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A confocal microscopic study of solitary pulmonary neuroendocrine cells in human airway epithelium Weichselbaum, Markus; Sparrow, Malcolm P; Hamilton, Elisha J; Thompson, Philip J; Knight, Darryl A Oct 10, 2005

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ralssBioMed CentRespiratory ResearchOpen AcceResearchA confocal microscopic study of solitary pulmonary neuroendocrine cells in human airway epitheliumMarkus Weichselbaum1,2, Malcolm P Sparrow1,2,3, Elisha J Hamilton1,2,4, Philip J Thompson1,2 and Darryl A Knight*1,2,5Address: 1Asthma and Allergy Research Institute, Sir Charles Gairdner Hospital, Nedlands, 6009, Western Australia, 2Centre for Asthma, Allergy and Respiratory Research, University of Western Australia, 6009, 3Department of Physiology, University of Western Australia, Nedlands, 6009, Western Australia, 4Heart Research Institute, Royal North Shore Hospital, The University of Sydney NSW 2006 Australia and 5James Hogg iCAPTURE center for Cardiovascular and Respiratory Research, St. Pauls Hospital, University of British Columbia, Vancouver, BC V6Z 1Y6, CanadaEmail: Markus Weichselbaum - weichsel@cyllene.uwa.edu.au; Malcolm P Sparrow - msparrow@cyllene.uwa.edu.au; Elisha J Hamilton - ejhamilton@mail.usyd.edu.au; Philip J Thompson - pjthomps@aari.uwa.edu.au; Darryl A Knight* - dknight@mrl.ubc.ca* Corresponding author    AbstractBackground: Pulmonary neuroendocrine cells (PNEC) are specialized epithelial cells that arethought to play important roles in lung development and airway function. PNEC occur either singlyor in clusters called neuroepithelial bodies. Our aim was to characterize the three dimensionalmorphology of PNEC, their distribution, and their relationship to the epithelial nerves in wholemounts of adult human bronchi using confocal microscopy.Methods: Bronchi were resected from non-diseased portions of a lobe of human lung obtainedfrom 8 thoracotomy patients (Table 1) undergoing surgery for the removal of lung tumors. Wholemounts were stained with antibodies to reveal all nerves (PGP 9.5), sensory nerves (calcitonin generelated peptide, CGRP), and PNEC (PGP 9.5, CGRP and gastrin releasing peptide, GRP). Theanalysis and rendition of the resulting three-dimensional data sets, including side-projections, wasperformed using NIH-Image software. Images were colorized and super-imposed using AdobePhotoshop.Results: PNEC were abundant but not homogenously distributed within the epithelium, withdensities ranging from 65/mm2 to denser patches of 250/mm2, depending on the individualwholemount. Rotation of 3-D images revealed a complex morphology; flask-like with the cell bodynear the basement membrane and a thick stem extending to the lumen. Long processes issuedlaterally from its base, some lumenal and others with feet-like processes. Calcitonin gene-relatedpeptide (CGRP) was present in about 20% of PNEC, mainly in the processes. CGRP-positive nerveswere sparse, with some associated with the apical part of the PNEC.Conclusion: Our 3D-data demonstrates that PNEC are numerous and exhibit a heterogeneouspeptide content suggesting an active and diverse PNEC population.Published: 10 October 2005Respiratory Research 2005, 6:115 doi:10.1186/1465-9921-6-115Received: 09 May 2005Accepted: 10 October 2005This article is available from: http://respiratory-research.com/content/6/1/115© 2005 Weichselbaum et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 11(page number not for citation purposes)Respiratory Research 2005, 6:115 http://respiratory-research.com/content/6/1/115BackgroundPulmonary neuroendocrine cells (PNEC) are specializedairway epithelial cells that occur as solitary cells or as clus-ters called neuroepithelial bodies (NEB) [1]. They arelocated in the nasal respiratory epithelium, laryngealmucosa [2] and throughout the entire respiratory tractfrom the trachea to the terminal airways [3]. In the fetallung they are frequently located at the branching points ofairway tubules, and in humans are present by 10 weeksgestation [4]. Neuroendocrine cells are bottle- or flask-likein shape, and reach from the basement membrane to thelumen. They can be distinguished by their profile of bio-active amines and