PERSPECTIVE
Clara Cells and Clara Cell 10 kD Protein (CC10)

Gurmukh Singh and Sikandar L. Katyal

Department of Pathology, Veterans Affairs Medical Center, Pittsburgh, Pennsylvania

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Clara cells are nonciliated secretory epithelial cells lining the pulmonary airways, distinct from mucous and serous secretory cells in morphology and their secretory products. These cells were first recognized as a distinct cell type based on morphology and histochemistry in 1881 by Kölliker (1). Max Clara described what appears to be the same cell type in human lungs in 1937 and has the honor of having the cells named for him (2; recent literature also cited in 2). It was surmised, on the basis of the presence of granules in the cells, that the cells were of the exocrine secretory type; however, the nature of the secretions was not identified. Kuhn demonstrated that the granules in Clara cells consisted of protein and thus recognized them as distinct from mucous secretory cells (3).

In 1967, Niden suggested that Clara cells secrete pulmonary surfactant and that the lamellar bodies seen in alveolar type II pneumocytes represented phagocytized surfactant (4). This postulate, though later proven to be incorrect, raised the standing of Clara cells in the biomedical community. Kuhn, in his continuing investigation of pulmonary cells, showed that surfactant lipids were synthesized by type II pneumocytes and that Clara cells were not secreting surfactant. However, it may be that Clara cells are not entirely uninvolved in pulmonary surfactant biology considering that three of the surfactant related proteins, namely, surfactant proteins A, B, and D, are synthesized by Clara cells and that Clara cell 10 kD protein (CC10) binds surfactant lipids.

Initial studies on Clara cells were centered primarily around their morphology and included discussion of mechanism of secretion based on histologic observations. There was a contested suggestion that Clara cells released their secretory granules in a merocrine fashion by shedding the apical part of the cells. Similarly, even the regulation of secretion was studied by morphometric determination of the volume density of granules in Clara cells. In a set of elegant experiments, Massaro demonstrated that Clara cell secretory process responded to adrenergic stimulation (2). Even the cholinergic secretogogue activity seen in intact animals was attributed to indirect sympathetic stimulation. In an extension of these investigations, Plopper conducted morphometric studies designed to quantitate the intracellular content of Clara cell 10 kD protein. These studies seem to indicate that the bulk of the Clara cell secretory activity is probably constitutive with minimal effect of secretogoges.

In an extensive study of the morphology of Clara cells in a number of species, Plopper published the variability in the ultrastructure of the Clara cells among species as well as differences in the distribution of Clara cells along the airways in a given species. The seminal paper of Clara described a unique population of cells in the epithelial lining of the distal airways. However, morphologically similar cells are present in the airways extending from the trachea to the respiratory bronchioles in at least some of the species. There are variations in the intracellular organelles among species, mainly in the content of rough versus smooth endoplasmic reticulum. Even at the light microscopic level, there is variation in the morphology of Clara cells among species as well as at different levels of the airways in the same species. The non-ciliated, non-mucous secretory cells with club-shaped cytoplasmic projection above the surrounding ciliated cells perhaps represent only the classic but a minority version of the cell. A population of cuboidal, non-ciliated non-mucous secretory cells in the terminal and respiratory bronchioles in human lungs, described by Bassett and colleagues, appears to represent a transitional cell population with characteristics of both airway and alveolar cells (5).

Despite extensive morphologic studies, the role of Clara cells in lung biology remains elusive. Not even a single definitive and exclusive, physiologic role has been ascribed to these cells. The best evidence for a function for these cells points to a role in repair of bronchial epithelium. A substantive investigation was carried out by Brody and associates in using isolated Clara cells (6). The isolated cells were used to seed denuded trachea that were implanted in nude mice and restoration of the epithelium was examined sequentially. Isolated Clara cells, while not 100% pure, led to the seeding and development of a more or less normal epithelium in the denuded trachea, suggesting that Clara cells could play the role of a progenitor cell for the respiratory epithelium in injured lungs. Their likely role as progenitor or reserve cell for other epithelial cells is bolstered by the studies of Plopper, Stripp, and Stahlman. In experimentally induced epithelial damage to the airways, the Clara cells lead the recovery of epithelium. The time course studies on epithelial recovery suggest that, at a minimum, ciliated cells develop from Clara cells. It is possible that Clara cells or transitional cells at the alveolar-bronchial interface are also involved in the repair of alveolar epithelium. This is suggested by the distal migration of Clara cells in the lungs of animals exposed to ozone and by the frequent observation of "bronchiolization" of alveoli in lungs with chronic injury. The role of basal cells as the definitive progenitor cells has not been excluded. Studies of the developing lung support the role of Clara cells in the development and, indirectly, repair of the airway epithelium. Clara cells appear to play an inductive role in the development and maturation of airway epithelium (7).

