Functional Morphology of PulmonaryNeuroepithelial Bodies: Extremely

Complex Airway ReceptorsDIRK ADRIAENSEN,* INGE BROUNS, JEROEN VAN GENECHTEN, AND

JEAN-PIERRE TIMMERMANSLaboratory of Cell Biology and Histology, Department of Biomedical Sciences,

University of Antwerp–RUCA, Antwerp, Belgium

ABSTRACTInnervated groups of neuroendocrine cells, called neuroepithelial bod-

ies (NEBs), are diffusely spread in the epithelium of intrapulmonary air-ways in many species. Our present understanding of the morphology ofNEBs in mammalian lungs is comprehensive, but none of the proposedfunctional hypotheses have been proven conclusively. In recent reviews onairway innervation, NEBs have been added to the list of presumed physi-ological lung receptors. Microscopic data on the innervation of NEBs, how-ever, have given rise to conflicting interpretations. Using neuronal tracing,denervation, and immunostaining, we recently demonstrated that the in-nervation of NEBs is much more complex than the almost unique vagalnodose sensory innervation suggested by other authors. The aim of thepresent work is to summarize our present understanding about the originand chemical coding of the profuse nerve terminals that selectively contactpulmonary NEBs. A thorough knowledge of the complex interactions be-tween the neuroendocrine cells and at least five different nerve fiber pop-ulations is essential for defining the position(s) of NEBs among the manypulmonary receptors characterized by lung physiologists. Anat Rec Part A270A:25–40, 2003. © 2003 Wiley-Liss, Inc.

Key words: NEBs; neuroepithelial bodies; innervation; airwayreceptors; lung; rat

Highly specialized neuroepithelial bodies (NEBs) (Lau-weryns et al., 1972), which consist of extensively inner-vated groups of pulmonary neuroendocrine cells (PNECs),are normal components of the epithelium of intrapulmo-nary airways in humans, mammals, and all air-breathingvertebrate species investigated so far.

The pulmonary neuroendocrine system was first re-ported more than 50 years ago (Frohlich, 1949), but espe-cially over the last 25 years detailed information has beenprovided about the distribution, ontogeny, and micro-scopic morphology of NEBs (for reviews see Scheuermann,1987; Sorokin and Hoyt, 1989; Adriaensen and Scheuer-mann, 1993; Sorokin et al., 1997).

PNECs belong to the diffuse neuroendocrine system(DNES) (Pearse and Takor Takor, 1979), members ofwhich have been assigned important roles in the periph-eral control of various organs. The pulmonary DNES inhealthy lungs appears to be characterized by the produc-tion of amines and several neuropeptides, including sero-tonin (5-HT), bombesin (gastrin-releasing peptide), calci-

tonin gene-related peptide (CGRP), calcitonin, enkephalin,somatostatin, cholecystokinin, and substance P (SP) (forreviews see Sorokin and Hoyt, 1989; Scheuermann et al.,1992; Adriaensen and Scheuermann, 1993). These bioac-tive substances are stored in secretory granules (60–200nm diameter) with typical endocrine-like characteristics,the so-called dense-cored vesicles (DCVs).

Interestingly, PNECs are by far the first cell type tofully differentiate in the human airway epithelium (before

*Correspondence to: Dirk Adriaensen, Laboratory of Cell Biol-ogy and Histology, University of Antwerp–RUCA, Groenenborg-erlaan 171, B-2020 Antwerp, Belgium. Fax: �32-3-218-0301.E-mail: dadria@ruca.ua.ac.be

Received 22 May 2002; Accepted 5 September 2002DOI 10.1002/ar.a.10007

THE ANATOMICAL RECORD PART A 270A:25–40 (2003)

© 2003 WILEY-LISS, INC.

the 8th week of gestation), and changes in PNECs/NEBshave been associated with many perinatal, neonatal, andadult lung diseases and disorders.

NEBs may have important functions in the regulation ofphysiological processes in lungs at all ages, but their exactrole is poorly understood. Mainly on the basis of incom-plete morphological data, several hypotheses about thefunction of pulmonary NEBs have been proposed (Sorokinand Hoyt, 1990). NEBs are now believed to have differentfunctions during specific periods in prenatal, early post-natal, and adult life. Oxygen-sensing (Youngson et al.,1993; Cutz and Jackson, 1999; Peers and Kemp, 2001) andeffector (O’Kelly et al., 1998, 1999) mechanisms have beenidentified in pulmonary neuroendocrine cells.

Recent reviews on afferent receptors in the lower air-ways have added NEBs to the list of presumed receptortypes, which included slowly (SARs) and rapidly adaptingstretch receptors (RARs) and C-fiber receptors (Widdi-combe, 2001). However, Widdicombe (2001) also statedthat so far there have been no claims for recordings fromsingle afferent fibers from NEBs. In our opinion, the mostlikely reason for that is a lack of knowledge of the func-tional morphology and the nervous connections of theseNEB “receptors.”

Because the present review is part of a special issue onairway receptors, it mainly focuses on the innervation ofpulmonary NEBs in general, and on the sensory compo-nents in particular. Our recent neuronal tracing, immu-nocytochemistry, and denervation experiments in rats,which provide good evidence for an extremely complexinnervation pattern of NEBs (Adriaensen et al., 1998,2001; Brouns et al., 2000, 2002a, b, 2003), are summa-rized, illustrated, and discussed in the light of the possiblereceptor function(s) of pulmonary NEBs.

Literature Data and Classical Concepts Aboutthe Innervation of Pulmonary NEBs

A selective innervation of NEBs was probably describedlong before the existence of the pulmonary neuroendocrinesystem itself was known. Several authors described intra-epithelial varicose nerve terminals that were concentratedin groups and irregularly distributed along the airways ofdifferent species, including humans (Berkley, 1894;Larsell, 1921; Larsell and Dow, 1933; Elftman, 1943). Itwas Frohlich (1949) who described delicate nerve termi-nals at the surface of, or between the cells of groupedneuroendocrine cells in the bronchial epithelium of rabbitsand cats. Since that time, many researchers have ob-served an indisputable innervation of NEBs in both lightand electron microscopic investigations.

Organ culture studies suggested that the nerve termi-nals contacting NEBs rapidly degenerate, but that dener-vated NEBs survive (Sonstegard et al., 1979), and thatsolitary PNECs may give rise to structured organoid bod-ies in vitro (Carabba et al., 1985). Three-dimensional re-construction of a single hamster NEB using electron mi-croscopic images of serial sections showed that also inintact animals, not every NEB is necessarily innervated(Pearsall et al., 1985).

A number of methods have been used to visualize nervefibers that contact NEBs. To this end, an unambiguousand simultaneous identification of PNECs and nerves wasconsidered 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 neuroen-docrine cells were described in the lungs of human new-borns (Frohlich, 1949; Lauweryns and Peuskens, 1972),rabbits (Lauweryns et al., 1972; Hung, 1980), rats(Wasano, 1977), and mice (Hung, 1984), as well as in lowervertebrates such as turtles (Scheuermann et al., 1983).Silver impregnation of the NEB cells, however, almostalways hampers a clear view of the nerve terminals con-tacting the NEB cells.

A regularly used method for studying lung innervationis the histochemical demonstration of acetylcholinesterase(AChE) activity. Networks of AChE-positive nerve fiberswere described in contact with pulmonary NEBs of fetaland 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 structuresand stains the NEB cells, resulting in a poor definition ofnerve endings at the contact sites.

Formaldehyde-induced fluorescence resulted in thedemonstration of a blue-green nerve plexus, with spectralcharacteristics of catecholamines, in the lamina propria ofairways, close to the base of NEBs in rabbits (Lauwerynset 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 character-istics of serotonin.

