Functional morphology of pulmonary neuroepithelial bodiesExtremely complex airway receptors.

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THE ANATOMICAL RECORD PART A 270A:25– 40 (2003)
Functional Morphology of Pulmonary
Neuroepithelial Bodies: Extremely
Complex Airway Receptors
DIRK ADRIAENSEN,* INGE BROUNS, JEROEN VAN GENECHTEN, AND
JEAN-PIERRE TIMMERMANS
Laboratory of Cell Biology and Histology, Department of Biomedical Sciences,
University of Antwerp–RUCA, Antwerp, Belgium
ABSTRACT
Innervated groups of neuroendocrine cells, called neuroepithelial bodies (NEBs), are diffusely spread in the epithelium of intrapulmonary airways in many species. Our present understanding of the morphology of
NEBs in mammalian lungs is comprehensive, but none of the proposed
functional hypotheses have been proven conclusively. In recent reviews on
airway innervation, NEBs have been added to the list of presumed physiological lung receptors. Microscopic data on the innervation of NEBs, however, have given rise to conﬂicting interpretations. Using neuronal tracing,
denervation, and immunostaining, we recently demonstrated that the innervation of NEBs is much more complex than the almost unique vagal
nodose sensory innervation suggested by other authors. The aim of the
present work is to summarize our present understanding about the origin
and chemical coding of the profuse nerve terminals that selectively contact
pulmonary NEBs. A thorough knowledge of the complex interactions between the neuroendocrine cells and at least ﬁve different nerve ﬁber populations is essential for deﬁning the position(s) of NEBs among the many
pulmonary receptors characterized by lung physiologists. Anat Rec Part A
270A:25– 40, 2003. © 2003 Wiley-Liss, Inc.
Key words: NEBs; neuroepithelial bodies; innervation; airway
receptors; lung; rat
Highly specialized neuroepithelial bodies (NEBs) (Lauweryns et al., 1972), which consist of extensively innervated groups of pulmonary neuroendocrine cells (PNECs),
are normal components of the epithelium of intrapulmonary airways in humans, mammals, and all air-breathing
vertebrate species investigated so far.
The pulmonary neuroendocrine system was ﬁrst reported more than 50 years ago (Fröhlich, 1949), but especially over the last 25 years detailed information has been
provided about the distribution, ontogeny, and microscopic morphology of NEBs (for reviews see Scheuermann,
1987; Sorokin and Hoyt, 1989; Adriaensen and Scheuermann, 1993; Sorokin et al., 1997).
PNECs belong to the diffuse neuroendocrine system
(DNES) (Pearse and Takor Takor, 1979), members of
which have been assigned important roles in the peripheral control of various organs. The pulmonary DNES in
healthy lungs appears to be characterized by the production of amines and several neuropeptides, including serotonin (5-HT), bombesin (gastrin-releasing peptide), calci©
2003 WILEY-LISS, INC.
tonin gene-related peptide (CGRP), calcitonin, enkephalin,
somatostatin, cholecystokinin, and substance P (SP) (for
reviews see Sorokin and Hoyt, 1989; Scheuermann et al.,
1992; Adriaensen and Scheuermann, 1993). These bioactive substances are stored in secretory granules (60 –200
nm diameter) with typical endocrine-like characteristics,
the so-called dense-cored vesicles (DCVs).
Interestingly, PNECs are by far the ﬁrst cell type to
fully differentiate in the human airway epithelium (before
*Correspondence to: Dirk Adriaensen, Laboratory of Cell Biology and Histology, University of Antwerp–RUCA, Groenenborgerlaan 171, B-2020 Antwerp, Belgium. Fax: ⫹32-3-218-0301.
E-mail: dadria@ruca.ua.ac.be
Received 22 May 2002; Accepted 5 September 2002
DOI 10.1002/ar.a.10007
26
ADRIAENSEN ET AL.
the 8th week of gestation), and changes in PNECs/NEBs
have been associated with many perinatal, neonatal, and
adult lung diseases and disorders.
NEBs may have important functions in the regulation of
physiological processes in lungs at all ages, but their exact
role is poorly understood. Mainly on the basis of incomplete morphological data, several hypotheses about the
function of pulmonary NEBs have been proposed (Sorokin
and Hoyt, 1990). NEBs are now believed to have different
functions during speciﬁc periods in prenatal, early postnatal, and adult life. Oxygen-sensing (Youngson et al.,
1993; Cutz and Jackson, 1999; Peers and Kemp, 2001) and
effector (O’Kelly et al., 1998, 1999) mechanisms have been
identiﬁed in pulmonary neuroendocrine cells.
Recent reviews on afferent receptors in the lower airways have added NEBs to the list of presumed receptor
types, which included slowly (SARs) and rapidly adapting
stretch receptors (RARs) and C-ﬁber receptors (Widdicombe, 2001). However, Widdicombe (2001) also stated
that so far there have been no claims for recordings from
single afferent ﬁbers from NEBs. In our opinion, the most
likely reason for that is a lack of knowledge of the functional morphology and the nervous connections of these
NEB “receptors.”
Because the present review is part of a special issue on
airway receptors, it mainly focuses on the innervation of
pulmonary NEBs in general, and on the sensory components in particular. Our recent neuronal tracing, immunocytochemistry, and denervation experiments in rats,
which provide good evidence for an extremely complex
innervation pattern of NEBs (Adriaensen et al., 1998,
2001; Brouns et al., 2000, 2002a, b, 2003), are summarized, illustrated, and discussed in the light of the possible
receptor function(s) of pulmonary NEBs.
Literature Data and Classical Concepts About
the Innervation of Pulmonary NEBs
A selective innervation of NEBs was probably described
long before the existence of the pulmonary neuroendocrine
system itself was known. Several authors described intraepithelial varicose nerve terminals that were concentrated
in groups and irregularly distributed along the airways of
different species, including humans (Berkley, 1894;
Larsell, 1921; Larsell and Dow, 1933; Elftman, 1943). It
was Fröhlich (1949) who described delicate nerve terminals at the surface of, or between the cells of grouped
neuroendocrine cells in the bronchial epithelium of rabbits
and cats. Since that time, many researchers have observed an indisputable innervation of NEBs in both light
and electron microscopic investigations.
Organ culture studies suggested that the nerve terminals contacting NEBs rapidly degenerate, but that denervated NEBs survive (Sonstegard et al., 1979), and that
solitary PNECs may give rise to structured organoid bodies in vitro (Carabba et al., 1985). Three-dimensional reconstruction of a single hamster NEB using electron microscopic images of serial sections showed that also in
intact animals, not every NEB is necessarily innervated
(Pearsall et al., 1985).
A number of methods have been used to visualize nerve
ﬁbers that contact NEBs. To this end, an unambiguous
and simultaneous identiﬁcation of PNECs and nerves was
considered essential.
With the use of different silver-staining techniques,
nonmyelinated nerve ﬁbers that contact the basement
membrane at the level of NEBs or surround the neuroendocrine cells were described in the lungs of human newborns (Fröhlich, 1949; Lauweryns and Peuskens, 1972),
rabbits (Lauweryns et al., 1972; Hung, 1980), rats
(Wasano, 1977), and mice (Hung, 1984), as well as in lower
vertebrates such as turtles (Scheuermann et al., 1983).
Silver impregnation of the NEB cells, however, almost
always hampers a clear view of the nerve terminals contacting the NEB cells.
A regularly used method for studying lung innervation
is the histochemical demonstration of acetylcholinesterase
(AChE) activity. Networks of AChE-positive nerve ﬁbers
were described in contact with pulmonary NEBs of fetal
and newborn rabbits (Sonstegard et al., 1982; Cutz et al.,
1985) and sheep (Cutz and Orange, 1977). Unfortunately,
this technique produces a blurred image of ﬁne structures
and stains the NEB cells, resulting in a poor deﬁnition of
nerve endings at the contact sites.
Formaldehyde-induced ﬂuorescence resulted in the
demonstration of a blue-green nerve plexus, with spectral
characteristics of catecholamines, in the lamina propria of
airways, close to the base of NEBs in rabbits (Lauweryns
et al., 1972; Hung, 1980), turtles (Scheuermann et al.,
1983), frogs (Wasano and Yamamoto, 1978), and toads
(Rogers and Haller, 1978). NEB cells can be differentiated,
displaying a yellow ﬂuorescence with spectral characteristics of serotonin.