The role of Clara cells in pulmonary pathology is no better defined than that in physiologic states. Most of the studies addressing Clara cells in pathologic states focus on pulmonary tumors. Clara cells have been described as the cells of origin of some of the pulmonary tumors in experimental animals and humans, based primarily on morphologic observations. An extensive study of the subject by Rehm showed that in rats and mice virtually all of the naturally occurring as well as experimentally induced tumors exhibit markers of type II pneumocytes (8). Human tumors express multiple cell markers in the same tumor and even in a given cell in a tumor, making it difficult for the observer to assign a specific cell of origin for the tumor solely on the basis of cell-type specific markers.

A Clara cell secretory product, uteroglobin/blastokinin, in rabbits and related species was identified to be the same as a low molecular weight protein secreted by the gravid endometrium. Most of the studies about the function of uteroglobin addressed its role in reproductive functions even though its expression in rabbit lung was identified early in the investigation. Rabbit uteroglobin binds progesterone and the uteroglobin gene is regulated by progesterone. However, the physiologic relevance of these findings is uncertain in view of the observed variability of the progesterone binding activity among different species, e.g., rabbit, hare and pica; and failure to detect progesterone bound to the native protein purified from the uterine secretions (9).

In an exploratory study of the proteins in lung lavage, Singh and Katyal identified a 10 kD protein that is primarily expressed by bronchial Clara cells in rodents and humans (10). The protein is structurally similar to rabbit uteroglobin and the two proteins appear to be closely related. The protein is referred to in the literature by various names, e.g., uteroglobin, Clara cell secretory protein (CCSP), Clara cell 16 kD protein, Clara cell 10 kD protein (CC10), human protein 1, urine protein 1, and polychlorinated biphenyl-binding protein. Human Clara cell 10 kD protein does not bind progesterone to any appreciable extent, but like rabbit uteroglobin is an inhibitor of phospholipase A₂. Examination of other properties of CC10, e.g., protease inhibition, and binding to other steroid hormones were not contributory. CC10 binds polychlorinated biphenyls (PCB), as shown by Anderson and associates, and this observation may explain chronic pulmonary disease in PCB-exposed subjects but does not provide any clues to the physiologic importance of the binding (11). X-ray diffraction studies of crystallized CC10 revealed a hydrophobic pocket in the dimer of the protein, similar to that observed in uteroglobin. The pocket may be naturally occupied by a lipid, since phosphatidylcholine and phosphatidylinositol are present in the purified preparation of human CC10. Crystallographic data and modeling of the moiety in the pocket of the protein were consistent with phosphatidylcholine and phosphatidylinositol occupying the hydrophobic pocket of the normal protein in native state (12).

The human CC10 gene has been localized to chromosome 11, p12-q13, a region occupied by other genes involved in regulation of inflammation (13). The sum of findings, i.e., the inhibition of phospholipase A₂ by CC10, the binding of phospholipase A₂ substrate (phosphatidylcholine) by CC10, and the location of the CC10 gene in the proximity of other genes involved in the regulation of inflammation, suggests that CC10 functions as a regulator of inflammation in the lung. Empirical evidence gathered by Mantile and colleagues also points to an anti-inflammatory role for CC10 (14). There is an obvious need to curtail unwarranted inflammatory response in the lung, as elsewhere. However, the need may be greater in the lung due to the persistent exposure to noxious/infectious agents that may elicit an inflammatory response and destruction of lung architecture. Although the CC10 gene knockout mice develop to adulthood normally, their response to lung injury is compromised in keeping with the hypothesis that CC10 regulates inflammation in the lung. The CC10 deficient mice demonstrate an increased susceptibility to hyperoxic injury and an exaggerated inflammatory response (15).

Despite extensive characterization of the structure of CC10, there is only indirect evidence of its physiologic role in the lung. Because CC10 deficient mice reproduce normally and seem to live a healthy existence in a clean environment, it would appear that duplicate/redundant measures exist to make up for CC10 in deficient mice. While investigation of its role as a regulator of inflammation is appropriate, we should keep an open mind to its potential role in transporting small hydrophobic molecules and in the biology of pulmonary surfactant.

	Footnotes

Address correspondence to: Gurmukh Singh, M.D., Department of Pathology, Veterans Affairs Medical Center, Pittsburgh, PA 15240.

