Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration

Abstract

Remodeling of the conducting airway epithelium is a common finding in the chronically injured lung and has been associated with increased risk for developing lung cancer. Pulmonary neuroendocrine cells and clusters of these cells termed neuroepithelial bodies (NEBs) play a central role in each of these processes. We previously developed an adult mouse model of airway injury and repair in which epithelial regeneration after naphthalene-induced Clara cell ablation occurred preferentially at airway branch points and gave rise to nascent Clara cells. Continued repair was accompanied by NEB hyperplasia. We now provide the following evidence that the NEB microenvironment serves as a source of airway progenitor cells that contribute to focal regeneration of the airway epithelium: 1) nascent Clara cells and NEBs localize to the same spatial domain; 2) within NEB, both Clara cell secretory protein- and calcitonin gene-related peptide-immunopositive cells are proliferative; 3) the NEB microenvironment of both the steady-state and repairing lung includes cells that are dually immunopositive for Clara cell secretory protein and calcitonin gene-related peptide, which were previously identified only within the embryonic lung; and 4) NEBs harbor variant Clara cells deficient in cytochrome P450 2F2-immunoreactive protein. These data suggest that the NEB microenvironment is a reservoir of pollutant-resistant progenitor cells responsive to depletion of an abundant airway progenitor such as the Clara cell.

Figure 1.

NEBs and nascent Clara cells localize to the same spatial domain. Adjacent serial sections (A and B, C and D, E and F) from animals 72 hours postnaphthalene treatment were stained for the neuroepithelial cell marker CGRP (A, C, E) or CCSP (B, D, F), using standard immunohistochemical techniques and DAB detection of the antigen-antibody complexes. A terminal bronchiole is represented in A and B, whereas C–F represent two different branch points. The frequency of NEBs associated with CCSP-IP cells was determined to be 87%. Original magnification, ×400.

Figure 2.

Two mitotic cell populations are localized to branchpoints. Adjacent serial sections from animals 72 hours postnaphthalene treatment were stained for CCSP (A) or for CGRP (B), using standard immunohistochemical techniques and DAB detection of the antigen-antibody complexes. Slides were then dipped in emulsion, and grains (black dots over nuclei) developed after 1 week. Original magnification, ×1000. C: The percent of all mitotic airway cells expressing either CGRP or CCSP was 16.06 ± 5.53 or 81.47 ± 3.00%, respectively.

Figure 3.

CCSP- and CGRP-IP cells are distinct populations. Dual immunofluorescence and laser scanning confocal microscopy were used to detect CCSP-IP cells (A, Cy2-green epifluorescence) and CGRP-IP cells (C, Cy5-red epifluorescence) in the same region of the same section. B: Image generated by merging A and C. The majority of cells in this section expressed either CCSP or CGRP (arrows). This pattern is representative of the majority of NEBs studied. The arrowhead indicates cells that may express both lineage markers simultaneously. Original magnification, ×630.

Figure 4.

A rare population of dual-positive cells is localized to NEBs. Dual immunofluorescence and laser scanning confocal microscopy were used to detect CCSP-IP cells (A, Cy2-green epifluorescence) and CGRP-IP cells (C, Cy5-red epifluorescence) in the same region of the same section. B: Image generated by merging A and C. Three cells (*) that express both antigens are located within this branch point NEB. Original magnification, ×630.

Figure 5.

Two variant Clara cell subpopulations are associated with NEBs in the normal lung. Sequential immunohistochemistry (Materials and Methods) was used to detect CGRP (A) and CCSP (B) on the same section. Amine-ethyl carbizol (pink-red) was used to detect CGRP, and DAB (gray) was used to detect CCSP immunocomplexes. C: Image generated by overlaying A and B. Long arrows in A–C indicate CGRP-IP/CCSP-immunonegative cells, whereas short arrows mark cells expressing both CGRP and CCSP. Similar methods were used to detect the Clara cell markers P450–2F2 (D, amine-ethyl carbizol detection) and CCSP (E, DAB detection) on the same section. F: Image generated by overlaying D and E. Short arrows in D–F indicate P450–2F2-IP/CCSP-IP cells, and long arrows mark P450–2F2 immunonegative/CCSP-IP cells. Original magnification, ×1000.