More recently, immunocytochemical methods that were
generally applied for the demonstration of NEBs often
also appeared to label contacting nerve ﬁbers. In most
cases, the problem of differentiating the nerve terminals
at the level of the NEBs remains. In rabbits, hamsters,
and rats, pulmonary NEBs and associated nerve ﬁbers
were described using antisera against protein gene product 9.5 (PGP9.5) (Lauweryns and Van Ranst, 1988). In
newborn cats (Adriaensen and Scheuermann, 1993), NSEimmunoreactive (IR) nerve ﬁbers were observed to contact
NSE-positive NEBs. CGRP-IR nerve ﬁbers connected to
CGRP-positive NEBs were reported in the lungs of cats
(Adriaensen and Scheuermann, 1993) and rats (Cadieux
et al., 1986; Van Ranst and Lauweryns, 1990; Terada et
al., 1992). In guinea pigs, a double immunoﬂuorescence
labeling technique, applying antisera against neuropeptides and general neuroendocrine markers (PGP9.5; chromogranin), resulted in the demonstration of solitary tracheal and bronchial PNECs spirally surrounded by CGRP/
SP-IR nerve terminals (Kummer et al., 1991). Using
additional pre-embedding electron microscopic immunocytochemistry, the latter nerve terminals were seen to form
direct contacts with the PNECs, but without the presence
of synaptic specializations. In the human lung, however,
where PNECs and NEBs were shown to be SP-IR, no
associated SP-positive nerve ﬁbers have been reported
(Gallego et al., 1990).
None of the techniques described so far demonstrated
unambiguously that the closely associated nerve ﬁbers
indeed provide a direct innervation of the neuroendocrine
cells in NEBs. Such information could only be obtained
using electron microscopy.
Nerve ﬁbers associated with NEBs were seen in the
mouse lung using scanning electron microscopy (Hung,
1984), but because this technique only reveals surface
structure, it was not possible to obtain a clear view of the
interactions between neuroendocrine cells and nerves.
NEBS: COMPLEX AIRWAY RECEPTORS
With the use of transmission electron microscopy
(TEM), nerve terminals were demonstrated directly contacting the basal part of PNECs, or running between and
looping around the cells in NEBs. The following references
were selected based mainly on their descriptions of synapse-like specializations between nerve terminals and
NEB cells. Direct innervation of pulmonary NEBs was
observed in humans (Lauweryns et al., 1970) and many
mammalian species, such as mice (Wasano, 1977; Wasano
and Yamamoto, 1981), rats (Van Lommel and Lauweryns,
1993a), hamsters (Sorokin and Hoyt, 1989), rabbits (Lauweryns et al., 1972; Lauweryns and Van Lommel, 1987;
Sorokin and Hoyt, 1989; Adriaensen and Scheuermann,
1993), and cats (Van Lommel and Lauweryns, 1993b). In
birds, similar NEB/nerve contacts were reported in the
lungs of chickens (Cook and King, 1969; Walsh and McLelland, 1978; Wasano and Yamamoto, 1979; López et al.,
1983), pigeons (McLelland and MacFarlane, 1986), and
quails (Adriaensen et al., 1994). In lower vertebrates, direct innervation was shown in reptiles, such as turtles
(Scheuermann et al., 1983), lizards (Ravazzola and Orci,
1981), and snakes (Wasano and Yamamoto, 1976); in amphibia, such as newts (Scheuermann et al., 1989; Goniakowska-Witalinska et al., 1992; Adriaensen and Scheuermann, 1993), frogs (Wasano and Yamamoto, 1978;
Goniakowska-Witalinska, 1981), and toads (Rogers and
Haller, 1980; Goniakowska-Witalinska et al., 1990); and
in lungﬁsh (Dipnoi) (Adriaensen et al., 1990). Nerve ﬁbers
have been reported to contact solitary PNECs and NEB
cells, although by far the most reports concern NEBs. In
several species, solitary PNECs apparently do not show
direct contacts with nerve ﬁbers, which was reason
enough for some authors to regard them as different entities from NEBs. It is clear, however, that the surface
membrane of all PNECs is functionally divided into basolateral and apical regions. Possible nerve contacts in this
respect provide further possibilities for functional integration. Using TEM, different types of morphologically characterized nerve terminals have been described in contact
with PNECs.
The type of nerve terminal most often reported in contact with NEBs, in many species, is packed with mitochondria and some small clear vesicles. They penetrate deep
between the NEB cells, often close to the luminal surface.
The nerve terminals reveal asymmetric synaptic contacts,
with an accumulation of DCVs near electron-dense coneshaped thickenings of the surface membrane of PNECs,
and are generally believed to be afferent (sensory) (Cook
and King, 1969; Wasano and Yamamoto, 1981; Lauweryns
et al., 1985; Lauweryns and Van Lommel, 1986, 1987;
Adriaensen and Scheuermann, 1993; Van Lommel and
Lauweryns, 1993a, b).
A second frequently reported type of nerve terminal
appears to be loaded with small clear vesicles (40 – 60 nm
diameter) and shows a few larger DCVs and mitochondria.
In several species they reveal asymmetric synaptic contacts with NEB cells, with an accumulation of the clear
vesicles near cone-shaped electron-dense specializations
of the axolemma of the nerve terminal, while the surface
membrane of the PNEC shows a uniform thickening;
these nerve terminals are generally considered efferent
(cholinergic-like) (Walsh and McLelland, 1978; Sonstegard et al., 1982; Stahlman and Gray, 1984; Adriaensen
and Scheuermann, 1993).
27
In some mammalian species, it has been demonstrated
that both morphologically afferent and efferent contacts
may be peripheral specializations of the same (sensory?)
nerve ﬁber (Lauweryns and Van Lommel, 1987; Van Lommel and Lauweryns, 1993a, b).
A few reports mention nerve endings ﬁlled with small
dense-cored granules (about 60 nm diameter) (Rogers and
Haller, 1978), typical of adrenergic nerves, and terminals
containing larger (80 –225 nm), moderately dense DCVs
(Stahlman and Gray, 1984), believed to belong to a peptidergic pathway, in close apposition to PNECs.
Although TEM probably offers a rather good morphological characterization of the direct innervation of a limited number of pulmonary NEBs in several species, it
leaves another important question unanswered, i.e., that
of the origin of the nerve ﬁber population(s) that selectively innervate(s) NEBs.
To date, most of the studies dealing with this aspect
have combined electron microscopy, for the evaluation of
nerve terminals contacting NEBs, and experimental vagotomy with or without an additional hypoxic stimulus (in
rabbits (Lauweryns et al., 1985; Lauweryns and Van Lommel, 1986) and rats (Van Lommel and Lauweryns,
1993a)). It was reported that, after unilateral infranodosal
vagotomy, the numbers of both morphologically afferent
and efferent nerve terminals in NEBs were reduced by
about 70% in lungs ipsilateral to the denervation, while no
changes were seen in the contralateral lungs. The NEB
innervation appeared to be intact after supranodosal vagotomy. Based on these ﬁndings, it was suggested that
NEBs are predominantly innervated by sensory nerve ﬁbers originating from neurons located in the vagal ganglion nodosum. The remaining 30% of nerve terminals in
the investigated NEBs were thought to be the result of
cross innervation from the contralateral vagus or of the
presence of another unknown nerve ﬁber population (e.g.,
sympathetic) contacting NEBs. Electrical stimulation of
the sectioned vagal nerve revealed a possible vagal inhibitory secretomotor inﬂuence on rabbit pulmonary NEBs,
since the basal secretion of DCVs was apparently decreased (Lauweryns et al., 1985).
More speciﬁcally for rat lung, the very extensive innervation of NEBs was suggested to be exclusively sensory,
originating from the vagal nodose ganglion (Van Lommel
and Lauweryns, 1993a). The electron microscopically
quantiﬁed efferent- and afferent-like terminals were interpreted as belonging to the same population of sensory
ﬁbers, since vagotomy experiments resulted in a clearly
reduced number of nerve terminals in NEBs, while the
ratio between efferent- and afferent-like terminals appeared unchanged in the selected NEBs. It was further
concluded that, unlike in rabbits, a separate motor innervation of NEBs is probably absent in rat lungs. However,
given our own more recent data on the innervation of rat
NEBs (see below), the question could be raised as to
whether the combination of experimental denervation and
electron microscopy, which is very labor-intensive and
therefore necessarily limited by the number of fully quantiﬁable specimens, is a reliable method by which to draw
conclusions on such a complex and inhomogeneous diffuse
population of NEBs. The latter point is strengthened by
the observation that NEBs in organ-cultured rat lungs
apparently receive an innervation from neurons located in
intrinsic airway ganglia (Sorokin et al., 1993).