More recently, immunocytochemical methods that weregenerally applied for the demonstration of NEBs oftenalso appeared to label contacting nerve fibers. In mostcases, the problem of differentiating the nerve terminalsat the level of the NEBs remains. In rabbits, hamsters,and rats, pulmonary NEBs and associated nerve fiberswere described using antisera against protein gene prod-uct 9.5 (PGP9.5) (Lauweryns and Van Ranst, 1988). Innewborn cats (Adriaensen and Scheuermann, 1993), NSE-immunoreactive (IR) nerve fibers were observed to contactNSE-positive NEBs. CGRP-IR nerve fibers connected toCGRP-positive NEBs were reported in the lungs of cats(Adriaensen and Scheuermann, 1993) and rats (Cadieuxet al., 1986; Van Ranst and Lauweryns, 1990; Terada etal., 1992). In guinea pigs, a double immunofluorescencelabeling technique, applying antisera against neuropep-tides and general neuroendocrine markers (PGP9.5; chro-mogranin), resulted in the demonstration of solitary tra-cheal and bronchial PNECs spirally surrounded by CGRP/SP-IR nerve terminals (Kummer et al., 1991). Usingadditional pre-embedding electron microscopic immunocy-tochemistry, the latter nerve terminals were seen to formdirect contacts with the PNECs, but without the presenceof synaptic specializations. In the human lung, however,where PNECs and NEBs were shown to be SP-IR, noassociated SP-positive nerve fibers have been reported(Gallego et al., 1990).

None of the techniques described so far demonstratedunambiguously that the closely associated nerve fibersindeed provide a direct innervation of the neuroendocrinecells in NEBs. Such information could only be obtainedusing electron microscopy.

Nerve fibers associated with NEBs were seen in themouse lung using scanning electron microscopy (Hung,1984), but because this technique only reveals surfacestructure, it was not possible to obtain a clear view of theinteractions between neuroendocrine cells and nerves.

26 ADRIAENSEN ET AL.

With the use of transmission electron microscopy(TEM), nerve terminals were demonstrated directly con-tacting the basal part of PNECs, or running between andlooping around the cells in NEBs. The following referenceswere selected based mainly on their descriptions of syn-apse-like specializations between nerve terminals andNEB cells. Direct innervation of pulmonary NEBs wasobserved in humans (Lauweryns et al., 1970) and manymammalian species, such as mice (Wasano, 1977; Wasanoand Yamamoto, 1981), rats (Van Lommel and Lauweryns,1993a), hamsters (Sorokin and Hoyt, 1989), rabbits (Lau-weryns et al., 1972; Lauweryns and Van Lommel, 1987;Sorokin and Hoyt, 1989; Adriaensen and Scheuermann,1993), and cats (Van Lommel and Lauweryns, 1993b). Inbirds, similar NEB/nerve contacts were reported in thelungs of chickens (Cook and King, 1969; Walsh and McLel-land, 1978; Wasano and Yamamoto, 1979; Lopez et al.,1983), pigeons (McLelland and MacFarlane, 1986), andquails (Adriaensen et al., 1994). In lower vertebrates, di-rect innervation was shown in reptiles, such as turtles(Scheuermann et al., 1983), lizards (Ravazzola and Orci,1981), and snakes (Wasano and Yamamoto, 1976); in am-phibia, such as newts (Scheuermann et al., 1989; Gonia-kowska-Witalinska et al., 1992; Adriaensen and Scheuer-mann, 1993), frogs (Wasano and Yamamoto, 1978;Goniakowska-Witalinska, 1981), and toads (Rogers andHaller, 1980; Goniakowska-Witalinska et al., 1990); andin lungfish (Dipnoi) (Adriaensen et al., 1990). Nerve fibershave been reported to contact solitary PNECs and NEBcells, although by far the most reports concern NEBs. Inseveral species, solitary PNECs apparently do not showdirect contacts with nerve fibers, which was reasonenough for some authors to regard them as different en-tities from NEBs. It is clear, however, that the surfacemembrane of all PNECs is functionally divided into baso-lateral and apical regions. Possible nerve contacts in thisrespect provide further possibilities for functional integra-tion. Using TEM, different types of morphologically char-acterized nerve terminals have been described in contactwith PNECs.

The type of nerve terminal most often reported in con-tact with NEBs, in many species, is packed with mitochon-dria and some small clear vesicles. They penetrate deepbetween the NEB cells, often close to the luminal surface.The nerve terminals reveal asymmetric synaptic contacts,with an accumulation of DCVs near electron-dense cone-shaped thickenings of the surface membrane of PNECs,and are generally believed to be afferent (sensory) (Cookand King, 1969; Wasano and Yamamoto, 1981; Lauwerynset al., 1985; Lauweryns and Van Lommel, 1986, 1987;Adriaensen and Scheuermann, 1993; Van Lommel andLauweryns, 1993a, b).

A second frequently reported type of nerve terminalappears to be loaded with small clear vesicles (40–60 nmdiameter) and shows a few larger DCVs and mitochondria.In several species they reveal asymmetric synaptic con-tacts with NEB cells, with an accumulation of the clearvesicles near cone-shaped electron-dense specializationsof the axolemma of the nerve terminal, while the surfacemembrane of the PNEC shows a uniform thickening;these nerve terminals are generally considered efferent(cholinergic-like) (Walsh and McLelland, 1978; Sonste-gard et al., 1982; Stahlman and Gray, 1984; Adriaensenand Scheuermann, 1993).

In some mammalian species, it has been demonstratedthat both morphologically afferent and efferent contactsmay be peripheral specializations of the same (sensory?)nerve fiber (Lauweryns and Van Lommel, 1987; Van Lom-mel and Lauweryns, 1993a, b).

A few reports mention nerve endings filled with smalldense-cored granules (about 60 nm diameter) (Rogers andHaller, 1978), typical of adrenergic nerves, and terminalscontaining larger (80–225 nm), moderately dense DCVs(Stahlman and Gray, 1984), believed to belong to a pepti-dergic pathway, in close apposition to PNECs.

Although TEM probably offers a rather good morpho-logical characterization of the direct innervation of a lim-ited number of pulmonary NEBs in several species, itleaves another important question unanswered, i.e., thatof the origin of the nerve fiber population(s) that selec-tively innervate(s) NEBs.

To date, most of the studies dealing with this aspecthave combined electron microscopy, for the evaluation ofnerve terminals contacting NEBs, and experimental va-gotomy with or without an additional hypoxic stimulus (inrabbits (Lauweryns et al., 1985; Lauweryns and Van Lom-mel, 1986) and rats (Van Lommel and Lauweryns,1993a)). It was reported that, after unilateral infranodosalvagotomy, the numbers of both morphologically afferentand efferent nerve terminals in NEBs were reduced byabout 70% in lungs ipsilateral to the denervation, while nochanges were seen in the contralateral lungs. The NEBinnervation appeared to be intact after supranodosal va-gotomy. Based on these findings, it was suggested thatNEBs are predominantly innervated by sensory nerve fi-bers originating from neurons located in the vagal gan-glion nodosum. The remaining 30% of nerve terminals inthe investigated NEBs were thought to be the result ofcross innervation from the contralateral vagus or of thepresence of another unknown nerve fiber population (e.g.,sympathetic) contacting NEBs. Electrical stimulation ofthe sectioned vagal nerve revealed a possible vagal inhib-itory secretomotor influence on rabbit pulmonary NEBs,since the basal secretion of DCVs was apparently de-creased (Lauweryns et al., 1985).

