A confocal microscopic study of solitary pulmonary neuroendocrine cells in human airway epithelium.

BACKGROUND: Pulmonary neuroendocrine cells (PNEC) are specialized epithelial cells that are thought to play important roles in lung development and airway function. PNEC occur either singly or in clusters called neuroepithelial bodies. Our aim was 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. METHODS: Bronchi were resected from non-diseased portions of a lobe of human lung obtained from 8 thoracotomy patients (Table 1) undergoing surgery for the removal of lung tumors. Whole mounts were stained with antibodies to reveal all nerves (PGP 9.5), sensory nerves (calcitonin gene related peptide, CGRP), and PNEC (PGP 9.5, CGRP and gastrin releasing peptide, GRP). The analysis and rendition of the resulting three-dimensional data sets, including side-projections, was performed using NIH-Image software. Images were colorized and super-imposed using Adobe Photoshop. RESULTS: PNEC were abundant but not homogenously distributed within the epithelium, with densities ranging from 65/mm2 to denser patches of 250/mm2, depending on the individual wholemount. Rotation of 3-D images revealed a complex morphology; flask-like with the cell body near the basement membrane and a thick stem extending to the lumen. Long processes issued laterally from its base, some lumenal and others with feet-like processes. Calcitonin gene-related peptide (CGRP) was present in about 20% of PNEC, mainly in the processes. CGRP-positive nerves were sparse, with some associated with the apical part of the PNEC. CONCLUSION: Our 3D-data demonstrates that PNEC are numerous and exhibit a heterogeneous peptide content suggesting an active and diverse PNEC population.

Ontogeny of airway smooth muscle: structure, innervation, myogenesis and function in the fetal lung.

Airway smooth muscle (ASM) is an integral component of the primordial lung. It differentiates from the mesenchyme as a ring of cells around the base of the epithelial bud that express smooth muscle-specific proteins. These rapidly form into interlocking bundles that progressively become wider and more compact along the bronchial tree to the trachea. Their orientation is perpendicular to the long axis of the airway. The ASM exhibits rhythmic contractility (i.e. it is a phasic-type smooth muscle) soon after formation, and the spontaneous airway narrowing shifts the lung liquid distally causing expansion of the tubule walls. This stretching is the mechanical stimulus to smooth muscle (SM) myogenesis and lung growth. Neural tissue, i.e. precursor ganglia interconnected by nerve trunks and smaller bundles, forms a sheath over the ASM layer with varicose fibres descending to the muscle. These are guided by glial-derived neurotrophic factor (GDNF) that appears to be produced by ASM. Maturation of neural tissue is slower than the ASM; functional cholinergic innervation is manifest by the early canalicular stage when most neurotransmitters appear.

Three-dimensional mapping of sensory innervation with substance p in porcine bronchial mucosa: comparison with human airways.

In asthma, neurogenic inflammation in bronchial airways may occur though the release of neuropeptides from C fibers via an axon reflex. Structural evidence for this neural pathway was sought in the pig and in humans by three-dimensional mapping of substance P-immunoreactive (SP-IR) nerves in whole mounts of mucosa using immunofluorescent staining and confocal microscopy. To show continuity, nerves were traced with 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate from their epithelial endings through the mucosa. The pan-neuronal marker protein gene product 9.5 revealed an extensive apical and basal plexus of nerves in the epithelium; 94% of these were varicose SP-IR fibers. Apical SP-IR fibers had a length density of 88 mm/mm(2). Varicose apical processes followed closely around the circumference of goblet cells. Calcitonin gene-related peptide was colocalized with SP-IR in varicosites. The epithelial fibers converged into bundles as they entered the lamina propria where lateral branches ran along arterioles, often contiguous with the vascular smooth muscle. 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate tracing showed that they projected to the epithelium. SP-IR fibers were rare near postcapillary venules. In human bronchial epithelium, protein gene product 9.5 revealed a similar apical and basal plexus of varicose fibers that weakly stained for SP-IR. Thus, a continuous sensory nerve pathway from the epithelium to arterioles provides structural support for a local axon reflex.

Development of neural tissue and airway smooth muscle in fetal mouse lung explants: a role for glial-derived neurotrophic factor in lung innervation.

We have characterized the distribution of neural tissue and its primary target tissue, airway smooth muscle (ASM), in an in vitro mouse model of early lung development comprising left lung lobes at embryonic Day 12, cultured for 2 or 5 d. Neural tissue was detected with antibodies to protein gene product 9.5 (PGP 9.5), synapsin, and p75NTR (the low-affinity neurotrophin receptor), and smooth muscle with an antibody to alpha-actin. Imaging by confocal microscopy revealed few PGP 9.5-positive neurons at the start of culture; after 2 d clusters of neurons and nerve fibers had appeared along the lobar bronchus and after 5 d along the secondary and tertiary branches. Neural tissue did not just follow the smooth muscle-covered tubules, as seen in vivo, but also grew outside the lobes onto a wide layer of alpha-actin-positive cells, suggesting that smooth muscle may express a trophic factor that attracts nerves. Explants cultured with glial-derived neurotrophic factor (GDNF) exhibited a striking increase in the amount of p75NTR- and PGP 9.5-positive tissue outside the lobes, whereas GDNF-impregnated beads attracted neuronal precursors and influenced the direction of neurite extension. We show that the mouse lung explant is suitable for investigating trophic signals involved in pulmonary innervation and that GDNF may have a role in the early innervation of the developing airways.