peptides namely serotonin, calcitonin,calcitonin gene-related peptide (CGRP), chromogranin A,gastrin-releasing peptide (GRP) and cholecystokinin[4,5]. NEB may play a role as hypoxic-sensitive airwaychemoreceptors [6], and an oxygen-sensitive potassiumchannel coupled to an oxygen sensory protein has beendemonstrated in their lumenal membrane in the rabbit[7]. They are also considered to be involved in regulatinglocalized epithelial cell growth and regeneration througha paracrine mechanism whereby their bioactive peptidesare released into the environment [8]. Peptides andamines released by PNEC are involved in normal fetallung development including branching morphogenesis[9]. The best-characterized peptides are GRP, the mamma-lian form of bombesin, and CGRP, which exert directmitogenic effects on epithelial cells and exhibit manygrowth factor-like properties [10].The majority of data available on the morphology, distri-bution, peptide expression and function of PNEC andNEB have been obtained from animal studies [11,12]. Inhuman airways, the morphology of NEB have been stud-ied ultrastructurally during the fetal and perinatal stage oflung development, and their peptides identified usingimmunogold-labeled antibodies where they are colocal-ized in the dense core vesicles in the cytoplasm [4,13-15].However, there is little data describing the three dimen-sional morphology and peptide distribution in adulthuman airways where both PNEC and NEB are reportedto be sparse [16,17]. It has been suggested that PNEC mayplay a role in mediating airway remodelling in normallungs and in naturally occurring pulmonary disease wherethey increase in number [8,18].The innervation of fetal and postnatal NEB has been alsostudied ultrastructurally in humans where both adrener-gic and cholinergic nerve endings have been observed [4],in rabbits [19] and rats and dogs [20,21]. In rats, vagalnodose afferents traced using the carbocyanine dye DiI,terminate within NEB, but they are not positive for thesensory nerve marker CGRP [22] whereas the epithelium[22,24] and pigs [25]. In guinea pigs most of these affer-ents arise in the jugular ganglia [26,27]. However, little isknown about the relationship between nerves and PNEC.The aims of this study were to characterize the threedimensional morphology of PNEC, their distribution,and their relationship to the epithelial nerves in wholemounts of adult human bronchi using confocal micros-copy. The peptides CGRP and GRP were examined fortheir consistency as markers of PNEC. Protein gene prod-uct 9.5 (PGP 9.5) was used as a marker of PNEC and forepithelial nerves, and CGRP for sensory nerves. The inves-tigation was restricted to the solitary PNEC because NEBappear to be extremely rare in adult human lung [17].MethodsHuman airway tissueA small section of bronchus was resected from the non-diseased portion of a lobe from a human lung obtained atthoracotomy from 8 patients undergoing surgery for theremoval of lung tumors (Table 1). Three subjects were life-long non-smokers. The sample was removed from thefreshly excised lobe on ice and fixed in Streck Tissue Fixa-tive (Streck Laboratories, US). The airway segment(s)ranged from 3 to 6 mm inner diameter and were up to 1cm in length. They were cut open lengthwise and the air-way wall carefully dissected to create thin sheets that com-prised epithelium and mucosa while discarding smoothmuscle and cartilage. As a standard antigen retrievalmethod, tissues were microwaved for 20 min in citratebuffer (pH 6.0) and afterwards blocked for one hour inPBS pH 7.4 containing 1% bovine serum albumin. Thetissue was cut into pieces of approximately 5 mm2 area -usually about 10 pieces – which are referred to as whole-mounts, and all further treatment was carried out in 96-well culture plates equipped with anti-evaporation lids.Preparation and staining of whole mountsWhole mounts were stained with antibodies to reveal allnerves (PGP 9.5), sensory nerves (CGRP), and PNEC(PGP 9.5, CGRP and GRP). The PGP 9.5 antibodies (mon-oclonal and polyclonal) were obtained from UltraClone,UK and used at a dilution of 1/100 and 1/500, respec-tively. Antibodies to GRP (polyclonal) and CGRP (mono-clonal and polyclonal) were purchased from Dako, NSW,Australia. The