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 ​(Table1)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.

Table 1

Patient Demographic Data

Patient	Gender	Age	Smoking status	Disease
1	Male	65	Smoker	SSC
2	Male	67	Smoker	LSC
3	Female	40	Non-smoker	Adeno
4	Female	37	Non-smoker	LSC
5	Female	61	Ex-Smoker	Adeno
6	Female	74	Smoker	SSC
7	Male	70	Smoker	SSC
8	Female	72	Smoker	SSC

Open in a separate windowAbbreviations: SSC, small cell carcinoma; LSC, Large cell carcinoma; Adeno, Adenocarcinoma.

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.