More specifically for rat lung, the very extensive inner-vation of NEBs was suggested to be exclusively sensory,originating from the vagal nodose ganglion (Van Lommeland Lauweryns, 1993a). The electron microscopicallyquantified efferent- and afferent-like terminals were in-terpreted as belonging to the same population of sensoryfibers, since vagotomy experiments resulted in a clearlyreduced number of nerve terminals in NEBs, while theratio between efferent- and afferent-like terminals ap-peared unchanged in the selected NEBs. It was furtherconcluded that, unlike in rabbits, a separate motor inner-vation of NEBs is probably absent in rat lungs. However,given our own more recent data on the innervation of ratNEBs (see below), the question could be raised as towhether the combination of experimental denervation andelectron microscopy, which is very labor-intensive andtherefore necessarily limited by the number of fully quan-tifiable specimens, is a reliable method by which to drawconclusions on such a complex and inhomogeneous diffusepopulation of NEBs. The latter point is strengthened bythe observation that NEBs in organ-cultured rat lungsapparently receive an innervation from neurons located inintrinsic airway ganglia (Sorokin et al., 1993).

27NEBS: COMPLEX AIRWAY RECEPTORS

To date, we know very little about the presence or originof different nerve fiber populations selectively innervatingNEBs in the human lung.

To conclude this historical perspective and at the sametime come back to the main point of the present shortreview, i.e., the characterization of pulmonary NEBs asputative airway receptors, it may be of interest to revisitthe scheme that was presented in our 1993 NEB review(Adriaensen and Scheuermann, 1993) (Fig. 1). Essen-tially, what is represented is the best possible view we hadat that time of the morphological organization of NEBsand directly related nerve terminals. It further aimed atexplaining a possible mechanism for the assumed reactionof NEBs to airway hypoxia. Most of the information rep-resented in the scheme was based on electron microscopicliterature data. It may be stressed that several more re-cent reviews on pulmonary NEBs still come to the sameconclusion, i.e., NEB innervation is mainly vagal nodoseand NEBs may act as hypoxia sensors via a vagal afferentpathway (Cutz and Jackson, 1999; Van Lommel et al.,1999), and that the same scheme was used as representa-tive for pulmonary NEBs in a recent review on airwayreceptors (Widdicombe, 2001). The interest in this concepthas been recently strengthened by the characterization ofa complete, carotid body-like, oxygen-sensing mechanismin NEBs, which implicates exocytosis of transmitters as aresult of hypoxia (for review see Peers and Kemp, 2001).In summary, many researchers in the field today believethat if the stimulus is strong enough, hypoxia may cause,in addition to local reflex actions, an afferent signal totravel toward the central nervous system (CNS) via thevagus nerve.

However, this leaves us with the essential question ofwhy lung physiologists have been unable to confirm thisapparently rather simple mechanism. Are NEBs reallythe lung receptors we would like them to be? If so, did weoverlook something? Having many years of experiencelooking at the functional morphology of NEBs, we evi-dently went for the latter possibility and tried to find outif it is really that simple. We realized that if NEBs areindeed airway receptors, the best way to try to understandthem would be to have a good look at their nervous con-nections, and we decided to focus on the rat lung.

Functional Morphological Characterization ofthe Selective Innervation of NEBs in Rat Lungs

Vagal nodose sensory component of the selectiveinnervation of pulmonary NEBs. After we consideredall available literature data, it was clear that an essentialfactor in the recognition of NEBs as airway receptorswould be the full confirmation and characterization oftheir vagal nodose innervation. The following questionsarose: Is the vagal nodose innervation of NEBs reallythere? How can it unambiguously be identified in the lightmicroscope? What are the neurochemical characteristics?How does this vagal nodose connection relate to what lungphysiologists know, or thought they knew?

Since several authors reported that in rat lungsCGRP-IR NEBs at all levels of the intrapulmonary air-ways are contacted by CGRP-positive nerve fibers(Cadieux et al., 1986; Shimosegawa and Said, 1991;Terada et al., 1992; Sorokin et al., 1997) (personal obser-vations), it was generally believed that they representedthe long-predicted vagal sensory fibers. However, after

performing some vagotomy experiments we realized thatthis CGRP-IR nerve fiber population is nonvagal, as isfurther characterized in the next section.

The problem of identifying a vagal nodose component ofthe innervation of NEBs was then addressed in the mostdirect way possible: the red fluorescent neuronal tracerDiI, a lipophilic carbocyanine dye (Honig, 1993), was in-jected unilaterally into rat nodose ganglia, and NEBs werevisualized in toto by using immunocytochemistry and con-focal microscopy on thick frozen sections (Adriaensen etal., 1998). The most striking finding was the extensiveintraepithelial terminal arborizations of DiI-labeled vagalnodose afferents in intrapulmonary airways, often locatedclose to the luminal surface of the airways and apparentlyalways co-appearing with CGRP-IR NEBs (Fig. 2). Not allNEBs received a traced nerve fiber. IntrapulmonaryCGRP-containing nerve fibers, including those innervat-ing NEBs, always appeared to belong to a nerve fiberpopulation different from the DiI-traced fibers. This wasthe first hard evidence demonstrating at the light micro-scopic level that at least some of the pulmonary NEBs inrats are indeed supplied with sensory nerve fibers thatoriginate in the vagal nodose ganglion, and that formbeaded ramifications between the NEB cells.

However, neuronal tracing is a labor-intensive andtime-consuming technique, and is hardly compatible witha routine application. Therefore, we tried to further char-acterize this vagal nodose population of nerve terminals inNEBs.

Combinations of neuronal tracing and/or vagal dener-vation experiments with immunolabeling taught us thatthe vagal nodose fibers contacting pulmonary NEBs canbe labeled selectively with antibodies against the calcium-binding protein calbindin D28k (Adriaensen et al., 1999;Brouns et al., 2000). Calbindin (CB) appears to label NEBcells, as well as the nodose fibers contacting them (Fig. 3),and hence can be regarded as a very interesting routinemarker for NEBs in the rat lungs. Apparently, a little lessthan half of the NEBs are contacted by such CB-IR nervefibers in control animals. The first NEBs contacted byCB-IR nerve fibers could be detected at gestational day(GD) 17. After unilateral infranodose vagotomy, no CB-IRnerves contacting NEBs were left in the ipsilateral lung,while no changes were observed in the contralateral lung.Because both NEBs and contacting nerve fibers arestained, this marker does not allow an evaluation of theintraepithelial terminals. Double immunocytochemicalstaining with antibodies against CB and CGRP clearlyrevealed that both substances mark a different nerve fiberpopulation, often contacting the same NEBs (Fig. 4).

To further characterize the different nerve fiber popu-lations related to NEBs in the rat lung, we performed asystemic capsaicin treatment (Brouns et al., 2003). Theresults revealed no changes in the CB-IR vagal nodoseinnervation of NEBs as compared to control rats, stronglysuggesting that a capsaicin-insensitive population is in-volved. The method of capsaicin treatment is time-con-suming, destroys certain nerve fiber populations, and isobviously not always the method of choice for the selectivedemonstration of nerve fiber populations in routine appli-cations. Therefore, we tried to confirm the results of thecapsaicin treatment using antibodies against the capsa-icin receptor (vanilloid receptor 1 (VR1)). Double stainingfor VR1 and CB indeed confirmed that the vagal nodosecomponent of the innervation of NEBs does not express

28 ADRIAENSEN ET AL.

Fig. 1. Conceptual scheme summarizing findings on the innervationof mammalian NEBs and their possible reaction to airway hypoxia.Hypoxic air stimulates (red dotted arrows) the NEB cells (yellow) todischarge the contents of their DCVs at afferent synaptic sites of intra-corpuscular sensory nerve endings (white arrowheads). This could resultin depolarization and the development of a generator potential thatspreads over the nerve fiber (white arrows). Given the possibility thatafferent and efferent nerve terminals might occur along the same nerveprocess, as was indeed demonstrated in serial sections of newbornrabbit NEBs, the efferent endings would develop synaptic activity anddischarge neurotransmitter (black arrowheads). This may in turn influ-ence the physiological state of the receptor cell (green arrows), thusmodulating the transduction of stimuli or regulating local paracrine se-cretion acting on nearby blood vessels (red arrows), or smooth musclebundles (blue arrows). The mechanism of local modulation of a neuro-receptor by efferent nerve endings formed at the periphery of sensorynerves could be called the “axon reflex.” When the generator potentialreaches a threshold value, an action potential is triggered (white arrows

with double arrowheads). Some nerve terminals appear to be exclusivelyefferent (double black arrowheads). Clara-like cells (CC); capillary (C);smooth muscle bundle (SM); airway lumen (L). Modified from Adriaensenand Scheuermann (1993).

