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Thorax 1983;38:333-340

Neurone-specific enolase and S-100: new markers fordelineating the innervation of the respiratory tract inman and other mammalsMN SHEPPARD, SS KURIAN, SC HENZEN-LOGMANS, F MICHETTI, D COCCHIA,P COLE, RA RUSH, PJ MARANGOS, SR BLOOM, JM POLAKFrom the Departments ofHistochemistry and Medicine, Royal Postgraduate Medical School, HammersmithHospital, London; the Department of Anatomy, Universitit Cattolica, Rome; the Cardiothoracic Institute,Brompton Hospital, London; the School of Medicine, Flinders University of South Australia; and theNational Institute of Mental Health, Bethesda, Maryland, USA

ABSTRACr Lung innervation has been studied in the past by methylene blue staining and silverimpregnation and more recently by histochemical methods. These techniques give only a partialpicture of the total innervation. We have delineated the innervation of the lung in man and threeother mammalian species by immunostaining with antibodies to two new markers of nervoustissue. These markers are neurone-specific enolase (NSE), an enzyme present in nerve cells inboth the central and the peripheral nervous systems, and S-100, a protein found in glial cells.Throughout the respiratory tract NSE was localised in ganglion cells and nerve fibres in all speciesexamined, while S-100 was found in the supporting glial cells of ganglia and in the Schwann cellsof peripheral nerves. The distribution of NSE immunoreactivity in serial sections was comparedwith that of acetylcholinesterase-containing, noradrenergic, and peptide-containing nerves. In allareas NSE was found to be a specific marker for all three types of nerves. Thus these twoantibodies provide an effective histological means of examining both the neuronal and the non-neuronal components of the lung innervation and should be of value in investigating this systemin lung disease.

The lung has a rich nerve supply, consisting of bothsensory and motor divisions. The methods used todemonstrate this innervation by light microscopy inearlier studies included methylene blue and silverstaining."3 More recently, histochemical techniqueshave been applied, for example, to localise acetyl-cholinesterase, which is thought to be a marker forcholinergic nerves,46 and to demonstrateformaldehyde-induced fluorescence in aminergicnerves.78 All these methods have their limitationsand give only a partial view of the total innervation.

Recently two highly acidic soluble proteins havebeen isolated from brain extracts and characterised.9The first, neurone-specific enolase (NSE), is anisoenzyme of the glycolytic enzyme enolase.'0 Anti-bodies raised against this enzyme and used asimmunochemical reagents show that it is specificallylocalised in neurones" in both the central and the

Address for reprint requests: Dr Julia M Polak, Department ofHistochemistry, Royal Postgraduate Medical School, Hammer-smith Hospital, London W12 OHS.

Accepted 7 January 1983

peripheral nervous systems'2 as well as in neuroen-docrine cells throughout the body.'314 The secondprotein is S-100 (so named because of its solubilityin 100% ammonium sulphate at neutral pH), acalcium-binding protein,'5 which is found byimmunocytochemical methods in glia of the centralnervous system'6 and glial cells of the peripheralganglia as well as Schwann cells in peripheralnerves.'7 18 In view of the localisation of NSE andS-100 in neurones and glial cells respectively inother tissues, we have studied the respiratory tractof man and three other mammalian species to seewhether its innervation can be delineated byimmunostaining with antibodies to these proteins.We have also compared the distribution of NSE andS-100 immunoreactivity with that of acetylcholin-esterase-positive nerves, noradrenergic nerves, andpeptide-containing nerves in all four species.

Methods

TissueThe respiratory tract was obtained from six adults

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Sheppard, Kurian, Henzen-Logmans, Michetti, Cocchia, Cole, Rush, Marangos, Bloom, Polak

(four men and two women aged 18-73 years, mean60 years) and four children (two boys and two girls,two aged 24 hours, one 6 months, and one 6 years)dying of non-cardiopulmonary causes. Only freshpostmortem material, obtained less than 24 hoursafter death, was examined. In each case the areasdissected out included the trachea, major bronchi,the inner lung zone containing minor bronchi, themiddle lung zone containing medium-sized bronchi,and the outer lung zone including alveoli. In addi-tion, fresh surgical biopsy specimens of nasalmucosa from the inferior turbinate area were takenat operation from eight adult patients (five men andthree women aged 18-73 years, mean 39 years)undergoing upper respiratory tract surgery for con-ditions not related to the nasal mucosa.

