Background
Pulmonary neuroendocrine cells (PNEC) are specialized airway epithelial cells that occur as solitary cells or as clusters called neuroepithelial bodies (NEB) [
1]. They are located in the nasal respiratory epithelium, laryngeal mucosa [
2] and throughout the entire respiratory tract from the trachea to the terminal airways [
3]. In the fetal lung they are frequently located at the branching points of airway tubules, and in humans are present by 10 weeks gestation [
4]. Neuroendocrine cells are bottle- or flask-like in shape, and reach from the basement membrane to the lumen. They can be distinguished by their profile of bioactive 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 airway chemoreceptors [
6], and an oxygen-sensitive potassium channel coupled to an oxygen sensory protein has been demonstrated in their lumenal membrane in the rabbit [
7]. They are also considered to be involved in regulating localized epithelial cell growth and regeneration through a paracrine mechanism whereby their bioactive peptides are released into the environment [
8]. Peptides and amines released by PNEC are involved in normal fetal lung development including branching morphogenesis [
9]. The best-characterized peptides are GRP, the mammalian form of bombesin, and CGRP, which exert direct mitogenic effects on epithelial cells and exhibit many growth factor-like properties [
10].
The majority of data available on the morphology, distribution, peptide expression and function of PNEC and NEB have been obtained from animal studies [
11,
12].
In human airways, the morphology of NEB have been studied ultrastructurally during the fetal and perinatal stage of lung development, and their peptides identified using immunogold-labeled antibodies where they are colocalized in the dense core vesicles in the cytoplasm [
4,
13‐
15]. However, there is little data describing the three dimensional morphology and peptide distribution in adult human airways where both PNEC and NEB are reported to be sparse [
16,
17]. It has been suggested that PNEC may play a role in mediating airway remodelling in normal lungs and in naturally occurring pulmonary disease where they increase in number [
8,
18].
The innervation of fetal and postnatal NEB has been also studied ultrastructurally in humans where both adrenergic and cholinergic nerve endings have been observed [
4], in rabbits [
19] and rats and dogs [
20,
21]. In rats, vagal nodose afferents traced using the carbocyanine dye DiI, terminate within NEB, but they are not positive for the sensory nerve marker CGRP [
22] whereas the epithelium is richly innervated with CGRP- and Substance P (SP)- containing nerve terminals in guinea pigs [
23], rats [
22,
24] and pigs [
25]. In guinea pigs most of these afferents arise in the jugular ganglia [
26,
27]. However, little is known about the relationship between nerves and PNEC.
The aims of this study were to characterize the three dimensional morphology of PNEC, their distribution, and their relationship to the epithelial nerves in whole mounts of adult human bronchi using confocal microscopy. The peptides CGRP and GRP were examined for their consistency as markers of PNEC. Protein gene product 9.5 (PGP 9.5) was used as a marker of PNEC and for epithelial nerves, and CGRP for sensory nerves. The investigation was restricted to the solitary PNEC because NEB appear to be extremely rare in adult human lung [
17].
Discussion
This is the first report characterizing PNEC morphology in three dimensions in human airways. The use of whole mounts of mucosal tissue enabled the direct demonstration of PNEC abundance over large areas of airway epithelium. Rotation of 3-D images revealed the complexity of the PNEC body and its processes that issue laterally from its base, some lumen directed, others feet-like that were directed toward the lamina propria. PGP 9.5 and GRP were reliable markers, both staining all PNEC, whereas CGRP was present in about 20% of the PNEC population. However, the distribution of staining varied widely among cells, with GRP and CGRP mainly present in the processes, but with GRP occurring also in the cell body to varying degrees. The variation exhibited in the morphology of the PNEC and its differing peptide profiles suggests that these cells may be in a dynamic state in the epithelium.
PNEC were abundant when stained with either PGP 9.5 or GRP. Previous studies in human airways that investigated PNEC density used conventional cross sections and revealed numbers ranging from 1.05 PNEC/cm basement membrane (corresponding to 4 PNEC per 10,000 epithelial cells) using neuron specific enolase [
17] to 12.5 PNEC/cm, or 41 PNEC per 10,000 epithelial cells, using chromogranin A as a marker [
30]. In the current study, our measurements reveal that the density of PNECs ranges from 65/mm
2 to 260/mm
2 within an individual wholemount. There did not appear to be a homogenous distribution of PNEC either within or between wholemounts. Areas of high PNEC density appeared to be juxtaposed to areas of sparse cell numbers. Direct comparisons between the numbers provided in our study and those reported previously are confounded by the considerable discrepancy between the proportions of PNEC revealed by markers used to identify PNEC [
17,
30]. We used PGP 9.5 to label the whole PNEC population, whereas Boers reported that only 14% of all PNEC showed PGP 9.5 immunoreactivity [
30]. Furthermore, our study indicates that all PNEC contain both PGP 9.5 and GRP, whereas Gosney et al., [
17] found GRP present in 65% of all PNEC stained with neuron specific enolase and Boers demonstrated GRP in 59% of all PNEC stained with chromogranin A [
30]. The reasons for these discrepancies are unknown, although the greater sensitivity and ability of confocal microscopy to resolve cell types and contents may account for the observed differences. Similarly, the size of the airway may also influence the PNEC distribution. In rat lungs at least, the density of NEB/PNEC appears to be dependent on airway size, with a greater density of cells observed in proximal airways compared to more distal generations [
31]. Only large cartilaginous airways (3–6 mm ID) were available for this study. Although multiple wholemount pieces were analysed from each airway, our study was limited to 1 piece of bronchial tissue per subject and as such we are unable to comment on the overall distribution of PNEC throughout the lungs.