28
ADRIAENSEN ET AL.
To date, we know very little about the presence or origin
of different nerve ﬁber populations selectively innervating
NEBs in the human lung.
To conclude this historical perspective and at the same
time come back to the main point of the present short
review, i.e., the characterization of pulmonary NEBs as
putative airway receptors, it may be of interest to revisit
the scheme that was presented in our 1993 NEB review
(Adriaensen and Scheuermann, 1993) (Fig. 1). Essentially, what is represented is the best possible view we had
at that time of the morphological organization of NEBs
and directly related nerve terminals. It further aimed at
explaining a possible mechanism for the assumed reaction
of NEBs to airway hypoxia. Most of the information represented in the scheme was based on electron microscopic
literature data. It may be stressed that several more recent reviews on pulmonary NEBs still come to the same
conclusion, i.e., NEB innervation is mainly vagal nodose
and NEBs may act as hypoxia sensors via a vagal afferent
pathway (Cutz and Jackson, 1999; Van Lommel et al.,
1999), and that the same scheme was used as representative for pulmonary NEBs in a recent review on airway
receptors (Widdicombe, 2001). The interest in this concept
has been recently strengthened by the characterization of
a complete, carotid body-like, oxygen-sensing mechanism
in NEBs, which implicates exocytosis of transmitters as a
result of hypoxia (for review see Peers and Kemp, 2001).
In summary, many researchers in the ﬁeld today believe
that if the stimulus is strong enough, hypoxia may cause,
in addition to local reﬂex actions, an afferent signal to
travel toward the central nervous system (CNS) via the
vagus nerve.
However, this leaves us with the essential question of
why lung physiologists have been unable to conﬁrm this
apparently rather simple mechanism. Are NEBs really
the lung receptors we would like them to be? If so, did we
overlook something? Having many years of experience
looking at the functional morphology of NEBs, we evidently went for the latter possibility and tried to ﬁnd out
if it is really that simple. We realized that if NEBs are
indeed airway receptors, the best way to try to understand
them would be to have a good look at their nervous connections, and we decided to focus on the rat lung.
Functional Morphological Characterization of
the Selective Innervation of NEBs in Rat Lungs
Vagal nodose sensory component of the selective
innervation of pulmonary NEBs. After we considered
all available literature data, it was clear that an essential
factor in the recognition of NEBs as airway receptors
would be the full conﬁrmation and characterization of
their vagal nodose innervation. The following questions
arose: Is the vagal nodose innervation of NEBs really
there? How can it unambiguously be identiﬁed in the light
microscope? What are the neurochemical characteristics?
How does this vagal nodose connection relate to what lung
physiologists know, or thought they knew?
Since several authors reported that in rat lungs
CGRP-IR NEBs at all levels of the intrapulmonary airways are contacted by CGRP-positive nerve ﬁbers
(Cadieux et al., 1986; Shimosegawa and Said, 1991;
Terada et al., 1992; Sorokin et al., 1997) (personal observations), it was generally believed that they represented
the long-predicted vagal sensory ﬁbers. However, after
performing some vagotomy experiments we realized that
this CGRP-IR nerve ﬁber population is nonvagal, as is
further characterized in the next section.
The problem of identifying a vagal nodose component of
the innervation of NEBs was then addressed in the most
direct way possible: the red ﬂuorescent neuronal tracer
DiI, a lipophilic carbocyanine dye (Honig, 1993), was injected unilaterally into rat nodose ganglia, and NEBs were
visualized in toto by using immunocytochemistry and confocal microscopy on thick frozen sections (Adriaensen et
al., 1998). The most striking ﬁnding was the extensive
intraepithelial terminal arborizations of DiI-labeled vagal
nodose afferents in intrapulmonary airways, often located
close to the luminal surface of the airways and apparently
always co-appearing with CGRP-IR NEBs (Fig. 2). Not all
NEBs received a traced nerve ﬁber. Intrapulmonary
CGRP-containing nerve ﬁbers, including those innervating NEBs, always appeared to belong to a nerve ﬁber
population different from the DiI-traced ﬁbers. This was
the ﬁrst hard evidence demonstrating at the light microscopic level that at least some of the pulmonary NEBs in
rats are indeed supplied with sensory nerve ﬁbers that
originate in the vagal nodose ganglion, and that form
beaded ramiﬁcations between the NEB cells.
However, neuronal tracing is a labor-intensive and
time-consuming technique, and is hardly compatible with
a routine application. Therefore, we tried to further characterize this vagal nodose population of nerve terminals in
NEBs.
Combinations of neuronal tracing and/or vagal denervation experiments with immunolabeling taught us that
the vagal nodose ﬁbers contacting pulmonary NEBs can
be labeled selectively with antibodies against the calciumbinding protein calbindin D28k (Adriaensen et al., 1999;
Brouns et al., 2000). Calbindin (CB) appears to label NEB
cells, as well as the nodose ﬁbers contacting them (Fig. 3),
and hence can be regarded as a very interesting routine
marker for NEBs in the rat lungs. Apparently, a little less
than half of the NEBs are contacted by such CB-IR nerve
ﬁbers in control animals. The ﬁrst NEBs contacted by
CB-IR nerve ﬁbers could be detected at gestational day
(GD) 17. After unilateral infranodose vagotomy, no CB-IR
nerves contacting NEBs were left in the ipsilateral lung,
while no changes were observed in the contralateral lung.
Because both NEBs and contacting nerve ﬁbers are
stained, this marker does not allow an evaluation of the
intraepithelial terminals. Double immunocytochemical
staining with antibodies against CB and CGRP clearly
revealed that both substances mark a different nerve ﬁber
population, often contacting the same NEBs (Fig. 4).
To further characterize the different nerve ﬁber populations related to NEBs in the rat lung, we performed a
systemic capsaicin treatment (Brouns et al., 2003). The
results revealed no changes in the CB-IR vagal nodose
innervation of NEBs as compared to control rats, strongly
suggesting that a capsaicin-insensitive population is involved. The method of capsaicin treatment is time-consuming, destroys certain nerve ﬁber populations, and is
obviously not always the method of choice for the selective
demonstration of nerve ﬁber populations in routine applications. Therefore, we tried to conﬁrm the results of the
capsaicin treatment using antibodies against the capsaicin receptor (vanilloid receptor 1 (VR1)). Double staining
for VR1 and CB indeed conﬁrmed that the vagal nodose
component of the innervation of NEBs does not express
Fig. 1. Conceptual scheme summarizing ﬁndings on the innervation
of mammalian NEBs and their possible reaction to airway hypoxia.
Hypoxic air stimulates (red dotted arrows) the NEB cells (yellow) to
discharge the contents of their DCVs at afferent synaptic sites of intracorpuscular sensory nerve endings (white arrowheads). This could result
in depolarization and the development of a generator potential that
spreads over the nerve ﬁber (white arrows). Given the possibility that
afferent and efferent nerve terminals might occur along the same nerve
process, as was indeed demonstrated in serial sections of newborn
rabbit NEBs, the efferent endings would develop synaptic activity and
discharge neurotransmitter (black arrowheads). This may in turn inﬂuence the physiological state of the receptor cell (green arrows), thus
modulating the transduction of stimuli or regulating local paracrine secretion acting on nearby blood vessels (red arrows), or smooth muscle
bundles (blue arrows). The mechanism of local modulation of a neuroreceptor by efferent nerve endings formed at the periphery of sensory
nerves could be called the “axon reﬂex.” When the generator potential
reaches a threshold value, an action potential is triggered (white arrows
with double arrowheads). Some nerve terminals appear to be exclusively
efferent (double black arrowheads). Clara-like cells (CC); capillary (C);
smooth muscle bundle (SM); airway lumen (L). Modiﬁed from Adriaensen
and Scheuermann (1993).