dilutions were as follows: GRP, 1/200; pol-yclonal CGRP, 1/400, monoclonal CGRP, 1/100. The sec-ondary antibodies (anti-mouse and anti-rabbit)conjugated to Alexa-488 and Alexa-543, respectively wereobtained from Molecular Probes, MA and used in a dilu-tion of 1/200. Typically, 10 µl of antibody solution wasused for each well. Control experiments to test for auto-fluorescence and non-specific staining were carried outPage 2 of 11(page number not for citation purposes)is richly innervated with CGRP- and Substance P (SP)-containing nerve terminals in guinea pigs [23], ratsusing non-immune rabbit and mouse sera as describedpreviously [28]. The tissues were incubated with primaryRespiratory Research 2005, 6:115 http://respiratory-research.com/content/6/1/115antibodies overnight (4°C) in the presence of 0.3% TritonX-100 to enhance permeabilization. After washing 3 × 20min in PBS, fluorophor-conjugated secondary antibodieswere applied overnight (4°C). After further washing withPBS, the preparations were mounted in 90% glycerol con-taining p-phenylethylenediamine (1 mg/ml) to reducebleaching of the fluorochromes. Custom-made slideswere used that enabled imaging the specimen from bothsides. The coverslips were raised with spacers (Imagingspacers, Sigma) in order to minimize compression of thespecimens. The edges of the coverslips were sealed withnail polish to prevent evaporation of the mountingmedium.Confocal microscopyWholemount pieces were double-stained in combina-tions of poly- and monoclonal antibodies and imagedusing confocal microscopy (Biorad MRC-1000, ComosSoftware 7.0) as previously described [29]. For highestmagnification, the focus depth was increased at 1 micronsteps during scanning. The analysis and rendition of theresulting three-dimensional data sets, including side-pro-jections, was performed using NIH-Image software (Ver-sion 1.61b12). Images were colorized and super-imposedwith Adobe Photoshop 5 to reveal the complex structureof these cells. The Image Processing Tool Kit plug-in(Raindeer Software) was used to measure PNEC density.From each patient, a minimum of four fields of adequatestaining quality and optimal signal to noise ratio, wereimaged at a magnification of × 10. The PNEC were manu-ally marked out and the software automatically calculatedthe PNEC density per area. Because the PNEC numberswere not homogeneously distributed in most of the bron-chi sampled, no attempt was made to calculate the meandensity of these cells for each bronchus.ResultsPNEC staining with PGP9.5stained with PGP 9.5 or GRP. The numbers were notevenly distributed, ranging from 65 to 100/mm2 over thearea of any one wholemount, but with denser patchescomprising 150 to 260/mm2 observed less frequently inseveral of the lungs (Figure 1). Staining with the neuralmarker PGP 9.5 typically revealed PNEC with a flask-likeshape when viewed from the side (Figure 2a, 90° rota-tion). The cell body was located near the basement mem-brane with the apical part of the cell comprising acharacteristic thick stem that extended to the lumen sur-face (Figure 2b and 2c). The overall height of the cellsaveraged 50.1 ± 6.7 µm (SD, n = 21) in four lungs. Proc-esses issued from the cell body along the basal region ofthe epithelium and also toward the lumen (Figure 2a, 90°rotation, Fig 2b and 2c). These processes have a dendritic-like appearance when viewed as a projection from thelumen because their three-dimensional morphology can-not be readily appreciated from this aspect (Figure 2a, 0°rotation; Figure 2d). Varicose nerves ascended in closeassociation with the PNEC stem to reach an apical nerveplexus (Figure 2 and 3a, 3b) that lies just below the lumi-nal surface of the epithelium. Figure 3a shows an exampleof an isolated patch of varicose epithelial nerves taken atlow power (lumen view, 0 degrees). The apical disposi-tion of these nerves is seen in the 90 degree rotation. Nocorrelation was found between the distribution of PNECand nerves in the epithelium.Co-localization of GRP and PGP9.5 in PNECAll PGP 9.5-positive PNEC were also positive for GRP,however, the PGP 9.5 staining intensity of individualPNEC varied considerably. GRP exhibited significantlyhigher detail of the processes whereas PGP 9.5 