Fig. 2. a and b: Detail of DiI-traced (red fluorescence) vagal sensorynerve terminals contacting a pulmonary NEB (calcitonin gene-relatedpeptide (CGRP)-immunofluorescence; FITC fluorescence) at the alveolarlevel in the adult rat lung. Maximum value projections (MVPs) of 36optical sections (1-�m interval). a: Red channel clearly demonstratingthe DiI-traced nerve fibers approaching the epithelium (arrows) andforming intraepithelial terminals (arrowheads). b: Combination of the redand green channels, showing the complete NEB and all of the labelednerve endings. The traced nerve endings run along a bronchiole (B) andenter the epithelium at the base of the NEB (open arrowheads).

Fig. 3. MVP of 14 confocal optical sections (2-�m interval) of an NEBin a bronchus of a neonatal rat. Thick calbindin D28k (CB)-IR (red Cy3fluorescence) nerve fibers (arrows) contact the CB-IR NEB cells.

capsaicin receptors (Fig. 5), while often another popula-tion that is VR1-IR appears to co-innervate NEBs (seebelow). In contrast to some literature data claiming thatcapsaicin treatment causes a depletion of CGRP from ratNEBs (Tjen-A-Looi et al., 1998), quantification of the num-ber of CGRP- and PGP9.5-IR NEBs after capsaicin treat-ment did not reveal significant differences with controllungs in our study. The latter observation was strength-ened by the absence of VR1 expression from NEBs (Figs. 5and 12), making a direct effect of capsaicin on the CGRPcontent of NEBs highly unlikely.

Another interesting feature of the vagal sensory compo-nent of the innervation of pulmonary NEBs, especially inthe light of the present efforts to characterize NEBs asairway receptors, can be visualized using antibodiesagainst the myelin basic protein (MBP), as a marker formyelinated nerve fibers in the lung. It was observed thatcombined with CB staining, from postnatal day 10 on, thevagal nodose fibers contacting NEBs are invariably my-elinated (Fig. 6) (Brouns et al., 2003). Myelinated nervefibers have been observed electron microscopically in thevicinity of NEBs (Van Lommel and Lauweryns, 1993a),

Figures 4–10.

30 ADRIAENSEN ET AL.

but no evidence or suggestions have linked these myelin-ated fibers to the vagal nodose intraepithelial nerve ter-minals situated between NEB cells.

Examination of rat lungs using specific antibodiesagainst P2X3 purinoreceptors (ATP receptors) revealedintraepithelial arborizations of P2X3 receptor-IR nerveterminals, which in all cases appeared to ramify betweenCGRP- or CB-labeled NEB cells (Brouns et al., 2000)(Figs. 7 and 8). The first NEBs contacted by P2X3 recep-tor-IR nerve terminals could be detected at GD 17. NEBcells did not express P2X3 receptors. It was further dem-onstrated that P2X3 receptor and CB IR completely colo-calize in the vagal nodose population of nerve fibers thatselectively contacts NEBs (Fig. 8), whereas CGRP-IR fi-bers clearly form a different population (Fig. 7). The dis-appearance of characteristic P2X3 receptor-IR nerve ter-minals in contact with NEBs after infranodosal vagaldenervation, and the colocalization of tracer and P2X3receptor-labeling in vagal nodose neuronal cell bodies inretrograde tracing experiments from the lungs, supportour hypothesis that the P2X3 receptor-expressing nervefibers contacting NEBs have their origin in the vagalnodose ganglia. The combination of MBP and P2X3 recep-tor immunostaining again clearly pointed to a myelinatedpopulation (Fig. 9) (Brouns et al., 2003). Further concen-trating on ATP and ATP receptors, we applied quinacrinehistochemistry, a method that has been shown to selec-tively label high concentrations of ATP, if accumulatedtogether with proteins in secretory granules. In rat lungs,the latter method has been shown to selectively labelNEBs (Brouns et al., 2000). Combination of quinacrine

histochemistry and P2X3 receptor-staining showed thatthe ATP receptor-expressing nerve terminals in rat lungsare exclusively associated with quinacrine-stained NEBs(Fig. 10). ATP may, therefore, act as a neurotransmitter inthe vagal sensory innervation of NEBs via a P2X3 recep-tor-mediated pathway. Given the extensive data obtainedin other systems (Burnstock, 1999a, b), the possibility thatat least some of the NEBs may be involved in vagal affer-ent respiratory mechanosensory transduction should beconsidered.

All available data on the vagal nodose sensory compo-nent of the innervation of NEBs are summarized in thescheme of Figure 20 (nerve fiber population shown inblue). Essentially, the sensory nerve fiber population in-volved reveals extensive intraepithelial terminals, has avagal nodose origin, can be marked by its CB IR, expressesP2X3 ATP receptors but no VR1 capsaicin receptors, and ismyelinated. Consequently, as predicted in the initialscheme (Fig. 1), NEBs are indeed supplied by a populationof vagal nodose sensory fibers that have now been fullycharacterized. However, the question remains: why has itso far been impossible for lung physiologists to link NEBsto measurable activities in vagal afferents? Is it perhapsmore complicated than was predicted?

Calcitonin gene-related peptide-immunoreac-tive component of the selective innervation of pul-monary NEBs. As noted above, the vagal sensory com-ponent of the innervation of rat pulmonary NEBsappeared to form a clearly different nerve fiber populationthan the sensory CGRP-IR innervation that contacts

Fig. 4. Immunocytochemical double-staining of an NEB in a bron-chiole of a 10-day-old rat labeled for CGRP (green FITC fluorescence)and CB (red Cy3 fluorescence). CGRP/CB-IR NEB cells are contacted bya nerve bundle composed of CB-IR nerve fibers (arrows) and thin vari-cose CGRP-IR nerve terminals (arrowheads). Note that in the NEB cells,CGRP labeling is strongest at the basal side while CB labeling appearsto 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 amarker for the vagal sensory subpopulation of nerve terminals contact-ing pulmonary NEBs. High magnification detail of the basal part of aCB-IR NEB, revealing that VR1 IR (open arrows) and CB IR (arrows) areexpressed by separate nerve fiber populations. MVP of six confocaloptical sections (1-�m interval).

Fig. 6. Immunocytochemical staining for CB (green FITC fluores-cence) and myelin basic protein (MBP; red Cy3 fluorescence) of abronchiolar NEB in a 21-day-old rat. Combination of the red and greenchannel shows that the CB-IR nerve fibers (arrows) are surrounded byMBP-IR myelin sheaths (open arrows), that are lost (arrowheads) justbefore the CB-IR fibers branch and contact the CB-IR NEB. MVP of 25confocal optical sections (1-�m interval).

Fig. 7. a and b: Immunocytochemical double-staining for the ATPreceptor P2X3 (green FITC-fluorescence) and CGRP (red Cy3-fluores-cence) revealing pulmonary NEBs in a rat bronchiole contacted by P2X3

receptor- and CGRP-expressing nerve fibers. MVPs of 16 confocaloptical sections (1-�m interval). a: Green channel showing two neigh-boring intraepithelial complexes of P2X3 receptor-IR nerve terminals.Note an approaching nerve fiber (arrows). b: Combination of the red andgreen channels, showing two CGRP-IR NEBs contacted by separatepopulations of CGRP-containing (open arrows) and P2X3 receptor-ex-pressing (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) andCB-IR nerve terminals. MVP of 10 confocal optical sections (1.5-�minterval). a: Green channel showing an intraepithelial P2X3 receptor-expressing 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 reveal-ing that nerve fibers in contact with the NEB express both P2X3 recep-tors and CB, although the staining intensity varies along the nerve fibers.