Fig. 2. a and b: Detail of DiI-traced (red ﬂuorescence) vagal sensory
nerve terminals contacting a pulmonary NEB (calcitonin gene-related
peptide (CGRP)-immunoﬂuorescence; FITC ﬂuorescence) at the alveolar
level in the adult rat lung. Maximum value projections (MVPs) of 36
optical sections (1-␮m interval). a: Red channel clearly demonstrating
the DiI-traced nerve ﬁbers approaching the epithelium (arrows) and
forming intraepithelial terminals (arrowheads). b: Combination of the red
and green channels, showing the complete NEB and all of the labeled
nerve endings. The traced nerve endings run along a bronchiole (B) and
enter the epithelium at the base of the NEB (open arrowheads).
Fig. 3. MVP of 14 confocal optical sections (2-␮m interval) of an NEB
in a bronchus of a neonatal rat. Thick calbindin D28k (CB)-IR (red Cy3
ﬂuorescence) nerve ﬁbers (arrows) contact the CB-IR NEB cells.
30
ADRIAENSEN ET AL.
capsaicin receptors (Fig. 5), while often another population that is VR1-IR appears to co-innervate NEBs (see
below). In contrast to some literature data claiming that
capsaicin treatment causes a depletion of CGRP from rat
NEBs (Tjen-A-Looi et al., 1998), quantiﬁcation of the number of CGRP- and PGP9.5-IR NEBs after capsaicin treatment did not reveal signiﬁcant differences with control
lungs in our study. The latter observation was strengthened by the absence of VR1 expression from NEBs (Figs. 5
and 12), making a direct effect of capsaicin on the CGRP
content of NEBs highly unlikely.
Another interesting feature of the vagal sensory component of the innervation of pulmonary NEBs, especially in
the light of the present efforts to characterize NEBs as
airway receptors, can be visualized using antibodies
against the myelin basic protein (MBP), as a marker for
myelinated nerve ﬁbers in the lung. It was observed that
combined with CB staining, from postnatal day 10 on, the
vagal nodose ﬁbers contacting NEBs are invariably myelinated (Fig. 6) (Brouns et al., 2003). Myelinated nerve
ﬁbers have been observed electron microscopically in the
vicinity of NEBs (Van Lommel and Lauweryns, 1993a),
Figures 4 –10.
NEBS: COMPLEX AIRWAY RECEPTORS
31
but no evidence or suggestions have linked these myelinated ﬁbers to the vagal nodose intraepithelial nerve terminals situated between NEB cells.
Examination of rat lungs using speciﬁc antibodies
against P2X3 purinoreceptors (ATP receptors) revealed
intraepithelial arborizations of P2X3 receptor-IR nerve
terminals, which in all cases appeared to ramify between
CGRP- or CB-labeled NEB cells (Brouns et al., 2000)
(Figs. 7 and 8). The ﬁrst NEBs contacted by P2X3 receptor-IR nerve terminals could be detected at GD 17. NEB
cells did not express P2X3 receptors. It was further demonstrated that P2X3 receptor and CB IR completely colocalize in the vagal nodose population of nerve ﬁbers that
selectively contacts NEBs (Fig. 8), whereas CGRP-IR ﬁbers clearly form a different population (Fig. 7). The disappearance of characteristic P2X3 receptor-IR nerve terminals in contact with NEBs after infranodosal vagal
denervation, and the colocalization of tracer and P2X3
receptor-labeling in vagal nodose neuronal cell bodies in
retrograde tracing experiments from the lungs, support
our hypothesis that the P2X3 receptor-expressing nerve
ﬁbers contacting NEBs have their origin in the vagal
nodose ganglia. The combination of MBP and P2X3 receptor immunostaining again clearly pointed to a myelinated
population (Fig. 9) (Brouns et al., 2003). Further concentrating on ATP and ATP receptors, we applied quinacrine
histochemistry, a method that has been shown to selectively label high concentrations of ATP, if accumulated
together with proteins in secretory granules. In rat lungs,
the latter method has been shown to selectively label
NEBs (Brouns et al., 2000). Combination of quinacrine
histochemistry and P2X3 receptor-staining showed that
the ATP receptor-expressing nerve terminals in rat lungs
are exclusively associated with quinacrine-stained NEBs
(Fig. 10). ATP may, therefore, act as a neurotransmitter in
the vagal sensory innervation of NEBs via a P2X3 receptor-mediated pathway. Given the extensive data obtained
in other systems (Burnstock, 1999a, b), the possibility that
at least some of the NEBs may be involved in vagal afferent respiratory mechanosensory transduction should be
considered.
All available data on the vagal nodose sensory component of the innervation of NEBs are summarized in the
scheme of Figure 20 (nerve ﬁber population shown in
blue). Essentially, the sensory nerve ﬁber population involved reveals extensive intraepithelial terminals, has a
vagal nodose origin, can be marked by its CB IR, expresses
P2X3 ATP receptors but no VR1 capsaicin receptors, and is
myelinated. Consequently, as predicted in the initial
scheme (Fig. 1), NEBs are indeed supplied by a population
of vagal nodose sensory ﬁbers that have now been fully
characterized. However, the question remains: why has it
so far been impossible for lung physiologists to link NEBs
to measurable activities in vagal afferents? Is it perhaps
more complicated than was predicted?
Fig. 4. Immunocytochemical double-staining of an NEB in a bronchiole of a 10-day-old rat labeled for CGRP (green FITC ﬂuorescence)
and CB (red Cy3 ﬂuorescence). CGRP/CB-IR NEB cells are contacted by
a nerve bundle composed of CB-IR nerve ﬁbers (arrows) and thin varicose CGRP-IR nerve terminals (arrowheads). Note that in the NEB cells,
CGRP labeling is strongest at the basal side while CB labeling appears
to be more uniform throughout the cells. MVP of 27 optical sections
(1-␮m interval).
Fig. 5. Immunocytochemical double-staining for capsaicin receptors
(VR1; green FITC ﬂuorescence) and CB (red Cy3 ﬂuorescence), as a
marker for the vagal sensory subpopulation of nerve terminals contacting pulmonary NEBs. High magniﬁcation detail of the basal part of a
CB-IR NEB, revealing that VR1 IR (open arrows) and CB IR (arrows) are
expressed by separate nerve ﬁber populations. MVP of six confocal
optical sections (1-␮m interval).
Fig. 6. Immunocytochemical staining for CB (green FITC ﬂuorescence) and myelin basic protein (MBP; red Cy3 ﬂuorescence) of a
bronchiolar NEB in a 21-day-old rat. Combination of the red and green
channel shows that the CB-IR nerve ﬁbers (arrows) are surrounded by
MBP-IR myelin sheaths (open arrows), that are lost (arrowheads) just
before the CB-IR ﬁbers branch and contact the CB-IR NEB. MVP of 25
confocal optical sections (1-␮m interval).
Fig. 7. a and b: Immunocytochemical double-staining for the ATP
receptor P2X3 (green FITC-ﬂuorescence) and CGRP (red Cy3-ﬂuorescence) revealing pulmonary NEBs in a rat bronchiole contacted by P2X3
receptor- and CGRP-expressing nerve ﬁbers. MVPs of 16 confocal
optical sections (1-␮m interval). a: Green channel showing two neighboring intraepithelial complexes of P2X3 receptor-IR nerve terminals.
Note an approaching nerve ﬁber (arrows). b: Combination of the red and
green channels, showing two CGRP-IR NEBs contacted by separate
populations of CGRP-containing (open arrows) and P2X3 receptor-expressing (arrows) nerve ﬁbers.
Fig. 8. a– c: Bronchial CB-IR (red Cy-3 ﬂuorescence) NEB contacted
by a complex network of P2X3 receptor-(green FITC-ﬂuorescence) and
CB-IR nerve terminals. MVP of 10 confocal optical sections (1.5-␮m
interval). a: Green channel showing an intraepithelial P2X3 receptorexpressing arborization originating from multiple nerve ﬁber endings
(arrows). b: Red channel showing CB-IR nerve ﬁbers (open arrowheads)
in contact with the CB-IR NEB. c: Combination of both channels revealing that nerve ﬁbers in contact with the NEB express both P2X3 receptors and CB, although the staining intensity varies along the nerve ﬁbers.