The majority of PNEC were not in close association with epithelial nerves, partly because nerves were generally sparse and tended to occur in patches as we reported recently [
25]. When present, PGP 9.5 positive nerve fibres were generally observed to be in close apposition with the PNEC cell body, its stem, apex, and processes. Unfortunately, the immunofluorescent staining used in this study does not permit the distance between the nerve fibres and the PNEC to be measured accurately, and side projections computed from data obtained by scanning from the luminal surface are subject to loss of resolution. Nonetheless, when images were viewed from the lumen through a series of rotations over 360 degrees, it was clear that the nerves lie very close to these structures, within one micrometre. These are likely to be sensory nerves because they are varicose, with enlarged varicosities in the terminal region [
25], and in most lungs stained positively though weakly for CGRP. CRRP-positive nerves endings appeared to terminate near or on the processes issuing from the base of the PNEC where bright terminal enlarged varicosities were seen. Afferent and efferent nerves have been characterized ultrastructurally on cells within NEB in fetal and neonate humans [
4] and adult animals [
21,
22] but as far as we are aware, not on single PNEC. This approach is hampered because of the apparent paucity of epithelial sensory nerves in humans. The infrequent patches of PGP 9.5-positive epithelial nerves stain very weakly in humans [
25,
32], or not at all [
33,
34] for SP or CGRP [
35] in contrast to rats [
24] and pigs [
25] where they are abundant.
In humans, NEB decrease in frequency with age and are rare in adult lung. Gosney et al.,[
16] observed only three NEB after searching through preparations from 5 post-mortem specimens. Our data support this finding, as only two NEB were observed and were confined to a single lung after scanning several hundred whole mount PIECES from eight adult lungs. Brouns and colleagues recently demonstrated a complex innervation pattern of pulmonary NEBs in rat airways, comprising both sensory vagal nerves as well as non-vagal CGRP/SP positive nerves [
36]. The physiological role of the innervation of NEB is not well understood. It has been proposed that the nerve endings at the base of the NEB subserve an axon reflex, presumably arising in the NEB itself and possibly penetrating to deeper tissues such as the airway smooth muscle [
4]. There may also be local reflex connections through peripheral ganglia. Hypoxia detected by the O
2 sensor in the NEB is presumed to release mediators that stimulate vagal afferents, but no central nervous reflexes have been identified [
37]. Recent advances in microscopic techniques with increased sensitivity may shed more light on the morphological basis for many of the suggested functions of NEB innervation.
In our study, we used immunofluorescently-labeled antibodies to PGP 9.5, GRP and CGRP to detect PNEC in adult airway epithelium. PGP 9.5 stained all the cell bodies fairly uniformly but was weaker in the processes so that their fine ends frequently were not revealed. All PGP 9.5 positive cells were also positive for GRP, suggesting that it may also be a reliable marker for identifying PNEC. It was predominantly observed in the processes, but in many cells it stained the cell body and stem region either partially or completely. However, less than a quarter of PNEC exhibited CGRP immunoreactivity, with the greatest intensity displayed in the thick processes. Although CGRP is often used in animal studies as a marker for quantitative studies of NEB and PNEC [
38], we have shown that CGRP is not a reliable marker of the PNEC populations in human adult epithelium, as only a subset exhibited CGRP immunoreactivity. These markers, used in conjunction with three dimensional imaging and image rotation, have revealed the overall morphology of the PNEC that hitherto has not been appreciated using conventional light and electron microscopy. PGP 9.5 revealed the variety of shapes that the cell body can attain, most often flask or bottle-shaped with the base at the basement membrane and its long stem extending to the lumen where its tip was often more brightly stained. Some PNEC exhibited branching of the main stem close to the lumen.
GRP staining revealed a striking PNEC morphology with thick processes issuing laterally from near the base of the cell body upwards toward the apical epithelium and along the basement membrane. In addition GRP also stained fine processes originating from the side of the PNEC body that were not readily detected with PGP 9.5. The thick and thin processes of the PNEC, revealed by our 3-D confocal microscopy, may be the conduits that effect delivery of the bioamines and peptides proposed to be secreted by PNEC.
Bioamines and peptides contained within the PNEC have been proposed to be secreted into the adjacent epithelium and lamina propria in response to such stimuli as hypoxia [
6,
11]. GRP and CGRP have been shown to have mitogenic and growth factor like influences [
39] and may have a direct influence on epithelial regeneration and an indirect one via local vasodilation of the adjacent bronchial vasculature. Our confocal microscopic study demonstrates PNEC with heterogeneous peptide content, suggesting an active and diverse PNEC population is present in adult human airway epithelium.
In this study, lung tissue samples were derived from a diverse group of patients ranging from 39 to 74 years of age that were undergoing thoracotomy for removal of lung tumors. The small number of patients precluded the correlation of our results to gender or smoking history. Thus it is difficult to determine to what extent data presented in this study represent the steady-state versus disease-specific remodeling of the airway epithelium.
Authors' contributions
MW, EJH carried out the sample preparation and confocal microscopy and reviewed the manuscript. MPS, PJT and DAK conceived of the program, participated in the design and coordination of this study, and drafted the manuscript.