stainedpredominantly the cell body with its prominent stem (Fig-ure 3a, 30° and 90° rotations). This is strikingly shown inthe PNEC enclosed in the boxed area of Figure 3a whichhas been rotated and enlarged. Most PNEC exhibited adominance of one marker over the other (Figure 3a, 90Table 1: Patient Demographic DataPatient Gender Age Smoking status Disease1 Male 65 Smoker SSC2 Male 67 Smoker LSC3 Female 40 Non-smoker Adeno4 Female 37 Non-smoker LSC5 Female 61 Ex-Smoker Adeno6 Female 74 Smoker SSC7 Male 70 Smoker SSC8 Female 72 Smoker SSCAbbreviations: SSC, small cell carcinoma; LSC, Large cell carcinoma; Adeno, Adenocarcinoma.Page 3 of 11(page number not for citation purposes)Solitary PNEC were abundant within the epithelium ofthe bronchi of all eight human lungs examined whendegree rotation, and Figure 3b). Individual staining of asingle PNEC for both markers is shown in Figure 3c whereRespiratory Research 2005, 6:115 http://respiratory-research.com/content/6/1/115detail of the thick and fine processes are revealed withGRP.Double staining for GRP and PGP9.5 in PNEC associated with nervesThe association of ascending and apical nerves with PNECin the epithelium was more readily appreciated with dou-ble staining. Figure 3d shows a nerve rising from the baseof a PNEC that extends upwards along the stem and termi-nates near the luminal surface. Figure 3e shows threePNEC, two of which are in a patch of nerves (arrows,boxed region). The right PNEC and its processes are inclose proximity to a network of varicose nerves. When arotation was performed on the field (boxed area, right)nerves arising from the base of this PNEC ascended alongaccompanied the GRP-stained processes towards thelumen.Double staining for CGRP and PGP 9.5When PNEC were double stained for CGRP and PGP 9.5,CGRP was present in 22 ± 9% (SD, 10 fields, 4 lungs) ofall PNEC stained (Figure 4a). CGRP typically stained theprocesses but was faint or absent in the cell body. Figure4a (boxed area, right) shows a PNEC side-view withlumen-directed processes where one is particularlystrongly stained for CGRP. It was also present in theshorter processes directed towards the lamina propria(Figure 4b, left) where three pairs of PNEC display quitediverse morphology. Unlike the staining pattern observedwith GRP, CGRP is chiefly present in thicker, more proxi-Whole mount of mucosa from a human bronchus stained with gastrin releasing peptide (GRP) and imaged from the luminal surfac with a confocal microscopeFigur  1Whole mount of mucosa from a human bronchus stained with gastrin releasing peptide (GRP) and imaged from the luminal surface with a confocal microscope. The low power projection reveals an abundance of pulmonary neuroendocrine cells (PNEC) in the epithelium. Bar = 500 µm. Inset: higher power view revealing the morphology of PNEC. Where the epithelial surface is flat (ie. parallel with the cover slip), the view is from the top looking down on the cell body and processes but where they lie on the edge of a mucosal fold their flask-like shape is revealed. Bar = 50 µm.Page 4 of 11(page number not for citation purposes)its stem to join the apical nerve plexus. Other nervestravelling from the lamina propria into the epitheliummal part of the processes.Respiratory Research 2005, 6:115 http://respiratory-research.com/content/6/1/115Staining for CGRP in nerves and PNECFaint staining of CGRP could be detected in PNEC cellbodies but it was much less than that in the processes.esses and the cell body. When PNEC were present in afield containing CGRP-positive nerves, they appeared tomake contact with the PNEC. These contacts were charac-(a). High power projection of two PNEC and associated nerves imaged from the lumen and stained with the neural marker, prote n gene p oduct 9.5 (PGP 9.5)Figure 2(a). High power projection of two PNEC and associated nerves imaged from the lumen and stained with the neural marker, protein gene product 9.5 (PGP 9.5). To reveal the shape of the cell body and the diverse structure of its processes the data set has been rotated to enable viewing the PNEC from the side. The upper panel is the conventional view from the lumen surface. The middle panel is rotated at 45 deg, and the lower panel shows the side view at 90 