Fig. 9. a and b: Confocal microscopic images of immunocytochem-ical double-staining for MBP (red Cy3 fluorescence) and P2X3 receptors(green FITC fluorescence) in the bronchus of a 21-day-old rat. MVP of 30optical sections (0.8-�m interval). a: MBP IR can be detected in myelinsheaths (open arrows) of nerve fibers in close proximity to the bronchialepithelium (E). b: Combination of the red and green channels, showingthat FITC-labeled vagal afferent nerve fibers expressing P2X3 receptors(arrows) approach the epithelium, branch, protrude between the epithe-lial cells, and give rise to intraepithelial nerve terminals, many of which(open arrowheads) are seen close to the luminal surface (L, lumen of thebronchus). The latter intraepithelial structure represents an NEB. TheP2X3 receptor-IR nerve fibers are myelinated and the MBP-IR myelinsheets are lost in close proximity to the target (arrowhead).

Fig. 10. a–c: Consecutive quinacrine histochemistry and P2X3 re-ceptor staining in a rat bronchus. a: Fluorescence micrograph of quin-acrine accumulation in an intraepithelial cell group (arrowhead), indicat-ing the presence of an ATP-storing NEB. b and c: P2X3 receptorimmunocytochemistry revealing the presence of extensive receptor-expressing nerve terminals at exactly the same spot as marked in part a(arrowhead). Note the P2X3 receptor IR in bronchial smooth musclebundles (SM). Single confocal optical section. c: High magnificationdetail of part b, clearly showing the P2X3 receptor-IR nerve fiber (arrow)that loops through the SM, branches (arrowheads), and gives rise to aterminal intraepithelial arborization. MVP of 20 confocal optical sections(0.95-�m interval).

31NEBS: COMPLEX AIRWAY RECEPTORS

NEBs (Shimosegawa and Said, 1991; Terada et al., 1992;Sorokin et al., 1997). Denervation studies and retrogradetracing from the lung (Springall et al., 1987) (personalobservations) strongly indicate that the CGRP-IR nervefibers that selectively contact NEBs in the rat lung belongto a spinal sensory population, originating in dorsal rootganglia (DRG) T1–T6. The first NEBs contacted byCGRP-IR nerve fibers could be detected at GD 19.

We then tried to further characterize this CGRP-IRpopulation (Brouns et al., 2003). The first observation wasthat CGRP-positive terminals contacting NEBs invariablycolocalize SP (Fig. 11). Using CGRP IR alone, it was im-possible to visualize the real contact sites between nerveterminals and the NEB cells that also show a strongCGRP staining. SP does not label rat NEBs. Therefore,CGRP/SP double labeling allows the differentiation of in-

Figures 11–19.

32 ADRIAENSEN ET AL.

dividual nerve terminals at the level of NEB cells. Itturned out that the spinal sensory CGRP�/SP� nerveterminals do not penetrate the epithelium, as was shownfor the vagal sensory endings, but that they form a plexusthat preferentially contacts the basal surface of NEBs(Figs. 11 and 12), clearly a population that was notpresent in the initial scheme (Fig. 1). Obviously, airwaysalso harbor a vagal CGRP/SP-IR nerve fiber population,but evidence suggests that in rat lungs these vagal fibersmainly originate from the jugular ganglia, and terminalscan be found in the epithelium of large-diameter bronchionly—apparently without any specific relationship withNEBs (personal observations). This population is not dis-cussed further.

We then studied the selective CGRP/SP-IR innervationof NEBs in lungs of capsaicin-treated rats (Brouns et al.,2003). After capsaicin treatment, the percentage of NEBscontacted by CGRP-positive nerve terminals was dramat-ically reduced (5.90%) compared to control lungs (51.80%),while the numbers of CGRP-IR NEBs revealed no signif-icant changes (see above). To further strengthen the cap-saicin data, immunocytochemistry with antibodiesagainst the capsaicin receptor VR1 was applied. The re-sults 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. TheVR1/CGRP-IR nerve fiber plexus mainly contacts the baseof the NEBs, and again the NEBs themselves appeared tobe VR1-negative.

All available data on the spinal sensory component ofthe selective innervation of NEBs are summarized in thescheme of Figure 20 (nerve fiber population shown ingreen). Essentially, the sensory nerve fiber populationinvolved forms a mainly basal plexus, most likely has itsorigin in the DRGs T1-T6, can be marked by its CGRP/SPIR, is capsaicin-sensitive, and expresses VR1s.

It is now firmly established that rat pulmonary NEBsmay receive at least two different sensory nerve fiberpopulations. Again, however, the question remains as towhether this double sensory innervation can explain thediscrepancy between the good evidence that NEBs arehypoxia sensors, and the lack of physiological evidence forthe vagal transmission of hypoxia-related stimuli. Onepossibility is that central hypoxic responses, originatingfrom NEB “receptors” are mediated via a spinal afferentinstead of a vagal pathway. However, it may be morecomplicated than that.

Fig. 11. a and b: Immunocytochemical double-staining for sub-stance P (SP; green FITC fluorescence) and CGRP (red Cy3 fluores-cence) 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 showingSP-IR nerve fibers (open arrows) contacting the base of the epithelium(open arrowheads). b: The combination of the green and red channelsclearly shows an extensive network of SP/CGRP-IR (yellow fluorescencebeing indicative of colocalization) nerve fibers (open arrows) contactingthe base (open arrowheads) of a CGRP-IR NEB.

Fig. 12. Immunocytochemical double-staining for VR1 (green FITCfluorescence) and CGRP (red Cy3 fluorescence), as a marker for thespinal sensory subpopulation of nerve terminals contacting pulmonaryNEBs. Bronchiolar CGRP-IR NEB in a 10-day-old rat contacted byextensively branching CGRP-containing nerve fibers that also expressVR1 (open arrows; the yellow fluorescence is indicative of colocaliza-tion). CGRP IR NEB cells do not show VR1 IR. Reconstruction of sevenoptical 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 redand green channels showing nNOS-IR neuronal cell bodies (arrowheads)that give rise to a nitrergic nerve plexus (arrows) in the lamina propria andto intraepithelial nerve terminals (open arrowheads) selectively contact-ing the CB-IR NEB (asterisk). The image is suggestive of a direct rela-tionship between the selective intraepithelial nitrergic innervation of theNEB, and the nearby nNOS-IR neurons. Note the absence of CB immu-nostaining from the nitrergic nerves (arrows). MVP of 27 confocal opticalsections (1.5-�m interval).

Fig. 14. a–c: Detail of a CGRP-IR (green FITC-fluorescence) NEB ina 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-ex-pressing arborization (open arrowheads) originating from subepithelialnerve fibers (arrows). b: Green channel showing CGRP-IR nerve fibers(open arrows) contacting the base of a CGRP-IR NEB. c. Combination ofboth channels revealing that the intraepithelial nitrergic nerve terminalsdo not contain CGRP, although the subepithelial CGRP-IR nerve fibersrun in very close proximity to the nNOS-IR fibers.

Fig. 15. High-magnification detail of a CB-IR NEB in a rat bronchioleat postnatal day 2. nNOS (arrows) and CB (open arrows) IR are presentin different nerve fiber populations. Clearly, high-resolution confocalimaging is necessary to differentiate the fine intertwined terminals of

both populations. MVPs of eight confocal optical sections (1.5-�m in-terval).