Eleven cats (six male and five female, 2-6-4-2 kg),eight Wistar rats (all female, 190-250 g), and sixDunkin Hartley guinea pigs (four male and twofemale, 350-450 g) were studied. The rats andguinea pigs were killed by cervical dislocation andthe cats with an intraperitoneal injection of sodiumpentobarbitone. In each case the areas examinedwere similar to the areas of human materialexamined but the nasal mucosa was also included inthe dissection.Each sample of tissue was fixed and processed for

conventional histology, immunocytochemistry, andhistochemistry.

Fixation Each piece of fresh tissue, about 1.5 x0*75 x 0.5 cm, was divided into three equal parts,each part being fixed by one of three methods. (1)Tissue was fixed in parabenzoquinone solution (0-4g benzoquinone in 100 ml 0-01 molI phosphate-buffered saline (PBS), pH 7.4) for 1-11/2 hours at40C.'920 After fixation the tissue was washed over-night in buffered sucrose (70 mg/ml sucrose, 0*1mg/ml sodium azide in PBS), then snap frozen andsectioned in a cryostat at -20°C. (2) Tissue wassnap frozen in Arcton (Freon) 12, precooled inliquid nitrogen at - 153°C, and freeze dried over-night at -40°C. The tissue was then fixed in para-benzoquinone vapour, derived from a solution of

parabenzoquinone in toluene, for three hours at60°C. After this the blocks were vacuum embeddedin paraffin wax. (3) Tissue was fixed in either 10%formalin solution or Bouin's solution (75 ml satu-rated aqueous picric acid, 25 ml commercial forma-lin, 1 ml acetic acid) for 6-12 hours, after which itwas dehydrated through alcohols to xylene andembedded in paraffin wax.

Multiple serial sections were cut from all blocks ata thickness of 3-7 gm for conventional histology(sections being stained with haematoxylin and eosinand examined to ensure that only normal specimensof the respiratory tract were included in the study)and for comparative immunocytochemistry and his-tochemistry.

Immunocytochemistry The immunocytochemicaltechniques were indirect immunofluorescence2' andthe peroxidase antiperoxidase (PAP) procedure.22The antibodies used were raised against the brainproteins NSE and S-100, the peptides vasoactiveintestinal polypeptide (VIP) and substance P, andthe enzyme dopamine-,8-hydroxylase (DBH), whichis concerned in catecholamine biosynthesis and is amarker for catecholamine-containing nerves.23 Inperipheral sympathetic nerves noradrenaline hasbeen found to be the predominant catecholaminecomponent, so throughout this study we refer toDBH-immunoreactive fibres as noradrenergic.Details of the antibodies and their region specificityare given in the table. The antisera to NSE, S-100and DBH were raised in rabbits.'7 23 2' Controlsincluded prior absorption of the antibody with itsspecific antigen (table). The antiserum to VIP (28amino acids) was completely absorbed by the wholeantigen molecule but did not absorb with the synthe-tic amino acid fragments 1-22 or 7-28. This suggeststhat the VIP antiserum reacts with the whole VIPmolecule. The antiserum to substance P wasabsorbed by its relevant antigen but not by thesynthetic amino acid fragment 7-11. Thus the anti-serum to substance P (11 amino acids) appears to bedirected towards the middle or N-terminal portionof the substance P molecule. Additional immuno-

Characteristics ofthe antibodies used

Antigen Antisera specificity Diluion A en concentraton neededto abolish staining fully

IF PAP (nmol/ml diluted antiserum)

Rat NSE Not known 1:800 1:4000 1-0Bovine S-100 Not known 1:800 1:4000 2-5Porcine VIP Whole molecule 1:1000 1:20 000 0 1Synthetic substance P Mid portion to N-terminal 1:2000 1:16000 10Bovine DBH Not known 1:400 1:4000 1-0

NSE-neurone-specific enolase; VIP-vasoactive intestinal polypeptide; DBH-dopamine-p3-hydroxylase; IF-indirectimmunofluorescence; PAP-peroxidase antiperoxidase.

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Brain proteins as markers of lung innervation

cytochemical controls included the use of normalrabbit serum (1: 2000 dilution) and of thefluorescein-isothiocyanate conjugate or PAP com-plex alone.