degrees. Nerves are present in close apposition to the PNEC. Bar = 20 µm. (b & c) Projections of typical flask-like PNEC stained with PGP 9.5 and imaged from the lumen over the edge of a mucosal fold. The cell bodies are seen from their sides (thus a cut off line for the surface of the epi-thelium with the lumen is not clearly seen). Processes of varying morphology arise from the cell bodies and varicose nerves are present near the base and apex of the PNEC with individual nerve fibers rising through the epithelium. The apex of the cell is brightly stained in (b). Bars = 10 µm. (d) Four PNEC viewed from a flat area of the airway lumen showing dendritic-like proc-esses. This low power projection extends through a depth of 50 µm and includes nerves that lie below the basement membrane.b cdaPage 5 of 11(page number not for citation purposes)Figure 5a demonstrates that one of the lumen-directedprocesses stains stronger for CGRP than the other proc-terized by brightly stained, enlarged terminal varicositiesRespiratory Research 2005, 6:115 http://respiratory-research.com/content/6/1/115(a) Representative views of airway mucosa from four lungs double-stained for protein gene product 9.5 (PGP9.5, green) and g strin l asing peptide (GRP, red)Figure 3(a) Representative views of airway mucosa from four lungs double-stained for protein gene product 9.5 (PGP9.5, green) and gastrin releasing peptide (GRP, red). The upper panel is the lumen view, the middle one is rotated through 30 deg and the lower one through 90 deg, ie view from the side. Strings of varicose nerves are present in the epithelium. The dark holes indi-cate the location of goblet cells. PNEC are inconspicuous in the upper view but become more apparent when the field is pro-jected at an angle. The lower panels demonstrate that GRP is present predominantly in the PNEC processes, whereas PGP 9.5 is restricted to the cell body with its apical stem. Nerves feature strongly in the apical epithelium. Bar = 50µm. Boxed area: This PNEC has been turned through 90 degrees so that the prominent processes now point upwards, and enlarged (right, upper panel). A 90 deg rotation of the projection reveals that the PNEC processes stain strongly for GRP whereas the cell body and apical stem stain mainly for PGP 9.5. Some of the processes are lumen-directed, other processes with feet-like appearance are directed toward the lamina propria. Bar = 10µm. (b) Three PNEC in a field from another lung shown from the lumen (upper) and rotated through 90 deg (lower). Two of the PNEC are predominantly GRP positive whereas the third PNEC stains strongly for PGP 9.5. The upper stem of the middle cell body stains yellow indicating that the PGP 9.5 and the GRP staining are about equal. Bar = 10 µm. (c) A single PNEC shown as individual fields: PGP9.5 only (left), GRP only (middle), composite PGP9.5 + GRP (right). GRP reveals fine processes that issue from the cell body. In contrast to PGP 9.5, GRP does not stain the cell nucleus. Bar = 10 µm. (d) A single PNEC in close association with a nerve terminal. A nerve rises from the base of the PNEC, climbs through the epithelium along the PNEC stem and spreads laterally in the apical epithelium where it exhibits enlarged terminal varicosities. Upper panel: lumen view, middle panel: 45 deg rotation, lower panel, 90 degree rotation. Bar 10 µm. (e) Lumen view of mucosa where the epithelium is tilted showing three PNEC from an angle. Patches of fine varicose nerves are present. Some of the nerves lie close to the stems of two of the PNEC (arrow heads). Bar = 25 µm. Boxed area(right): High power view after rotating shows two PNEC within a patch of nerves. The left PNEC is strongly PGP9.5 posi-tive. The right PNEC has several processes in close apposition to nerves that rise through the epithelium to form an apical RRRRRa b                decPage 6 of 11(page number not for citation purposes)nerve plexus. Nerves in the apical epithelium lie close to the central stems of both PNEC. Bar = 10 µm.Respiratory Research 2005, 6:115 http://respiratory-research.com/content/6/1/115(a) Lumen view of a small area of epithelium representative of a whole mount of airway mucosa double-stained with calcitonin gene-related peptide (CGRP, red) and PGP9.5 (gre )Figur  4(a) Lumen view of a small area of epithelium representative of a whole mount of airway mucosa double-stained with calcitonin gene-related peptide (CGRP, red) and PGP9.5 (green). CGRP is present in terminal processes of some PNEC. Bar = 100 µm. Boxed area (right): Side projection of the PNEC where two lumen-directed processes and the long stem of the cell body (green) ascend to the apical epithelium. From this view the right process is strongly CGRP positive. Bar = 20 µm. (b) Wholemount of airway mucosa double-stained for CGRP (red) and PGP 9.5 (green) showing diverse morphology of PNEC. Upper panel is the lumen view, middle is a rotated through 45 degrees and the lower through 90 degrees. From left to right: A pair of PNEC with CGRP in the processes. The next two have fine processes that arise from the cell body and the apex of the stem. The right abRRRPage 7 of 11(page number not for citation purposes)hand pair of PNEC exhibits branching of the main stem close to the lumen. Bar = 50 µm.Respiratory Research 2005, 6:115 http://respiratory-research.com/content/6/1/115(a) Two PNEC stained with CGRP, shown as lumen view (upper panels) and rotated through 90 deg (lower panels)Figure 5(a) Two PNEC stained with CGRP, shown as lumen view (upper panels) and rotated through 90 deg (lower panels). Each of the latter reveal a brightly staining process that issues from their cell body toward lumen whereas the cell bodies (left side of each panel) and other processes show weaker staining. Bar = 20 µm. (b) Wholemount of airway mucosa stained for CGRP. A net-work of weakly staining CGRP varicose nerves and a single PNEC is present. The top panel is the lumen view (0°), followed by rotations of 30°, 60° and 90°. One fiber exhibits brightly stained varicosities at the apparent point of contact with the PNEC. Rotating the field demonstrates that nerves run from below the base of the PNEC prior to ascending to it. Other ascending fib-ers are present in the left side of the field. The horizontal lines are a consequence of the limited number of Z steps in the con-D E cRRRRPage 8 of 11(page number not for citation purposes)focal data set. Bar = 20 µm.Respiratory Research 2005, 6:115 http://respiratory-research.com/content/6/1/115indicative of nerve endings, suggestive of innervation ofPNEC by CGRP-positive nerves (Figure 5b).DiscussionThis is the first report characterizing PNEC morphology inthree dimensions in human airways. The use of wholemounts of mucosal tissue enabled the direct demonstra-tion of PNEC abundance over large areas of airway epithe-lium. Rotation of 3-D images revealed the complexity ofthe PNEC body and its processes that issue laterally fromits base, some lumen directed, others feet-like that weredirected toward the lamina propria. PGP 9.5 and GRPwere reliable markers, both staining all PNEC, whereasCGRP was present in about 20% of the PNEC population.However, the distribution of staining varied widelyamong cells, with GRP and CGRP mainly present in theprocesses, but with GRP occurring also in the cell body tovarying degrees. The variation exhibited in the morphol-ogy of the PNEC and its differing peptide profiles suggeststhat these cells may be in a dynamic state in theepithelium.PNEC were abundant when stained with either PGP 9.5 orGRP. Previous studies in human airways that investigatedPNEC density used conventional cross sections andrevealed numbers ranging from 1.05 PNEC/cm basementmembrane (corresponding to 4 PNEC per 10,000 epithe-lial cells) using neuron specific enolase [17] to 12.5PNEC/cm, or 41 PNEC per 10,000 epithelial cells, usingchromogranin A as a marker [30]. In the current study, ourmeasurements reveal that the density of PNECs rangesfrom 65/mm2 to 260/mm2 within an individual wholem-ount. There did not appear to be a homogenous distribu-tion of PNEC either within or between wholemounts.Areas of high PNEC density appeared to be juxtaposed toareas of sparse cell numbers. Direct comparisons betweenthe numbers provided in our study and those reportedpreviously are confounded by the considerable discrep-ancy between the proportions of PNEC revealed by mark-ers used to identify PNEC [17,30]. We used PGP 9.5 tolabel the whole PNEC