Fig. 9. a and b: Confocal microscopic images of immunocytochemical double-staining for MBP (red Cy3 ﬂuorescence) and P2X3 receptors
(green FITC ﬂuorescence) in the bronchus of a 21-day-old rat. MVP of 30
optical sections (0.8-␮m interval). a: MBP IR can be detected in myelin
sheaths (open arrows) of nerve ﬁbers in close proximity to the bronchial
epithelium (E). b: Combination of the red and green channels, showing
that FITC-labeled vagal afferent nerve ﬁbers expressing P2X3 receptors
(arrows) approach the epithelium, branch, protrude between the epithelial cells, and give rise to intraepithelial nerve terminals, many of which
(open arrowheads) are seen close to the luminal surface (L, lumen of the
bronchus). The latter intraepithelial structure represents an NEB. The
P2X3 receptor-IR nerve ﬁbers are myelinated and the MBP-IR myelin
sheets are lost in close proximity to the target (arrowhead).
Fig. 10. a– c: Consecutive quinacrine histochemistry and P2X3 receptor staining in a rat bronchus. a: Fluorescence micrograph of quinacrine accumulation in an intraepithelial cell group (arrowhead), indicating the presence of an ATP-storing NEB. b and c: P2X3 receptor
immunocytochemistry revealing the presence of extensive receptorexpressing nerve terminals at exactly the same spot as marked in part a
(arrowhead). Note the P2X3 receptor IR in bronchial smooth muscle
bundles (SM). Single confocal optical section. c: High magniﬁcation
detail of part b, clearly showing the P2X3 receptor-IR nerve ﬁber (arrow)
that loops through the SM, branches (arrowheads), and gives rise to a
terminal intraepithelial arborization. MVP of 20 confocal optical sections
(0.95-␮m interval).
Calcitonin gene-related peptide-immunoreactive component of the selective innervation of pulmonary NEBs. As noted above, the vagal sensory component of the innervation of rat pulmonary NEBs
appeared to form a clearly different nerve ﬁber population
than the sensory CGRP-IR innervation that contacts
32
ADRIAENSEN ET AL.
NEBs (Shimosegawa and Said, 1991; Terada et al., 1992;
Sorokin et al., 1997). Denervation studies and retrograde
tracing from the lung (Springall et al., 1987) (personal
observations) strongly indicate that the CGRP-IR nerve
ﬁbers that selectively contact NEBs in the rat lung belong
to a spinal sensory population, originating in dorsal root
ganglia (DRG) T1–T6. The ﬁrst NEBs contacted by
CGRP-IR nerve ﬁbers could be detected at GD 19.
We then tried to further characterize this CGRP-IR
population (Brouns et al., 2003). The ﬁrst observation was
that CGRP-positive terminals contacting NEBs invariably
colocalize SP (Fig. 11). Using CGRP IR alone, it was impossible to visualize the real contact sites between nerve
terminals and the NEB cells that also show a strong
CGRP staining. SP does not label rat NEBs. Therefore,
CGRP/SP double labeling allows the differentiation of in-
Figures 11–19.
NEBS: COMPLEX AIRWAY RECEPTORS
33
dividual nerve terminals at the level of NEB cells. It
turned out that the spinal sensory CGRP⫹/SP⫹ nerve
terminals do not penetrate the epithelium, as was shown
for the vagal sensory endings, but that they form a plexus
that preferentially contacts the basal surface of NEBs
(Figs. 11 and 12), clearly a population that was not
present in the initial scheme (Fig. 1). Obviously, airways
also harbor a vagal CGRP/SP-IR nerve ﬁber population,
but evidence suggests that in rat lungs these vagal ﬁbers
mainly originate from the jugular ganglia, and terminals
can be found in the epithelium of large-diameter bronchi
only—apparently without any speciﬁc relationship with
NEBs (personal observations). This population is not discussed further.
We then studied the selective CGRP/SP-IR innervation
of NEBs in lungs of capsaicin-treated rats (Brouns et al.,
2003). After capsaicin treatment, the percentage of NEBs
contacted by CGRP-positive nerve terminals was dramatically reduced (5.90%) compared to control lungs (51.80%),
while the numbers of CGRP-IR NEBs revealed no significant changes (see above). To further strengthen the capsaicin data, immunocytochemistry with antibodies
against the capsaicin receptor VR1 was applied. The results clearly showed that all CGRP-stained nerve ﬁbers in
the vicinity of and contacting NEBs express VR1s (Fig. 12)
and may therefore be considered capsaicin-sensitive. The
VR1/CGRP-IR nerve ﬁber plexus mainly contacts the base
of the NEBs, and again the NEBs themselves appeared to
be VR1-negative.
All available data on the spinal sensory component of
the selective innervation of NEBs are summarized in the
scheme of Figure 20 (nerve ﬁber population shown in
green). Essentially, the sensory nerve ﬁber population
involved forms a mainly basal plexus, most likely has its
origin in the DRGs T1-T6, can be marked by its CGRP/SP
IR, is capsaicin-sensitive, and expresses VR1s.
It is now ﬁrmly established that rat pulmonary NEBs
may receive at least two different sensory nerve ﬁber
populations. Again, however, the question remains as to
whether this double sensory innervation can explain the
discrepancy between the good evidence that NEBs are
hypoxia sensors, and the lack of physiological evidence for
the vagal transmission of hypoxia-related stimuli. One
possibility is that central hypoxic responses, originating
from NEB “receptors” are mediated via a spinal afferent
instead of a vagal pathway. However, it may be more
complicated than that.
Fig. 11. a and b: Immunocytochemical double-staining for substance P (SP; green FITC ﬂuorescence) and CGRP (red Cy3 ﬂuorescence) of an NEB in the intrapulmonary bronchus of a 10-day-old rat.
MVP of 31 optical sections (0.8-␮m interval). a: Green channel showing
SP-IR nerve ﬁbers (open arrows) contacting the base of the epithelium
(open arrowheads). b: The combination of the green and red channels
clearly shows an extensive network of SP/CGRP-IR (yellow ﬂuorescence
being indicative of colocalization) nerve ﬁbers (open arrows) contacting
the base (open arrowheads) of a CGRP-IR NEB.
Fig. 12. Immunocytochemical double-staining for VR1 (green FITC
ﬂuorescence) and CGRP (red Cy3 ﬂuorescence), as a marker for the
spinal sensory subpopulation of nerve terminals contacting pulmonary
NEBs. Bronchiolar CGRP-IR NEB in a 10-day-old rat contacted by
extensively branching CGRP-containing nerve ﬁbers that also express
VR1 (open arrows; the yellow ﬂuorescence is indicative of colocalization). CGRP IR NEB cells do not show VR1 IR. Reconstruction of seven
optical sections (1-␮m interval).
Fig. 13. Bronchial NEB in rat lung at PD10 double-stained for nNOS
(red Cy3 ﬂuorescence) and CB (green FITC ﬂuorescence). Combined red
and green channels showing nNOS-IR neuronal cell bodies (arrowheads)
that give rise to a nitrergic nerve plexus (arrows) in the lamina propria and
to intraepithelial nerve terminals (open arrowheads) selectively contacting the CB-IR NEB (asterisk). The image is suggestive of a direct relationship between the selective intraepithelial nitrergic innervation of the
NEB, and the nearby nNOS-IR neurons. Note the absence of CB immunostaining from the nitrergic nerves (arrows). MVP of 27 confocal optical
sections (1.5-␮m interval).
Fig. 14. a– c: Detail of a CGRP-IR (green FITC-ﬂuorescence) NEB in
a respiratory area, contacted by separate nNOS- (red Cy3 ﬂuorescence)
and CGRP-IR nerve terminals. MVP of 30 confocal optical sections
(0.9-␮m interval). a: Red channel showing an intraepithelial nNOS-expressing arborization (open arrowheads) originating from subepithelial
nerve ﬁbers (arrows). b: Green channel showing CGRP-IR nerve ﬁbers
(open arrows) contacting the base of a CGRP-IR NEB. c. Combination of
both channels revealing that the intraepithelial nitrergic nerve terminals
do not contain CGRP, although the subepithelial CGRP-IR nerve ﬁbers
run in very close proximity to the nNOS-IR ﬁbers.