Histochemistry Benzoquinone-fixed cryostat sec-tions, serial to those on which immunostaining wascarried out, were cut and allowed to dry. They werethen post-fixed in 10% formalin in PBS for 15minutes at room temperature and stained for acetyl-cholinesterase activity according to the method ofEl-Badawi and Schenk25 for four to six hours at37°C. All sections from each area of the respiratorytract were viewed with a Reichert-Jung Polyvarmicroscope equipped with an ultraviolet source andbright-field Nomarski optics.

ResultsNSE IMMUNOSTAININGImmunostaining with antibodies to NSE allowed thesimultaneous demonstration of a large number ofganglion cells and an extensive network of nervefibres throughout the respiratory tract of all speciesexamined. In general, the distribution of NSE wasrealted chiefly to the airways and blood vessels, par-ticularly in the upper respiratory tract, with agradual decrease towards the lung periphery. Occa-

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sional NSE-positive endocrine cells were found scat-tered in the respiratory epithelium of all the adultspecies, but they were most frequent in the humaninfant lung.

Ganglion cellsNSE-immunoreactive ganglion cells were located ingroups of two to 20 cells, mainly in the adventitia ofthe tracheobronchial wall close to the blood vessels.Smaller groups and occasional single cells were seenin the submucosa of the larger airways. In thetrachea and major bronchi ganglion cells were mostcommon in the posterior wall outside the trachealismuscle and cartilage ring. At the hilum they becamemore evenly distributed around the entire adventitiaof the bronchus and gradually diminished in numberas the bronchioles were reached. The NSE-immunoreactive ganglion cells in the submucosa ofthe larger airways were in close proximity to theseromucous glands and blood vessels and becameless frequent as the airways got smaller. No NSE-immunoreactive ganglion cells were seen in the wallsof the pulmonary vessels in any species, but theywere often noted in the connective tissue betweenthe bronchi and pulmonary arteries within the lung,chiefly at the hilum. No NSE-positive ganglion cellswere observed in the nasal mucosa of any species.

Fig 1 A Neurone-specific enolase (NSE)-immunoreactive nerve fibres surrounding the acini ofseromucous glands inthe human adult nasal mucosa. (Indirect immunofpuorescence, x 375.) B NSE-4mmunoreactive nerve fibres in theadventitia ofa blood vessel in the human adult nasal mucosa. (Indirect immunofluorescence, x 375.)



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Nerve fibresNSE-immunoreactive nerve fibres formed a com-plex network around the airways and pulmonaryblood vessels in all species. As already stated, fibreswere most numerous in the upper respiratory tract.In the nasal mucosa NSE-containing nerve fibreswere found beneath the epithelium, aroundseromucous glands, and in the adventitia of bloodvessels (fig 1). In the main airways the fibres formedtwo networks, one outside and the other within thecartilage plates. In the extrachondral network NSE-immunoreactive fibres formed large bundles often indirect continuity with the ganglion cells. In thetrachea and major bronchi the fibres were locatedchiefly in the posterior region, with fewer fibresanteriorly. At the hilum the nerve fibres, like theganglion cells, had a more even distribution in theadventitia of the bronchial wall. Within the lungNSE-immunoreactive fibres became fewer towardsthe respiratory portions of the airways, with smallerbundles and single fibres lying in the adventitia ofthe bronchioles and only an occasional single fibrebeing identified distal to this. Within the cartilageplates the NSE-immunoreactive fibres formed smal-ler bundles and surrounded the acini of seromucousglands, the blood vesels, and the smooth muscle.Fibres also extended towards the respiratoryepithelium, often lying beneath it, with occasional

fine fibres within the epithelium itself. This sub-chondral network of nerve fibres gradually disap-peared as the airways got smaller.The pulmonary vessels, both arteries and veins,

contained NSE-immunoreactive nerve fibres locatedin the adventitia and extending towards theadventitial-medial junction. These fibres were pres-ent down to the level of the small arterioles andvenules in the lung periphery in all species.