population, whereas Boers reportedthat only 14% of all PNEC showed PGP 9.5 immunoreac-tivity [30]. Furthermore, our study indicates that all PNECcontain both PGP 9.5 and GRP, whereas Gosney et al.,[17] found GRP present in 65% of all PNEC stained withneuron specific enolase and Boers demonstrated GRP in59% of all PNEC stained with chromogranin A [30]. Thereasons for these discrepancies are unknown, althoughthe greater sensitivity and ability of confocal microscopyto resolve cell types and contents may account for theobserved differences. Similarly, the size of the airway mayalso influence the PNEC distribution. In rat lungs at least,the density of NEB/PNEC appears to be dependent on air-[31]. Only large cartilaginous airways (3–6 mm ID) wereavailable for this study. Although multiple wholemountpieces were analysed from each airway, our study was lim-ited to 1 piece of bronchial tissue per subject and as suchwe are unable to comment on the overall distribution ofPNEC throughout the lungs.The majority of PNEC were not in close association withepithelial nerves, partly because nerves were generallysparse and tended to occur in patches as we reportedrecently [25]. When present, PGP 9.5 positive nerve fibreswere generally observed to be in close apposition with thePNEC cell body, its stem, apex, and processes. Unfortu-nately, the immunofluorescent staining used in this studydoes not permit the distance between the nerve fibres andthe PNEC to be measured accurately, and side projectionscomputed from data obtained by scanning from the lumi-nal surface are subject to loss of resolution. Nonetheless,when images were viewed from the lumen through aseries of rotations over 360 degrees, it was clear that thenerves lie very close to these structures, within onemicrometre. These are likely to be sensory nerves becausethey are varicose, with enlarged varicosities in the termi-nal region [25], and in most lungs stained positivelythough weakly for CGRP. CRRP-positive nerves endingsappeared to terminate near or on the processes issuingfrom the base of the PNEC where bright terminal enlargedvaricosities were seen. Afferent and efferent nerves havebeen characterized ultrastructurally on cells within NEB infetal and neonate humans [4] and adult animals [21,22]but as far as we are aware, not on single PNEC. Thisapproach is hampered because of the apparent paucity ofepithelial sensory nerves in humans. The infrequentpatches of PGP 9.5-positive epithelial nerves stain veryweakly in humans [25,32], or not at all [33,34] for SP orCGRP [35] in contrast to rats [24] and pigs [25] wherethey are abundant.In humans, NEB decrease in frequency with age and arerare in adult lung. Gosney et al.,[16] observed only threeNEB after searching through preparations from 5 post-mortem specimens. Our data support this finding, as onlytwo NEB were observed and were confined to a single lungafter scanning several hundred whole mount PIECES fromeight adult lungs. Brouns and colleagues recently demon-strated a complex innervation pattern of pulmonary NEBsin rat airways, comprising both sensory vagal nerves aswell as non-vagal CGRP/SP positive nerves [36]. The phys-iological role of the innervation of NEB is not well under-stood. It has been proposed that the nerve endings at thebase of the NEB subserve an axon reflex, presumablyarising in the NEB itself and possibly penetrating todeeper tissues such as the airway smooth muscle [4].Page 9 of 11(page number not for citation purposes)way size, with a greater density of cells observed inproximal airways compared to more distal generationsThere may also be local reflex connections throughperipheral ganglia. Hypoxia detected by the O2 sensor inRespiratory Research 2005, 6:115 http://respiratory-research.com/content/6/1/115the NEB is presumed to release mediators that stimulatevagal afferents, but no central nervous reflexes have beenidentified [37]. Recent advances in microscopic tech-niques with increased sensitivity may shed more light onthe morphological basis for many of the suggested func-tions of NEB innervation.In our study, we used immunofluorescently-labeled anti-bodies to PGP 9.5, GRP and CGRP to detect PNEC inadult airway epithelium. PGP 9.5 stained all the cell bod-ies