Fig. 15. High-magniﬁcation detail of a CB-IR NEB in a rat bronchiole
at postnatal day 2. nNOS (arrows) and CB (open arrows) IR are present
in different nerve ﬁber populations. Clearly, high-resolution confocal
imaging is necessary to differentiate the ﬁne intertwined terminals of
both populations. MVPs of eight confocal optical sections (1.5-␮m interval).
Fig. 16. a– c: Immunocytochemical double-staining for VIP (red Cy3ﬂuorescence) and nNOS (green FITC-ﬂuorescence). Detail of an intrapulmonary bronchus at PD10. MVP of 16 confocal optical sections (1.5-␮m
interval). a: Red channel showing a VIP-IR intraepithelial arborization
(open arrowheads), suggestive of the presence of an NEB. The lamina
propria of the bronchus harbors a weakly VIP-IR neuronal cell body
(open arrow). b: Green channel showing that the VIP-IR neuronal cell
body is strongly stained for nNOS (arrow). In contrast, the VIP-IR intraepithelial arborization shows weak nNOS IR (arrowheads). c: Combination of both channels revealing the discrepancy in staining intensity
between VIP (open arrowheads) and nNOS (arrow).
Fig. 17. Confocal detail of nNOS-IR (green FITC-ﬂuorescence) neurons located in a small ganglion situated in the lamina propria of a
bronchiole of a 10-day-old rat. Varicose CGRP-IR (red Cy3-ﬂuorescence) nerve ﬁbers intimately surround the two nNOS-IR neurons (asterisks) in this ganglion. Processes of the nitrergic neurons (arrows)
follow a course conspicuously similar to that of the CGRP-IR nerve ﬁbers
(open arrows). MVP of 17 optical sections (1-␮m interval).
Fig. 18. a and b: Immunocytochemical double-staining for VIP (green
FITC-ﬂuorescence) and CGRP (red Cy3-ﬂuorescence). MVPs of 14 confocal optical sections (1-␮m interval). a: VIP-IR nerve ﬁbers (open arrows)
give rise to an extensive intraepithelial arborization (open arrowheads). b:
Combination of green and red channels revealing that VIP-IR nerve
terminals protrude between CGRP-IR NEB cells. Note that the VIP-IR
nerve ﬁbers do not contain CGRP and can be seen to overlay apical NEB
cells, close to the luminal surface.
Fig. 19. a– c: Immunocytochemical double-staining for VAChT (red
Cy3 ﬂuorescence) and CGRP (green FITC ﬂuorescence) in a bronchiole
of a 10-day-old rat. MVPs of 15 confocal optical sections (0.9-␮m
interval). a: Red channel showing strong VAChT-IR nerve ﬁbers that are
abundant in the subepithelial region (SE). Epithelial cells (arrowheads)
show a very faint VAChT IR, while stronger VAChT-IR “terminals” (open
arrowheads) can be observed intraepithelially (E). VAChT-IR nerve ﬁbers
(arrows) appear to contact the epithelium. b: Green channel demonstrating CGRP-IR spinal sensory nerve terminals (open arrows) contacting a
CGRP-IR NEB. c. The combination of both channels shows that the
epithelial VAChT staining is present in NEB cells. VAChT-IR nerve terminals appear to contact the NEBs.
34
ADRIAENSEN ET AL.
Intrinsic motor component of the selective innervation of pulmonary NEBs. It is well known that
nitric oxide (NO) plays an important role in lung physiology and pathophysiology. In relation to hypoxia, especially
neuronal NO and endothelial NO appear to be important.
Chronic hypoxia in rats induces an upregulation of neuronal NO synthase (nNOS) expression (Xue et al., 1994;
Shaul et al., 1995; Gess et al., 1997). Therefore, we examined the possibility of a relationship between intrapulmonary nitrergic structures and pulmonary NEBs. Previous
conventional immunocytochemical studies of nNOS, however, reported only very few nNOS-IR nerve ﬁbers in rat
lungs (Kobzik et al., 1993).
Observing in greater detail and using a much more
sensitive detection method, we recently found that part of
the NEBs in rat lungs are selectively innervated by nitrergic (nNOS-IR) nerve terminals that penetrate in the
epithelium, between the NEB cells, and apparently originate from intrinsic pulmonary nitrergic neurons (Brouns
et al., 2002a). From postnatal day 2 onward, nNOS-IR
neurons, present mainly in small ganglia close to the
mucosa at all levels of intrapulmonary airways, were seen
to give rise to complex intraepithelial terminals that invariably colocalize with NEBs (Fig. 13). nNOS IR was
absent from the spinal afferent CGRP-IR (Fig. 14) and
from the vagal nodose afferent CB-IR (Fig. 15) nerve ﬁber
populations that were previously shown to selectively contact NEBs. Quantitative analysis revealed that all NEBs
receiving nNOS-IR terminals were also contacted by
CGRP-positive nerve ﬁbers, while about 55% were additionally contacted by CB-IR nerves. nNOS-positive ﬁbers
approaching NEBs are often very closely related to some
of the CGRP-IR ﬁbers contacting the same NEB. The
reported nitrergic neurons appeared to colocalize vasoactive intestinal polypeptide (VIP) (Fig. 16), did not express
cholinergic markers, and were always surrounded by a
basket of CGRP-IR nerve terminals. These nerve terminals originate from CGRP ﬁbers that spiral around the
axons of the nitrergic neurons (Fig. 17), and presumably
represent collaterals of the spinal CGRP-IR nerve ﬁbers
that selectively innervate NEBs.
The available data on the intrinsic nitrergic component
of the selective innervation of NEBs are summarized in
the scheme of Figure 20 (nerve ﬁber population shown in
red). Essentially, the nitrergic nerve ﬁber population concerned provides intraepithelial terminals between the
NEB cells, has its origin in neurons located in airway
ganglia, and reveals a very strong interaction with the
spinal afferent CGRP-IR component of the NEB innervation.
The characterization of this rather extensive population
of highly likely intrinsic intraepithelial motor terminals
raises some serious questions about the validity of the
mainly electron microscopic data contained in the initial
scheme of Figure 1, and about the interpretation of the
quantitative electron microscopic data of NEB innervation
after vagotomy in rat lungs (Van Lommel and Lauweryns,
1993a).
Simultaneous demonstration of vagal sensory,
spinal sensory, and intrinsic nitrergic nerve terminals in pulmonary NEBs. The previous chapters
revealed that pulmonary NEBs can be contacted by vagal
(CB-IR), spinal (CGRP-IR), and nitrergic (nNOS-IR) nerve
terminals. However, the evidence that NEBs receive all
three of the fully characterized (to date) nerve ﬁber populations is indirect. Recently, we described a reliable multiple immunolabeling method (Brouns et al., 2002b) using
unconjugated primary polyclonal antibodies raised in the
same species. In this way, simultaneous detection in a
single preparation of CB, CGRP, and nNOS in three different nerve ﬁber populations that selectively contact pulmonary NEBs can be achieved (Fig. 21). In the reported
procedure, nNOS was visualized using TSA enhancement
(PerkinElmer Life Sciences, Boston, MA), followed by detection of CGRP via a ﬂuorophore-coupled Fab secondary
antibody, and the subsequent labeling of CB with a ﬂuorophore-coupled “conventional” secondary antibody. All
possible remaining binding sites of the second primary
antibody were blocked using unlabeled anti-rabbit Fab
fragments between the second and third steps.
Triple-labeling immunocytochemistry thus provided evidence that part of the NEBs in rat lungs selectively receive at least three different populations of nerve terminals, the characteristics of which are schematized in
Figure 20.
Other components of the selective innervation of
pulmonary neuroepithelial bodies. The functional
morphology of rat pulmonary NEBs is clearly much more
complex than was predicted by electron microscopic studies (Van Lommel and Lauweryns, 1993a). Moreover, we
have now good evidence that several other nerve ﬁber
populations, which are not fully characterized yet, provide
additional nervous connections of NEBs.
A considerable number of NEBs appear to be contacted
by profuse beaded VIP-IR intraepithelial nerve terminals
(Fig. 18). Although VIP IR was seen to be localized in the
intrinsic nitrergic neurons that give rise to nitrergic terminals contacting NEBs, we believe that an additional
population of VIP-expressing nerve endings with an as yet
unidentiﬁed origin may be involved in the selective innervation of NEBs.