Fig. 16. a–c: Immunocytochemical double-staining for VIP (red Cy3-fluorescence) and nNOS (green FITC-fluorescence). Detail of an intrapul-monary bronchus at PD10. MVP of 16 confocal optical sections (1.5-�minterval). a: Red channel showing a VIP-IR intraepithelial arborization(open arrowheads), suggestive of the presence of an NEB. The laminapropria of the bronchus harbors a weakly VIP-IR neuronal cell body(open arrow). b: Green channel showing that the VIP-IR neuronal cellbody is strongly stained for nNOS (arrow). In contrast, the VIP-IR intra-epithelial arborization shows weak nNOS IR (arrowheads). c: Combina-tion of both channels revealing the discrepancy in staining intensitybetween VIP (open arrowheads) and nNOS (arrow).

Fig. 17. Confocal detail of nNOS-IR (green FITC-fluorescence) neu-rons located in a small ganglion situated in the lamina propria of abronchiole of a 10-day-old rat. Varicose CGRP-IR (red Cy3-fluores-cence) nerve fibers intimately surround the two nNOS-IR neurons (as-terisks) 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 (greenFITC-fluorescence) and CGRP (red Cy3-fluorescence). MVPs of 14 con-focal 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 nerveterminals protrude between CGRP-IR NEB cells. Note that the VIP-IRnerve fibers do not contain CGRP and can be seen to overlay apical NEBcells, close to the luminal surface.

Fig. 19. a–c: Immunocytochemical double-staining for VAChT (redCy3 fluorescence) and CGRP (green FITC fluorescence) in a bronchioleof a 10-day-old rat. MVPs of 15 confocal optical sections (0.9-�minterval). a: Red channel showing strong VAChT-IR nerve fibers that areabundant in the subepithelial region (SE). Epithelial cells (arrowheads)show a very faint VAChT IR, while stronger VAChT-IR “terminals” (openarrowheads) can be observed intraepithelially (E). VAChT-IR nerve fibers(arrows) appear to contact the epithelium. b: Green channel demonstrat-ing CGRP-IR spinal sensory nerve terminals (open arrows) contacting aCGRP-IR NEB. c. The combination of both channels shows that theepithelial VAChT staining is present in NEB cells. VAChT-IR nerve ter-minals appear to contact the NEBs.

33NEBS: COMPLEX AIRWAY RECEPTORS

Intrinsic motor component of the selective in-nervation of pulmonary NEBs. It is well known thatnitric oxide (NO) plays an important role in lung physiol-ogy and pathophysiology. In relation to hypoxia, especiallyneuronal NO and endothelial NO appear to be important.Chronic hypoxia in rats induces an upregulation of neu-ronal NO synthase (nNOS) expression (Xue et al., 1994;Shaul et al., 1995; Gess et al., 1997). Therefore, we exam-ined the possibility of a relationship between intrapulmo-nary nitrergic structures and pulmonary NEBs. Previousconventional immunocytochemical studies of nNOS, how-ever, reported only very few nNOS-IR nerve fibers in ratlungs (Kobzik et al., 1993).

Observing in greater detail and using a much moresensitive detection method, we recently found that part ofthe NEBs in rat lungs are selectively innervated by ni-trergic (nNOS-IR) nerve terminals that penetrate in theepithelium, between the NEB cells, and apparently origi-nate from intrinsic pulmonary nitrergic neurons (Brounset al., 2002a). From postnatal day 2 onward, nNOS-IRneurons, present mainly in small ganglia close to themucosa at all levels of intrapulmonary airways, were seento give rise to complex intraepithelial terminals that in-variably colocalize with NEBs (Fig. 13). nNOS IR wasabsent from the spinal afferent CGRP-IR (Fig. 14) andfrom the vagal nodose afferent CB-IR (Fig. 15) nerve fiberpopulations that were previously shown to selectively con-tact NEBs. Quantitative analysis revealed that all NEBsreceiving nNOS-IR terminals were also contacted byCGRP-positive nerve fibers, while about 55% were addi-tionally contacted by CB-IR nerves. nNOS-positive fibersapproaching NEBs are often very closely related to someof the CGRP-IR fibers contacting the same NEB. Thereported nitrergic neurons appeared to colocalize vasoac-tive intestinal polypeptide (VIP) (Fig. 16), did not expresscholinergic markers, and were always surrounded by abasket of CGRP-IR nerve terminals. These nerve termi-nals originate from CGRP fibers that spiral around theaxons of the nitrergic neurons (Fig. 17), and presumablyrepresent collaterals of the spinal CGRP-IR nerve fibersthat selectively innervate NEBs.

The available data on the intrinsic nitrergic componentof the selective innervation of NEBs are summarized inthe scheme of Figure 20 (nerve fiber population shown inred). Essentially, the nitrergic nerve fiber population con-cerned provides intraepithelial terminals between theNEB cells, has its origin in neurons located in airwayganglia, and reveals a very strong interaction with thespinal afferent CGRP-IR component of the NEB innerva-tion.

The characterization of this rather extensive populationof highly likely intrinsic intraepithelial motor terminalsraises some serious questions about the validity of themainly electron microscopic data contained in the initialscheme of Figure 1, and about the interpretation of thequantitative electron microscopic data of NEB innervationafter vagotomy in rat lungs (Van Lommel and Lauweryns,1993a).

Simultaneous demonstration of vagal sensory,spinal sensory, and intrinsic nitrergic nerve ter-minals in pulmonary NEBs. The previous chaptersrevealed 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 allthree of the fully characterized (to date) nerve fiber pop-ulations is indirect. Recently, we described a reliable mul-tiple immunolabeling method (Brouns et al., 2002b) usingunconjugated primary polyclonal antibodies raised in thesame species. In this way, simultaneous detection in asingle preparation of CB, CGRP, and nNOS in three dif-ferent nerve fiber populations that selectively contact pul-monary NEBs can be achieved (Fig. 21). In the reportedprocedure, nNOS was visualized using TSA enhancement(PerkinElmer Life Sciences, Boston, MA), followed by de-tection of CGRP via a fluorophore-coupled Fab secondaryantibody, and the subsequent labeling of CB with a flu-orophore-coupled “conventional” secondary antibody. Allpossible remaining binding sites of the second primaryantibody were blocked using unlabeled anti-rabbit Fabfragments between the second and third steps.

Triple-labeling immunocytochemistry thus provided ev-idence that part of the NEBs in rat lungs selectively re-ceive at least three different populations of nerve termi-nals, the characteristics of which are schematized inFigure 20.

Other components of the selective innervation ofpulmonary neuroepithelial bodies. The functionalmorphology of rat pulmonary NEBs is clearly much morecomplex than was predicted by electron microscopic stud-ies (Van Lommel and Lauweryns, 1993a). Moreover, wehave now good evidence that several other nerve fiberpopulations, which are not fully characterized yet, provideadditional nervous connections of NEBs.

A considerable number of NEBs appear to be contactedby profuse beaded VIP-IR intraepithelial nerve terminals(Fig. 18). Although VIP IR was seen to be localized in theintrinsic nitrergic neurons that give rise to nitrergic ter-minals contacting NEBs, we believe that an additionalpopulation of VIP-expressing nerve endings with an as yetunidentified origin may be involved in the selective inner-vation of NEBs.

As mentioned above, electron microscopy showed thatNEBs in rat lungs are contacted by nerve endings thatcontain small, clear cholinergic-like synaptic vesicles, andoften reveal synaptic contacts with NEB cells. Therefore,antibodies against the vesicular acetylcholine transporter(VAChT; a marker for cholinergic nerves) were used tovisualize these possible cholinergic nerve terminals in di-rect relation to NEBs. Weakly VAChT-IR intraepithelialcell groups, characterized as NEBs after multiple immu-nostaining (Fig. 19), appeared to be contacted byVAChT-IR cholinergic nerve fibers. Although the latternerve fiber population is not yet fully characterized, wehave evidence suggesting that cholinergic motor fibers,originating from preganglionic parasympathetic neurons(and hence an as yet uncharacterized population) may beinvolved.