fairly uniformly but was weaker in the processes so thattheir fine ends frequently were not revealed. All PGP 9.5positive cells were also positive for GRP, suggesting that itmay also be a reliable marker for identifying PNEC. It waspredominantly observed in the processes, but in manycells it stained the cell body and stem region either par-tially or completely. However, less than a quarter of PNECexhibited CGRP immunoreactivity, with the greatestintensity displayed in the thick processes. Although CGRPis often used in animal studies as a marker for quantitativestudies of NEB and PNEC [38], we have shown that CGRPis not a reliable marker of the PNEC populations inhuman adult epithelium, as only a subset exhibited CGRPimmunoreactivity. These markers, used in conjunctionwith three dimensional imaging and image rotation, haverevealed the overall morphology of the PNEC that hith-erto has not been appreciated using conventional lightand electron microscopy. PGP 9.5 revealed the variety ofshapes that the cell body can attain, most often flask orbottle-shaped with the base at the basement membraneand its long stem extending to the lumen where its tip wasoften more brightly stained. Some PNEC exhibitedbranching of the main stem close to the lumen.GRP staining revealed a striking PNEC morphology withthick processes issuing laterally from near the base of thecell body upwards toward the apical epithelium and alongthe basement membrane. In addition GRP also stainedfine processes originating from the side of the PNEC bodythat were not readily detected with PGP 9.5. The thick andthin processes of the PNEC, revealed by our 3-D confocalmicroscopy, may be the conduits that effect delivery of thebioamines and peptides proposed to be secreted byPNEC.Bioamines and peptides contained within the PNEC havebeen proposed to be secreted into the adjacent epitheliumand lamina propria in response to such stimuli as hypoxia[6,11]. GRP and CGRP have been shown to havemitogenic and growth factor like influences [39] and mayhave a direct influence on epithelial regeneration and anindirect one via local vasodilation of the adjacent bron-chial vasculature. Our confocal microscopic study demon-suggesting an active and diverse PNEC population ispresent in adult human airway epithelium.In this study, lung tissue samples were derived from adiverse group of patients ranging from 39 to 74 years ofage that were undergoing thoracotomy for removal oflung tumors. The small number of patients precluded thecorrelation of our results to gender or smoking history.Thus it is difficult to determine to what extent data pre-sented in this study represent the steady-state versus dis-ease-specific remodeling of the airway epithelium.ConclusionOur 3D-data demonstrates that PNEC are numerous andexhibit a heterogeneous peptide content suggesting anactive and diverse PNEC population. Valuable insightsinto the biology of cells identified in this study may comefrom a better understanding of their abundance, mor-phology and innervation comparing normal lung tissueversus injured or diseased lungs.Authors' contributionsMW, EJH carried out the sample preparation and confocalmicroscopy and reviewed the manuscript. MPS, PJT andDAK conceived of the program, participated in the designand coordination of this study, and drafted themanuscript.AcknowledgementsThe authors would like to thank the cardiothoracic surgeons and theatre staff at Sir Charles Gairdner Hospital and Pathology staff at the PathCentre for provision of lung specimens. This work was supported by the University of Western Australia Research Grants Scheme and the Sir Charles Gaird-ner Hospital Research Foundation.References1. Lauweryns JM, Van Ranst L: Protein gene product 9.5 expressionin the lungs of humans and other mammals. Immunocyto-chemical detection in neuroepithelial bodies, neuroendo-crine cells and nerves.  Neurosci Lett 1988, 85:311-6.2. Johnson EW, Eller PM, Jafek PW: Protein gene product 9.5-likeand calbindin-like immunoreactivity in the nasal respiratorymucosa of perinatal humans.  Anat Rec 1997, 247:38-45.3. Adriaensen D, Scheuermann DW: Neuroendocrine cells andnerves of the lung.  Anat Rec 1993, 236:70-85.4. 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