As mentioned above, electron microscopy showed that
NEBs in rat lungs are contacted by nerve endings that
contain small, clear cholinergic-like synaptic vesicles, and
often reveal synaptic contacts with NEB cells. Therefore,
antibodies against the vesicular acetylcholine transporter
(VAChT; a marker for cholinergic nerves) were used to
visualize these possible cholinergic nerve terminals in direct relation to NEBs. Weakly VAChT-IR intraepithelial
cell groups, characterized as NEBs after multiple immunostaining (Fig. 19), appeared to be contacted by
VAChT-IR cholinergic nerve ﬁbers. Although the latter
nerve ﬁber population is not yet fully characterized, we
have evidence suggesting that cholinergic motor ﬁbers,
originating from preganglionic parasympathetic neurons
(and hence an as yet uncharacterized population) may be
involved.
Preliminary data further revealed that tyrosine hydroxylase-IR nerve terminals selectively contact some rat pulmonary NEBs at their basal pole. These nerve ﬁbers likely
have their origin in sympathetic ganglia.
CONCLUDING REMARKS
Selective Innervation of NEBs in the Rat Lung
As touched on in the Introduction, it has been suggested
that pulmonary NEBs are predominantly, if not exclusively, contacted by (vagal nodose) sensory nerve termi-
NEBS: COMPLEX AIRWAY RECEPTORS
Fig. 20. Schematic representation of the three fully characterized
nerve ﬁber populations that selectively contact pulmonary NEBs.
Fig. 21. a– c: Confocal images of an NEB present in the epithelium of
an intrapulmonary bronchus of a 10-day-old rat, triple-stained for nNOS
(red Cy3 ﬂuorescence), CGRP (green FITC ﬂuorescence), and CB (blue
pseudocolor of Cy5 emission in far-red). The CGRP/CB-IR NEB cells are
contacted by three different nerve ﬁber populations. In many places, the
nerve ﬁbers of the different populations are so close together that they
35
could only be distinguished by confocal laser scanning microscopy.
MVP of 33 confocal optical sections (1-␮m interval). a: Red nNOS-IR
nerve ﬁbers (arrowheads) run in the lamina propria and penetrate between the epithelial cells, apparently almost reaching (open arrowheads)
the airway lumen (L). b: Green channel showing thin varicose CGRP-IR
nerve ﬁbers (open arrows) contacting the basal side of CGRP-IR NEB
cells. c: Blue channel showing a bundle of CB-IR nerve ﬁbers (arrows)
giving rise to a CB-IR nerve plexus contacting CB-IR NEB cells.
36
ADRIAENSEN ET AL.
nals (Van Lommel et al., 1998, 1999; Cutz and Jackson,
1999; Widdicombe, 2001) in several mammalian species,
and in particular in rats (Van Lommel and Lauweryns,
1993a). Most of the evidence referred to in these works
was based on data obtained from electron microscopic
studies, and consequently often from a very limited number of NEBs.
In our investigations, neuronal tracing, chemical or mechanical denervation, and (immuno)cytochemistry, in
combination with confocal microscopy, have proven to be
valuable tools with which to study the overall pattern of
NEBs innervation. The application of these techniques
has resulted in extensive evidence that NEBs in rat lungs
may be selectively contacted by at least ﬁve distinct nerve
ﬁber populations that are both sensory and motor in nature, and have different origins. Multiple immunocytochemical staining with presently well-characterized
markers revealed that at least part of the pulmonary
NEBs are simultaneously contacted by several nerve ﬁber
populations. Caution is recommended, however, when dividing NEBs into subpopulations on the basis of the light
microscopic data reported in the present review only. It is
clear that none of the nerve ﬁber populations contact all
NEBs. Moreover, division into subgroups requires a thorough knowledge of the interrelationships between the various nerve ﬁber populations contacting pulmonary NEBs,
and thus necessitates reliable, multiple staining procedures. Moreover, it should be taken into account that the
use of other markers might reveal even more nerve ﬁber
populations related to pulmonary NEBs.
In a previous electron microscopic study of rat NEBs,
Van Lommel and Lauweryns (1993a) reported “clusters”
of nerve ﬁbers located between the neuroendocrine cells of
rat NEBs, but stressed that all of these terminals belong
to a single population of vagal nodose afferents presenting
intraepithelial efferent-like collaterals. In our studies of
rat lungs, however, vagal nodose sensory, intrinsic nitrergic, and VIP-IR motor terminals (and possibly extrinsic cholinergic motor terminals) all appeared to penetrate
between the neuroendocrine cells of NEBs, thus providing
evidence for the existence of more than just one intraepithelial nerve ﬁber population. Therefore, the nerve endings that were reported intact in the ispilateral lung after
infranodosal vagotomy (Van Lommel and Lauweryns,
1993a) do not necessarily reﬂect a crossing-over of intact
contralateral vagal ﬁbers (Lauweryns and Van Lommel,
1986; Van Lommel and Lauweryns, 1993a).
Development of the Innervation of Pulmonary
NEBs
The migration of neuroblasts from the neural crest to
the trachea in rats starts at around GD12 (Morikawa et
al., 1978). Neuroblasts can be identiﬁed at the late bronchial bud stage (GD14) by their PGP9.5 IR, after which a
primitive nerve network, associated with the airway walls
and to a lesser degree with the vasculature, is seen to
extend rapidly (Sorokin et al., 1997). It has been suggested
that neuroendocrine cell groups are ﬁrst contacted by
these postganglionic parasympathetic nerves, thereby becoming real NEBs, and only much later (just before birth)
receive sensory nerve endings (Sorokin et al., 1997). These
conclusions were based on observations that intrinsic
PGP9.5-IR nerve terminals contact pulmonary NEBs
around GD17 in in vitro fetal rat lung, while CGRP-IR
nerve terminals contacting NEBs in vivo are not common
until the end of postnatal week 1 (Sorokin et al., 1997).
However, from our studies it is clear that CGRP IR labels
spinal, and not vagal, sensory nerve terminals in contact
with NEBs, and that the CGRP-negative vagal nodose
sensory innervation most likely has been overlooked. It
was also conﬁrmed that the ﬁrst CGRP-IR nerve ﬁbers
contacting CGRP-IR NEBs could be detected later, at
GD19. Our studies further showed that from GD15-16
onward, a large number of subepithelial nerve ﬁbers seen
in rat airways express CB, a marker for the vagal nodose
sensory nerve ﬁber population that selectively innervates
NEBs from GD17 on. The latter observations were conﬁrmed by an ontogenetic study using P2X3 receptor immunolabeling as a marker for the vagal sensory component of NEB innervation (personal observations).
On the other hand, pulmonary NEBs do receive part of
their innervation from neurons intrinsic to the lungs (Sorokin et al., 1993, 1997). The present study showed that
from GD17 onward, PGP9.5-IR NEB cells are contacted by
PGP9.5-IR nerve ﬁbers, apparently originating from intrinsic PGP9.5-IR cell bodies that do not reveal nNOS IR
at that time. It is likely that at least part of the initial
intrapulmonary PGP9.5-IR neuroblasts or neurons, located along the pulmonary epithelial tubes, differentiate
into nNOS-IR and/or VIP-IR neurons at a later stage of
development and are at least partly responsible for the
speciﬁc intraepithelial nitrergic nerve terminals in NEBs.
An ontogenetic study (Brouns et al., 2002a) found that the
nitrergic nerve terminals selectively contacting rat NEBs
can only be visualized from postnatal day 2 onward, supporting the hypothesis that the innervation of rat NEBs
undergoes further maturation after birth.
Although previous studies proposed that ingrowth of
autonomic and sensory ﬁbers converts clusters of neuroendocrine cells into bona ﬁde NEBs (Sorokin et al., 1997),
that non-innervated PNEC clusters may be interpreted as
developing NEBs, and that the entire pulmonary neuroendocrine system should be regarded as progressing toward
complete capture by the nervous system (Sorokin and
Hoyt, 1990), our data indicate that even in postnatal lungs
the innervation pattern is far from identical for all NEBs,
and it is likely that part of the NEBs do not receive a
selective innervation. Nevertheless, all NEBs in postnatal
rat lungs, regardless of whether they receive a demonstrable innervation or not, apparently produce a similar palette of amines, purines, and peptides, and do not degenerate after birth. It may be that the ingrowth of motor and
sensory nerve ﬁbers into pulmonary NEBs does not determine their reaction to certain stimuli. The multicomponent innervation may regulate the sensitivity of NEB cells
to stimuli, and serve as a tool for exerting additional local
intrapulmonary and CNS reﬂexes.
