The respiratory epithelium is lined by diverse cell types that vary along the cephalo-caudal axis during development and after acute and chronic injuries. The mechanisms controlling formation and repair of this cellular and functional diversity are relatively unknown at present. This study demonstrates that ciliated epithelial cells are capable of remarkable phenotypic plasticity, rapidly undergoing squamous metaplasia and redifferentiating into cuboidal and then columnar cell types to contribute to the restoration of the complex airway epithelium after an acute Clara cell injury to the bronchioles. These findings demonstrate that the early repair process is independent of cell proliferation. The findings are not consistent with a significant role for extrapulmonary cells (e.g., bone marrow–derived cells or other mesenchymal derived cells) in repair of the respiratory epithelium after injury. These findings also challenge the view that ciliated cells are “terminally” differentiated cell type. The potential role of ciliated cells in repair of bronchiolar epithelium was further supported by the finding that, in the pneumonectomy model, ciliated cells were among epithelial cells participating in compensatory growth of the respiratory epithelium. The concept that this relatively abundant subset of cells (ciliated cells) can rapidly spread and redifferentiate to participate in repair of the complex airway epithelium provides a basis for the rapid repair of the lung after infection, exposure to toxicants, or lung resection.
After naphthalene injury, ciliated cells sequentially underwent squamous to cuboidal to columnar morphologic transition as the complex bronchiolar epithelium was restored. Rapid spreading of ciliated cells that occurred after injury likely plays a critical role in maintaining an intact epithelial barrier. Subsequently, the squamous cells differentiated into cuboidal and columnar cells (both ciliated and nonciliated cells), demonstrating remarkable plasticity during the process of redifferentiation. Thus ciliated cells underwent transdifferentiation during repair of the bronchiolar epithelium. Squamous metaplasia and spreading of once ciliated cells occurred before proliferation, which was maximal 2–4 d after injury. Regeneration of the bronchiolar epithelium after naphthalene injury is thought to be completed after 14–20 d (1
), and is also associated with proliferation and migration of naphthalene-resistant Clara cells from protected niches near airway branch points (1
). From a stochastic view, however, it is not likely that the rapid restoration of the bronchiolar surface results from proliferation of rare naphthalene-resistant Clara cells, but primarily from spreading of squamous progenitor cells that were derived from ciliated cells. Formal proof for this concept will require cell-specific lineage tracing that is not feasible at present. The present study demonstrates that the early rapid restoration of the bronchiolar epithelium after Clara cell injury is mediated by spreading and squamous metaplasia of ciliated cells, which maintain the epithelial barrier during repair. Thus, repair of naphthalene injured bronchiolar epithelium consists of two major phases: (1
) an early phase during which ciliated cells transdifferentiate to maintain and restore the bronchiolar epithelium, and (2
) a proliferative phase during which cell number and differentiated phenotypes are restored.
The regeneration of bronchiolar epithelium in both naphthalene injury and pneumonectomy models was accompanied by dynamic changes in Sox family proteins, Sox17 and Sox2, and transcription factors known to play important roles in lung morphogenesis and cell differentiation, including Foxa1, Foxa2, Foxj1, TTF-1, and β-catenin (22
). In naphthalene-induced injury, selective loss of nonciliated bronchiolar cells occurs without apparent injury to or proliferation of alveolar epithelial cells, whereas in the pneumonectomy model marked hyperplasia and proliferation occurs in both airways and alveoli, involving multiple epithelial and nonepithelial cell types (25
). Nevertheless, dynamic changes in the same transcription factors were associated with regeneration of the bronchiolar epithelium after both naphthalene injury and pneumonectomy models. These observations support the concept that the initial repair of the bronchiolar epithelium, at least in part, recapitulates transcriptional programs that coordinate respiratory epithelial cell differentiation during normal lung normal development. Since multiple cell types proliferate and differentiate after injury or during compensatory growth, it is anticipated that distinct transcriptional programs will influence these processes in diverse cell types.
Immunostaining showed that expression of Sox17 and Sox2 was selectively enhanced in ciliated cells before injury and that the Sox proteins and β-catenin were coexpressed in the squamous and cuboidal cells during repair of epithelium. Multiple Sox proteins, including Sox2 and Sox17, interact with Wnt/ β-catenin signaling to regulate diverse developmental processes, including cell type specification and stem/progenitor cell maintenance. Sox2 was shown to interact with β-catenin to regulate Wnt/β-catenin signaling in the differentiation of osteoblast (34
). Sox17 and β-catenin are known to interact to regulate a subset of genes, including Foxa1 and Foxa2, in the early endoderm (35
). Foxa1, Foxa2, and Foxj1 were dynamically regulated after injury and restoration of the bronchiolar epithelium, the highest levels of expression being observed in ciliated cells and their derivatives early in the repair process. These Fox transcription factors are known to influence gene expression and epithelial cell differentiation in the lung (23
The present study provides cellular evidence that ciliated cells actively participate in repair of the bronchiolar epithelium after acute injury through rapid squamous metaplasia and redifferentiation into mature, columnar cell types. Thus, ciliated cells are capable of remarkable plasticity, undergoing dynamic changes in cell shape and gene expression during repair. These findings support previous electronmicroscopic studies demonstrating rapid spreading of ciliated cells after naphthalene exposure (15
). Taken together, repair of the bronchiolar epithelium after naphthalene share biological processes with repair of other tissue. Cell spreading/migration, redifferentiation, and proliferation play a critical role in wound healing of the skin, wherein keratinocytes migrate to denuded areas, and undergo cell shape changes that precede proliferation (36