Preliminary data further revealed that tyrosine hydrox-ylase-IR nerve terminals selectively contact some rat pul-monary NEBs at their basal pole. These nerve fibers likelyhave their origin in sympathetic ganglia.

CONCLUDING REMARKSSelective Innervation of NEBs in the Rat Lung

As touched on in the Introduction, it has been suggestedthat pulmonary NEBs are predominantly, if not exclu-sively, contacted by (vagal nodose) sensory nerve termi-

34 ADRIAENSEN ET AL.

Fig. 20. Schematic representation of the three fully characterizednerve fiber populations that selectively contact pulmonary NEBs.

Fig. 21. a–c: Confocal images of an NEB present in the epithelium ofan intrapulmonary bronchus of a 10-day-old rat, triple-stained for nNOS(red Cy3 fluorescence), CGRP (green FITC fluorescence), and CB (bluepseudocolor of Cy5 emission in far-red). The CGRP/CB-IR NEB cells arecontacted by three different nerve fiber populations. In many places, thenerve fibers of the different populations are so close together that they

could only be distinguished by confocal laser scanning microscopy.MVP of 33 confocal optical sections (1-�m interval). a: Red nNOS-IRnerve fibers (arrowheads) run in the lamina propria and penetrate be-tween the epithelial cells, apparently almost reaching (open arrowheads)the airway lumen (L). b: Green channel showing thin varicose CGRP-IRnerve fibers (open arrows) contacting the basal side of CGRP-IR NEBcells. 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.

35NEBS: COMPLEX AIRWAY RECEPTORS

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 workswas based on data obtained from electron microscopicstudies, and consequently often from a very limited num-ber of NEBs.

In our investigations, neuronal tracing, chemical or me-chanical denervation, and (immuno)cytochemistry, incombination with confocal microscopy, have proven to bevaluable tools with which to study the overall pattern ofNEBs innervation. The application of these techniqueshas resulted in extensive evidence that NEBs in rat lungsmay be selectively contacted by at least five distinct nervefiber populations that are both sensory and motor in na-ture, and have different origins. Multiple immunocyto-chemical staining with presently well-characterizedmarkers revealed that at least part of the pulmonaryNEBs are simultaneously contacted by several nerve fiberpopulations. Caution is recommended, however, when di-viding NEBs into subpopulations on the basis of the lightmicroscopic data reported in the present review only. It isclear that none of the nerve fiber populations contact allNEBs. Moreover, division into subgroups requires a thor-ough knowledge of the interrelationships between the var-ious nerve fiber populations contacting pulmonary NEBs,and thus necessitates reliable, multiple staining proce-dures. Moreover, it should be taken into account that theuse of other markers might reveal even more nerve fiberpopulations 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 ofrat NEBs, but stressed that all of these terminals belongto a single population of vagal nodose afferents presentingintraepithelial efferent-like collaterals. In our studies ofrat lungs, however, vagal nodose sensory, intrinsic ni-trergic, and VIP-IR motor terminals (and possibly extrin-sic cholinergic motor terminals) all appeared to penetratebetween the neuroendocrine cells of NEBs, thus providingevidence for the existence of more than just one intraepi-thelial nerve fiber population. Therefore, the nerve end-ings that were reported intact in the ispilateral lung afterinfranodosal vagotomy (Van Lommel and Lauweryns,1993a) do not necessarily reflect a crossing-over of intactcontralateral vagal fibers (Lauweryns and Van Lommel,1986; Van Lommel and Lauweryns, 1993a).

Development of the Innervation of PulmonaryNEBs

The migration of neuroblasts from the neural crest tothe trachea in rats starts at around GD12 (Morikawa etal., 1978). Neuroblasts can be identified at the late bron-chial bud stage (GD14) by their PGP9.5 IR, after which aprimitive nerve network, associated with the airway wallsand to a lesser degree with the vasculature, is seen toextend rapidly (Sorokin et al., 1997). It has been suggestedthat neuroendocrine cell groups are first contacted bythese postganglionic parasympathetic nerves, thereby be-coming real NEBs, and only much later (just before birth)receive sensory nerve endings (Sorokin et al., 1997). Theseconclusions were based on observations that intrinsicPGP9.5-IR nerve terminals contact pulmonary NEBsaround GD17 in in vitro fetal rat lung, while CGRP-IR

nerve terminals contacting NEBs in vivo are not commonuntil the end of postnatal week 1 (Sorokin et al., 1997).However, from our studies it is clear that CGRP IR labelsspinal, and not vagal, sensory nerve terminals in contactwith NEBs, and that the CGRP-negative vagal nodosesensory innervation most likely has been overlooked. Itwas also confirmed that the first CGRP-IR nerve fiberscontacting CGRP-IR NEBs could be detected later, atGD19. Our studies further showed that from GD15-16onward, a large number of subepithelial nerve fibers seenin rat airways express CB, a marker for the vagal nodosesensory nerve fiber population that selectively innervatesNEBs from GD17 on. The latter observations were con-firmed by an ontogenetic study using P2X3 receptor im-munolabeling as a marker for the vagal sensory compo-nent of NEB innervation (personal observations).

On the other hand, pulmonary NEBs do receive part oftheir innervation from neurons intrinsic to the lungs (So-rokin et al., 1993, 1997). The present study showed thatfrom GD17 onward, PGP9.5-IR NEB cells are contacted byPGP9.5-IR nerve fibers, apparently originating from in-trinsic PGP9.5-IR cell bodies that do not reveal nNOS IRat that time. It is likely that at least part of the initialintrapulmonary PGP9.5-IR neuroblasts or neurons, lo-cated along the pulmonary epithelial tubes, differentiateinto nNOS-IR and/or VIP-IR neurons at a later stage ofdevelopment and are at least partly responsible for thespecific intraepithelial nitrergic nerve terminals in NEBs.An ontogenetic study (Brouns et al., 2002a) found that thenitrergic nerve terminals selectively contacting rat NEBscan only be visualized from postnatal day 2 onward, sup-porting the hypothesis that the innervation of rat NEBsundergoes further maturation after birth.

Although previous studies proposed that ingrowth ofautonomic and sensory fibers converts clusters of neuroen-docrine cells into bona fide NEBs (Sorokin et al., 1997),that non-innervated PNEC clusters may be interpreted asdeveloping NEBs, and that the entire pulmonary neuroen-docrine system should be regarded as progressing towardcomplete capture by the nervous system (Sorokin andHoyt, 1990), our data indicate that even in postnatal lungsthe innervation pattern is far from identical for all NEBs,and it is likely that part of the NEBs do not receive aselective innervation. Nevertheless, all NEBs in postnatalrat lungs, regardless of whether they receive a demonstra-ble innervation or not, apparently produce a similar pal-ette of amines, purines, and peptides, and do not degen-erate after birth. It may be that the ingrowth of motor andsensory nerve fibers into pulmonary NEBs does not deter-mine their reaction to certain stimuli. The multicompo-nent innervation may regulate the sensitivity of NEB cellsto stimuli, and serve as a tool for exerting additional localintrapulmonary and CNS reflexes.