S-100 IMMUNOREACTIVITYSerial sections stained with antibodies to NSE andS-100 showed that in all species S-100 wasimmunostained only in the supporting glial cells sur-rounding the NSE-immunoreactive ganglion cells(fig 2), with no immunostaining of the ganglion cellbodies. The S-100-immunoreactive glial cells andtheir processes formed a dense network around theindividual ganglion cells. In all areas where NSE-immunoreactive ganglion cells were found S-100-immunoreactive glial cells surrounded them. Inserial sections, throughout the respiratory tract of allspecies nerve bundles which were found to containNSE were also S-100 immunoreactive. S-100immunoreactivity was found in cells with elongateddendritic processes surrounding peripheral nerves.Their morphology and distribution indicated thatthey were Schwann cells. S-100 immunoreactivity

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A BFig 2 A Two NSE-immunoreactive ganglion cells (arrows) in the adventitlia ofthe human adult major bronchus.(Peroxidase-antiperoxidase method, x 500.) B Section serial to 2A, showing S-I00-immunoreactive glial cellssurrounding the two negative ganglion cell bodies (arrows). (Peroxidase-antiperoxidase method, x 500.)

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A 13Fig 3 A NSE-4mmunoreactive ganglion cell (arrow) in the submucosa ofthe trachea in the human adult.(PAP method x 450.) B Section serial to 3A, showing the same ganglion cell containing acetylcholinesteraseactvity (arrow). (Cholinesterase stain, x 450.)

was also noted in chondrocytes within the cartilageplates, as previously reported.26

ACETYLCHOLINESTERASE-CONTAINING NERVECELLS AND FIBRESIn serial 6-,um cryostat sections we compared thedistributions of NSE-immunoreactive ganglion cellsand nerve fibres and those containing acetyl-cholinesterase activity. The same ganglion cellscould be examined with the two stains because mostganglion cells are more than 20 ,um in diameter, sothat several serial sections passed through the samecell, while confident identification of a single nervefibre in serial sections is not yet possible with currenttechniques. Thus only the general distribution ofnerve fibre bundles could be compared in the serialsections.

In all four species all acetylcholinesterase-positiveganglion cells were also immunoreactive for NSE(fig 3), while acetylcholinesterase-containing nervefibres had a distribution similar to that of NSE-positive nerve fibres throughout the respiratorytract. In general, NSE-immunoreactive ganglioncells and their fibres were present in larger numbersthan those which could be demonstrated by acetyl-cholinesterase staining.

NORADRENERGIC INNERVATIONWith antibodies to DBH noradrenergic nerve fibres

were found mainly in the upper respiratory tract, inthe airway smooth muscle, and in the adventitia ofthe tracheobronchial blood vessels. In the lung,fibres were fewer and were related to the adventitiaof the bronchial and pulmonary vessels, with fewfibres in the airway smooth muscle. Species variationwas noted, more fibres being observed in the catthan in the other three species. In serial sections thedistribution of noradrenergic nerves was found to besimilar to that of NSE-immunoreactive nerves, butNSE-containing nerves were present in much grea-ter numbers. No DBH-positive ganglion cells wereidentified in any species.

PEPTIDERGIC INNERVATIONVasoactive intestinal polypeptide and substance Pare the two peptides so far demonstrated byimmunocytochemistry in the pulmonary nervoussystem.27'30 VIP immunoreactivity, though presentin all species, is found most abundantly in the cat,within both ganglion cells and nerve fibres. In serialsections immunostained with antibodies to NSE andVIP all VIP-immunoreactive ganglion cells werealso immunoreactive for NSE (fig 4) but the NSEganglion cells were more abundant. In addition, allthe VIP-immunoreactive ganglion cells containedacetylcholinesterase but there were more acetyl-cholinesterase-positive ganglion cells than ganglioncells containing VIP. VIP-immunoreactive nerve

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Fig 4 A Two NSE-immunoreactive ganglion cells (arrows) in the adventita ofthe cat major bronchus.(Indirect immunofluoresence, x 475.) B Section serial to 4A, showing vasoactive intestinalpolypeptide (VIP)immunoreactivity in the same two ganglion cells (arrows). (Indirect immunofiuorescence, x 475.)

locations could not be differentiated from NSE-immunoreactive nerves, being chiefly seen aroundseromucous glands and blood vessels and in the air-way smooth muscle.

Substance P immunoreactivity was found withinnerve fibres, particularly beneath the respiratoryepithelium, in the airway smooth muscle, andaround blood vessels. They were seen throughoutthe respiratory tract of all species but were mostprominent in the guinea pig. Serial sectionsimmunostained with antibodies to NSE and sub-stance P showed that wherever nerve fibres contain-ing substance P were located NSE-positive fibreswere also stained in adjacent sections.We noted that the number of NSE-

immunoreactive ganglion cells and nerve fibresgreatly exceeded those that could be demonstratedby peptide immunocytochemistry.