(Received in original form March 18, 1997 and in revised form April 8, 1997).

Acknowledgments: The writers are grateful to Dr. William E. Brown, Carnegie-Mellon University, Pittsburgh, PA and Dr. Martin Sax, VA Medical Center, Pittsburgh, PA for their invaluable contribution to the studies on CC10. They wish to gratefully acknowledge the support from the Pathology Education and Research Foundation and the Department of Veterans Affairs.

	References

Top Article References

1. Kölliker, A. 1881-1882. Zur Kenntniss des Baues der Lunge des Menschen. Verhandl Phys-Med. Ges. Würzburg. 16:1.

2. Plopper, C. G. 1997. Clara cells. In Lung Biology in Health and Disease, Vol. 100. J. A. McDonald, editor. Marcel Dekker, New York.

3. Kuhn, C., L. A. Callaway, and F. B. Askin. 1975. The formation of granules in the bronchiolar Clara cells of rats. II. Enzyme cytochemistry. J. Ultrastruc. Res. 53: 66-76 [Medline].

4. Niden, A. H.. 1967. Bronchiolar and large alveolar cell in pulmonary phospholipid metabolism. Science 158: 1323-1324 [Abstract/Free Full Text].

5. Bassett, F., J. Poirier, M. LeCrome, and J. Turiaf. 1971. Etude ultrastructurale de l'epithelium bronchiolaire humain. Z. Zellforsch. 116: 425-442 [Medline].

6. Brody, A. R., G. Hook, G. S. Cameron, A. M. Jetten, C. J. Butterick, and P. Nettesheim. 1987. The differentiation capacity of Clara cells isolated from the lungs of rabbits. Lab Invest. 57: 219-229 [Medline].

7. Khoor, A., M. E. Gray, G. Singh, and M. T. Stahlman. 1996. Ontogeny of Clara cell-specific protein and its mRNA: their association with neuroepithelial bodies in human fetal lung and in bronchopulmonary dysplasia. J. Histochem. Cytochem. 44: 1429-1438 [Abstract].

8. Rehm, S., J. M. Ward, A. A. W. Ten, Have-Opbroek, L. M. Anderson, G. Singh, S. L. Katyal, and J. M. Rice. 1988. Mouse papillary lung tumors transplacentally induced by N-Nitrosoethylurea originate from alveolar type II cells: a light microscopic, ultrastructural and immunohistochemical study. Cancer Res. 48: 148-160 [Abstract/Free Full Text].

9. Wolf, M., J. Klug, R. Hackenberg, M. Gessler, K. G. Grzeschik, M. Beato, and G. Suske. 1992. Human CC10, the homologue of rabbit uteroglobin: genomic cloning, chromosomal localization and expression in endometrial cell lines. Hum. Mol. Genet. 1: 371-378 [Abstract/Free Full Text].

10. Singh, G., and S. L. Katyal. 1991. Secretory proteins of Clara cells and type II cells. In Treatise on Pulmonary Toxicology, Vol. I. Richard A. Parent, editor. CRC Press, Baton Rouge, LA. 93-103.

11. Anderson, O., L. Nordlund-Moller, H. J. Barnes, and J. Lund. 1994. Heterologous expression of human uteroglobin/polychlorinated biphenyl-binding protein: determination of ligand binding parameters and mechanism of phospholipase A2 inhibition in vitro. J. Biol. Chem. 269: 19081-19087 [Abstract/Free Full Text].

12. Umland, T. C., S. Swaminathan, G. Singh, V. Warty, W. Furey, J. Pletcher, and M. Sax. 1994. Structure of a human Clara cell phospholipid-binding protein-ligand complex at 1.9 Å resolution. Nature Structural Biology 1: 538-545 [Medline].

13. Hay, J. G., C. Danel, C. S. Chu, and R. G. Crystal. 1995. Human CC10 gene expression in airway epithelium and subchromosomal locus suggest linkage to airway disease. Am. J. Physiol. 268: L565-L575 [Abstract/Free Full Text].

14. Mantile, G., L. Miele, E. Cordella-Miele, G. Singh, S. L. Katyal, and A. B. Mukherjee. 1993. Human Clara cell 10 kDa protein is counterpart of rabbit uteroglobin. J. Biol. Chem. 268: 20343-20351 [Abstract/Free Full Text].

15. Johnston, C. J., G. W. Mango, J. N. Finkelstein, and B. R. Stripp. 1997. Altered pulmonary response to hyperoxia in Clara cell secretory protein deficient mice. Am. J. Respir. Cell Mol. Biol. 17: 147-155 [Abstract/Free Full Text].