFTR protein in lung epithelium
and other tissues (2, 4-7, 15, 16, 21, 24-27, 29, 31,
37, 39, 43, 76), including fetal rabbit (40). In
agreement with the above studies, CFTR protein was also localized to
the apical region of postnatal rabbit lung epithelial cells.
Enhancement with the CARD system identified abundant CFTR protein in
epithelial cells and was necessary to demonstrate expression in NEB
cells. Overall, CFTR protein levels in NEBs appeared lower compared
with that in surrounding mucociliary epithelium. In isolated NEBs and
in single NEB cells, RT-PCR confirmed expression of CFTR mRNA.
Patch-clamp analysis confirmed the presence of a CFTR Cl
conductance in NEB cells. Thus pulmonary NEBs should now be included among a growing number of cell types that express functional CFTR, including thyroid (16), endothelium (58),
pancreas (37, 76), intestine (21, 25), and
brain (26, 43). Taken together, the patterns of
tissue-specific CFTR expression suggest that this Cl
channel function may be involved in a variety of physiological processes.
From a physiological point of view, PNECs, both single cells and NEBs, are of great interest, since a wide variety of neuropeptides and amines are expressed in these cells (11, 55, 60). Several of the peptide products, particularly BN, exhibit a range of physiological and cellular functions, including mitogenesis, bronchial smooth muscle contraction, vasoactivity (34), mucus secretion (3), branching morphogenesis (33), and as a chemoattractant (14). How the expression of the neuropeptides in PNECs and NEBs is controlled is not known, but recent evidence suggests that epithelial-mesenchymal interactions are important as well as hypoxia (11, 49). We have shown that NEBs possess an O2 sensor function that consists of an O2-sensitive NADPH oxidase complex coupled to a H2O2-sensitive K+ channel (65, 73, 74). Thus it is postulated that this signaling complex depolarizes the NEB cell under conditions of hypoxia to trigger Ca2+ influx and subsequent release of neuropeptides and 5-HT in the surrounding tissues and vasculature (12).
The potential involvement of PNECs in CF lung disease processes is
still not understood. However, reports of PNEC hyperplasia in lungs of
CF patients (30) and detection of significant amounts of
BN-like peptide (BLP) in urine from CF patients (46)
suggest an overactive neuroendocrine cell system. In contrast to the
age-dependent decrease of BLP in urine of normal humans, BLP levels in
urine from CF patients were found to be elevated for up to 5 yr of age (46). Thus BLP in urine from CF patients may reflect
increased PNEC activity. Because BN/GRP possesses mitogenic properties
on lung mesenchyme (48, 56) and regeneration of pulmonary
epithelium (e.g., Clara cells) appears to focus around NEBs that are
located at bifurcations (51), it is possible that, in
lungs of infants with CF, NEBs via secretion of neuropeptides are
essential for maintenance of the bronchopulmonary epithelium either
directly or through interaction with adjacent mesenchyme. Receptors for GRP can be found in the surrounding mesenchymal cells and also in the
submuscosal gland ductal cells in the lung (64). A defect in CFTR function is thought to lead to thickening of the mucus produced
by the submucosal glands as it tries to exit the ducts, whereas ductal
plugging is seen in CF lungs (17). Thus BN/GRP acting
through its receptor may be involved in this process. During this early
phase in CF lung disease, inflammatory changes without infection are
evident (18, 42), with cytokines such as inteleukin-8 playing a role in recruitment of neutrophils (42). Immune
cells have been noted to associate with NEBs (62), and
other factors such as tumor necrosis factor- may be involved in PNEC
differentiation (22).
Pulmonary disease is the major cause of morbidity and mortality in patients with CF and is largely the result of a scenario of overwhelming infections, compromised host defense mechanisms, and ineffective antibiotic treatment (44). The pathogenesis underlying this sequence of events is thought to involve hyperactive mucous cells and submucosal glands (35, 52), impaired mucociliary clearance of a thickened mucus, and possibly a compromised pulmonary cell defense system, all resulting from loss of CFTR functions (44). This may facilitate overgrowth of nonpathogenic bacterial strains in healthy lungs (38). Although bacterial infection is a prominent feature of CF lung disease, during the early stages of the disease (i.e., postnatal period), infection is minimal, and other factors may play more important roles in establishing the pathogenic process (32).
Our observations that CFTR mRNA and protein are expressed in NEBs
postnatally, that NEBs possess Cl conductance channels
with CFTR characteristics, and the demonstration that CFTR may modulate
other channels (36, 53) all lend support to the idea that
CFTR could have a physiological role in NEBs. Further studies are
needed to determine if NEB function, such as the autocrine/paracrine
modulation that figures prominently within the first few months of
life, is compromised by defective CFTR at a time when the infectious
process is minimal and mucociliary functions are maturing.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Pavirani (Transgene, Strasbourg, France) for the gift of anti-cystic fibrosis transmembrane conductance regulator MATG antibodies.
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FOOTNOTES |
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The studies were supported by funds provided by the Canadian Cystic Fibrosis Foundation.
Address for reprint requests and other correspondence: H. Yeger, Dept. of Paediatric Laboratory Medicine, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada (E-mail: hermie{at}sickkids.on.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 July 2000; accepted in final form 3 April 2001.
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