TISSUE FIXATIONBoth NSE and S-100 immunoreactivity were excel-lently demonstrated in the formalin and Bouin'sfixed paraffin-embedded tissues of all speciesexamined. The tissue fixed in benzoquinone vapourdid show denser immunostaining but the distribu-tion of both NSE-containing and S-100-containingnerves did not differ from those seen in the routinely

fixed tissues. For the demonstration ofacetylcholinesterase-containing, noradrenergic, andpeptidergic nerves, however, tissue fixed in benzo-quinone solution and made into cryostat blocks gavethe best results.

Discussion

The prominence of the nervous system in the controlof normal physiological and pathological processesin the lung has been evident for some years.3132Nevertheless its morphological appearance hasreceived comparatively little attention. This isperhaps due to the lack of specific and reliable mar-kers for nerve cells and their supporting elements.Earlier studies made on the respiratory tract'-353used methylene blue and silver impregnation tech-niques to demonstrate all nervous structures non-specifically. These techniques, however, are knownto give variable results and produce artefacts.23Fishers stated that both techniques can give variableresults depending on the fixation method used andhe described nerve ending artefacts, similar to thosedescribed by previous authors, which were due tooverstaining and trauma.

Later studies using light microscopy have incorpo-rated histochemical methods, mainly acetylcholines-

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terase staining for cholinergic nerves andformaldehyde-induced fluorescence for adrenergicnerves.5 8 3' Both of these methods only give a partialview of the total innervation and have certain draw-backs. In the interpretation of acetylcholinesterasestaining it should be noted that acetylcholinesteraseitself has not been considered a truly specific markerfor cholinergic nerves since 19573S and recently theenzyme has been shown to hydrolyse substance p.36Diffusion artefacts at nerve endings with cholines-terase staining were shown in extrapulmonary ves-sels by Fisher.5 The formaldehyde-induced fluores-cence method for demonstrating aminergic nerves3'may give difficulties in interpretation because of theautofluorescence of tissue elastic. Antibodies toDBH will immunostain only catecholamine-containing nerves and will not demonstratedopamine-containing structures like the form-aldehyde-induced fluorescence method.23

In our study, using immunocytochemical techni-ques with antibodies to NSE and S-100, we havedemonstrated very clearly both the neuronal com-ponents and the supporting cells of the pulmonaryinnervation in man and three other mammalianspecies. NSE has already been shown to be a reliableand specific enzymatic marker for ganglion cells andnerve fibres in many tissues,12 14 including the humanfetal lung.38 Immunostaining with antibodies toS-100 is an excellent means of demonstrating thesupporting glial cells of pulmonary ganglia and theSchwann cells of peripheral nerves, both of whichhave been poorly described in the respiratory tractat the light microscopic level.An important finding was that the distribution of

NSE immunoreactivity was similar to that ofacetylcholinesterase-positive nerve cells and fibres,the noradrenergic innervation, and the peptide-containing nerve cells and fibres, so that NSEappears to be a specific marker for all the compo-nents of the lung innervation. In addition, both NSEand S-100 immunoreactivity showed more thanhad hitherto been seen by either the histochemicalmethods or peptide immunocytochemistry. The pre-sence of NSE-immunoreactive nerve cells and fibresin larger numbers than had been apparent with theother methods may indicate that there are still othernerve cells and fibres, whose neurotransmitter hasnot yet been discovered. Another advantage ofusing these two immunocytochemical markers todelineate the lung innervation is that their antigenic-ity is preserved after fixation in both formalin andBouin's solutions. Thus retrospective studies on thelung innervation are possible.Our results are particularly applicable to studies

of lung pathology. The nervous system of the lungplays a major part in the control of airway smooth

muscle,3132 pulmonary blood flow,39 and glandularsecretion.40 Thus its evaluation in pathological statesis essential and immunostaining with antibodies toNSE and S-100 provides a simple histological meansfor rapid assessment. Separate components of thenervous system can then be examined by the techni-ques of enzyme histochemistry or peptide immuno-cytochemistry.

This work was supported by the Medical ResearchCouncil and the United States Council for TobaccoResearch. MNS has a Wellcome research fellowship.

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