Functional ImplicationsThe data presented herein demonstrate that pulmonary

NEBs represent an extensive population of very complexintraepithelial receptors that probably can accommodatevarious sensory modalities. Although the physiologicalsignificance of the complex innervation pattern of NEBs isstill a matter of speculation, the sensory nature of NEBs isbeyond dispute. According to recent reviews, no physiolog-ical recordings have been obtained yet from single vagalafferent fibers from NEBs, and in general all recordings ofthe effect of airway hypoxia have yielded negative results

36 ADRIAENSEN ET AL.

(Widdicombe, 2001). However, it seems very unlikely thatlung physiologists, in performing many thousands of sin-gle fiber recordings in the vagus nerve to identify airwayreceptor populations in rat lungs, have never made regis-trations from the many hundreds of myelinated vagalnodose neurons that selectively contact pulmonary NEBsin each rat lung. More likely, the populations in questionhave not yet been recognized among the existing data.Undoubtedly, a considerable part of the already “charac-terized” airway receptors, a large part of which are mech-anoreceptors, will eventually turn out to be related toNEBs.

None of the nerve fiber populations characterized so farcontacts all pulmonary NEBs. However, the observationthat only half (or even 10%) of the NEBs receive a certainnerve fiber population does not mean they should be con-sidered as less important. After all, the “different” NEBpopulations do account for at least a few hundred receptorpoints each. The potential importance of NEBs is sup-ported by the fact that the total number of NEB cells andassociated nerve fibers in an animal easily outnumberscarotid body cells and their selective innervation (Sorokinand Hoyt, 1990).

Receptosecretory NEB cells are excellent candidates forregistering properties of the airway environment. Al-though there is accumulating evidence for a completefunctional system for oxygen sensing in PNECs (Peers andKemp, 2001), it should be stressed that the exact nature ofthe possible physiological stimulus modalities of NEBcells in healthy lungs is still unknown, and that it becomesincreasingly likely that other stimuli, such as mechanicalstimuli, may also be involved. Upon stimulation, NEBcells reveal changes in the exocytosis of DCVs, and pre-sumably release the amines, peptides, ACh, and ATPstored in these secretory granules. In addition to acting onnerve endings in contact with NEBs, the released bioac-tive substances may exert local actions on, for instance,nearby airway or vascular smooth muscle (paracrine), orcould be taken up by nearby blood vessels (endocrine).

It is tempting to look at NEBs as local regulators ofairway functions that do not necessarily require signalingto the CNS. The main sensor/effector action to hypoxiacould be local, and a possible central transduction of hy-poxic stimuli may be mediated by spinal rather than vagalafferents in rat lungs. Because they are located at strate-gic points along the intrapulmonary airways, NEBs ap-pear to be ideally placed to effectuate and coordinate thefine-tuning of local blood flow to local aeration, a physio-logical function that cannot be performed by the carotidbody or central hypoxia receptors. In this respect, pulmo-nary NEBs can be regarded as inexhaustible local pools ofvasoactive transmitters, such as the potent pulmonaryvasoconstrictor serotonin and vasodilator CGRP. Contin-uous release of endogenous CGRP from NEB cells may beresponsible for at least part of the homeostatic control ofblood vessel relaxation in normoxic lung areas. It has beenestablished that intact primary sensory CGRP/SP-con-taining and capsaicin-sensitive nerve fibers are requiredfor endogenous CGRP to modulate pulmonary vasculartone in hypoxic pulmonary hypertension (Tjen-A-Looi etal., 1998). Because hypoxia apparently inhibits CGRP se-cretion from rat NEBs (Springall and Polak, 1993), localphysiological hypoxia may result in local inhibition ofvasodilation, implying local adjustment of pulmonary per-fusion to ventilation. As demonstrated in the present

work, all NEBs with an intraepithelial nNOS-IR innerva-tion also reveal basal contacts with CGRP-IR afferents,the presumable collaterals of which appear to follow thecourse of nitrergic axons and finally form baskets aroundthe nitrergic neurons in the lamina propria. This nitrergicinnervation may be a necessary component for the hypoxicinhibition of CGRP release from NEB cells, requiringCGRP-IR afferents for activation of the mechanism.

On the other hand, a system that prevents NEB recep-tors from continuously transmitting hypoxic informationto the CNS would be crucial for avoiding exaggeratedcentral actions that may eventually lead to a generalpulmonary vasoconstriction and hypertension. Therefore,a similar mode of action of NO on the transduction ofhypoxic stimuli, as reported for carotid body cells (forreview, see Prabhakar, 1999), may be proposed for NEBs.Release of NO from the terminals of intrinsic pulmonarynitrergic neurons in NEBs may result in an inhibition ofthe sensory discharge of NEBs in response to, e.g., localhypoxia. Such a mechanism may keep NEB receptors“quiet” as far as the CNS is concerned, creating time andspace for local actions. In this way, afferent signaling tothe CNS may be limited to powerful stimuli that necessi-tate central regulation of respiration.

In addition to their receptor function(s), PNECs andNEBs may exert many other functions (independent oftheir innervation?) during prenatal, perinatal, and earlyneonatal life (for review, see Sorokin and Hoyt, 1989).Because the vagal nodose sensory component of the selec-tive innervation of NEBs appears to be fully differentiatedwell before birth, it may be essential for neonatal respira-tory adaptation.

Future ProspectsAlthough the innervation pattern of pulmonary NEBs

in rats appears to be fairly complex, there is no conclusiveevidence for assuming that the detailed functional mor-phological investigation is complete. We even have good(unreported) evidence that other markers for nerve fiberpopulations will provide additional features of the nervefiber populations that have already been characterized, orwill reveal additional nerve fiber populations contactingpulmonary NEBs. Further characterization of receptorsfor important neurotransmitters or neuromodulators,present in NEB cells (e.g., 5-HT and CGRP) and in nervefiber endings associated with NEBs (e.g., VIP, CGRP, SP,and ACh), will be necessary to further elucidate the func-tional significance of these substances.

It is clear that PNECs show some marked species-spe-cific differences in their palette of substances (Polak et al.,1993), and that the origin and chemical coding of nerveendings selectively contacting pulmonary NEBs may varyfrom species to species. Obviously, it will be necessary tovalidate the data obtained in rats (as discussed in thepresent review) in other species as well. As mentionedabove, to date very little is known about the presence ororigin of the different nerve fiber populations selectivelyinnervating human pulmonary NEBs.

During the last decade, various methods have been de-veloped to study PNECs/NEBs in vitro (Speirs and Cutz,1993). Because the close relationship between NEB cellsthemselves, and between NEBs and nerve fibers, is com-pletely lost in isolated cell suspensions of PNECs, morerecent in vitro studies have focused on the use of lungslices (Fu et al., 1999). This technique offers a more real-

37NEBS: COMPLEX AIRWAY RECEPTORS

istic natural environment for NEBs, and in several casesprobably does not even affect the relationship betweenintrinsic neuronal cell bodies and pulmonary NEBs. Phys-iological reactions of pulmonary NEBs and their selectiveinnervation to administered neurotransmitters/modula-tors and (ant)agonists, or on environmental stimuli (e.g.,hypoxia and nicotine), could be measured electrophysi-ologically or by microscopic visualization of physiologicparameters (e.g., calcium concentration and membranepotential) in these lung slices. In addition to investiga-tions on healthy rat lungs, genetic models that reveal lungdisorders, or even specific abnormalities of the pulmonaryDNES, should be included in these studies.

In vivo experiments in which laboratory animals areexposed to stimuli such as hypoxia, hyperoxia, hypercap-nia, or to allergens, ozone, cigarette smoke, and otherpollutants, combined with morphological or in vitro phys-iological studies, will be necessary to further elucidate thereactions of pulmonary NEBs to external stimuli.

In conclusion, a multidisciplinary approach, in whichmodern histological and microscopic techniques are com-bined with cellular/molecular biological and electrophysi-ological methods, will be crucial to achieve further insightinto the precise working mechanisms and roles of PNECs/NEBs in both the healthy and the diseased lung.

LITERATURE CITEDAdriaensen D, Scheuermann DW, Timmermans J-P, De Groodt-Las-

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