Functional Implications
The data presented herein demonstrate that pulmonary
NEBs represent an extensive population of very complex
intraepithelial receptors that probably can accommodate
various sensory modalities. Although the physiological
signiﬁcance of the complex innervation pattern of NEBs is
still a matter of speculation, the sensory nature of NEBs is
beyond dispute. According to recent reviews, no physiological recordings have been obtained yet from single vagal
afferent ﬁbers from NEBs, and in general all recordings of
the effect of airway hypoxia have yielded negative results
NEBS: COMPLEX AIRWAY RECEPTORS
(Widdicombe, 2001). However, it seems very unlikely that
lung physiologists, in performing many thousands of single ﬁber recordings in the vagus nerve to identify airway
receptor populations in rat lungs, have never made registrations from the many hundreds of myelinated vagal
nodose neurons that selectively contact pulmonary NEBs
in each rat lung. More likely, the populations in question
have not yet been recognized among the existing data.
Undoubtedly, a considerable part of the already “characterized” airway receptors, a large part of which are mechanoreceptors, will eventually turn out to be related to
NEBs.
None of the nerve ﬁber populations characterized so far
contacts all pulmonary NEBs. However, the observation
that only half (or even 10%) of the NEBs receive a certain
nerve ﬁber population does not mean they should be considered as less important. After all, the “different” NEB
populations do account for at least a few hundred receptor
points each. The potential importance of NEBs is supported by the fact that the total number of NEB cells and
associated nerve ﬁbers in an animal easily outnumbers
carotid body cells and their selective innervation (Sorokin
and Hoyt, 1990).
Receptosecretory NEB cells are excellent candidates for
registering properties of the airway environment. Although there is accumulating evidence for a complete
functional system for oxygen sensing in PNECs (Peers and
Kemp, 2001), it should be stressed that the exact nature of
the possible physiological stimulus modalities of NEB
cells in healthy lungs is still unknown, and that it becomes
increasingly likely that other stimuli, such as mechanical
stimuli, may also be involved. Upon stimulation, NEB
cells reveal changes in the exocytosis of DCVs, and presumably release the amines, peptides, ACh, and ATP
stored in these secretory granules. In addition to acting on
nerve endings in contact with NEBs, the released bioactive substances may exert local actions on, for instance,
nearby airway or vascular smooth muscle (paracrine), or
could be taken up by nearby blood vessels (endocrine).
It is tempting to look at NEBs as local regulators of
airway functions that do not necessarily require signaling
to the CNS. The main sensor/effector action to hypoxia
could be local, and a possible central transduction of hypoxic stimuli may be mediated by spinal rather than vagal
afferents in rat lungs. Because they are located at strategic points along the intrapulmonary airways, NEBs appear to be ideally placed to effectuate and coordinate the
ﬁne-tuning of local blood ﬂow to local aeration, a physiological function that cannot be performed by the carotid
body or central hypoxia receptors. In this respect, pulmonary NEBs can be regarded as inexhaustible local pools of
vasoactive transmitters, such as the potent pulmonary
vasoconstrictor serotonin and vasodilator CGRP. Continuous release of endogenous CGRP from NEB cells may be
responsible for at least part of the homeostatic control of
blood vessel relaxation in normoxic lung areas. It has been
established that intact primary sensory CGRP/SP-containing and capsaicin-sensitive nerve ﬁbers are required
for endogenous CGRP to modulate pulmonary vascular
tone in hypoxic pulmonary hypertension (Tjen-A-Looi et
al., 1998). Because hypoxia apparently inhibits CGRP secretion from rat NEBs (Springall and Polak, 1993), local
physiological hypoxia may result in local inhibition of
vasodilation, implying local adjustment of pulmonary perfusion to ventilation. As demonstrated in the present
37
work, all NEBs with an intraepithelial nNOS-IR innervation also reveal basal contacts with CGRP-IR afferents,
the presumable collaterals of which appear to follow the
course of nitrergic axons and ﬁnally form baskets around
the nitrergic neurons in the lamina propria. This nitrergic
innervation may be a necessary component for the hypoxic
inhibition of CGRP release from NEB cells, requiring
CGRP-IR afferents for activation of the mechanism.
On the other hand, a system that prevents NEB receptors from continuously transmitting hypoxic information
to the CNS would be crucial for avoiding exaggerated
central actions that may eventually lead to a general
pulmonary vasoconstriction and hypertension. Therefore,
a similar mode of action of NO on the transduction of
hypoxic stimuli, as reported for carotid body cells (for
review, see Prabhakar, 1999), may be proposed for NEBs.
Release of NO from the terminals of intrinsic pulmonary
nitrergic neurons in NEBs may result in an inhibition of
the sensory discharge of NEBs in response to, e.g., local
hypoxia. Such a mechanism may keep NEB receptors
“quiet” as far as the CNS is concerned, creating time and
space for local actions. In this way, afferent signaling to
the CNS may be limited to powerful stimuli that necessitate central regulation of respiration.
In addition to their receptor function(s), PNECs and
NEBs may exert many other functions (independent of
their innervation?) during prenatal, perinatal, and early
neonatal life (for review, see Sorokin and Hoyt, 1989).
Because the vagal nodose sensory component of the selective innervation of NEBs appears to be fully differentiated
well before birth, it may be essential for neonatal respiratory adaptation.
Future Prospects
Although the innervation pattern of pulmonary NEBs
in rats appears to be fairly complex, there is no conclusive
evidence for assuming that the detailed functional morphological investigation is complete. We even have good
(unreported) evidence that other markers for nerve ﬁber
populations will provide additional features of the nerve
ﬁber populations that have already been characterized, or
will reveal additional nerve ﬁber populations contacting
pulmonary NEBs. Further characterization of receptors
for important neurotransmitters or neuromodulators,
present in NEB cells (e.g., 5-HT and CGRP) and in nerve
ﬁber endings associated with NEBs (e.g., VIP, CGRP, SP,
and ACh), will be necessary to further elucidate the functional signiﬁcance of these substances.
It is clear that PNECs show some marked species-speciﬁc differences in their palette of substances (Polak et al.,
1993), and that the origin and chemical coding of nerve
endings selectively contacting pulmonary NEBs may vary
from species to species. Obviously, it will be necessary to
validate the data obtained in rats (as discussed in the
present review) in other species as well. As mentioned
above, to date very little is known about the presence or
origin of the different nerve ﬁber populations selectively
innervating human pulmonary NEBs.
During the last decade, various methods have been developed to study PNECs/NEBs in vitro (Speirs and Cutz,
1993). Because the close relationship between NEB cells
themselves, and between NEBs and nerve ﬁbers, is completely lost in isolated cell suspensions of PNECs, more
recent in vitro studies have focused on the use of lung
slices (Fu et al., 1999). This technique offers a more real-
38
ADRIAENSEN ET AL.
istic natural environment for NEBs, and in several cases
probably does not even affect the relationship between
intrinsic neuronal cell bodies and pulmonary NEBs. Physiological reactions of pulmonary NEBs and their selective
innervation to administered neurotransmitters/modulators and (ant)agonists, or on environmental stimuli (e.g.,
hypoxia and nicotine), could be measured electrophysiologically or by microscopic visualization of physiologic
parameters (e.g., calcium concentration and membrane
potential) in these lung slices. In addition to investigations on healthy rat lungs, genetic models that reveal lung
disorders, or even speciﬁc abnormalities of the pulmonary
DNES, should be included in these studies.
In vivo experiments in which laboratory animals are
exposed to stimuli such as hypoxia, hyperoxia, hypercapnia, or to allergens, ozone, cigarette smoke, and other
pollutants, combined with morphological or in vitro physiological studies, will be necessary to further elucidate the
reactions of pulmonary NEBs to external stimuli.
In conclusion, a multidisciplinary approach, in which
modern histological and microscopic techniques are combined with cellular/molecular biological and electrophysiological methods, will be crucial to achieve further insight
into the precise working mechanisms and roles of PNECs/
NEBs in both the healthy and the diseased lung.
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