MINIREVIEW
The Attenuated Fibroblast Sheath of the Respiratory Tract Epithelial-Mesenchymal Trophic Unit

Michael J. Evans, Laura S. Van Winkle, Michelle V. Fanucchi, and Charles G. Plopper

School of Veterinary Medicine, Department of Anatomy, Physiology and Cell Biology; and the Center for Comparative Respiratory Biology and Medicine, University of California, Davis, California

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Interactions between epithelial, mesenchymal, and neural tissue and also the extracellular matrix are necessary to initiate numerous cellular functions of the lung (1). The most common of these functions include differentiation during lung growth, repair of damaged tissue, and regulation of the inflammatory response. Each of these processes requires a localized response to a specific stimulus. Fibroblasts, especially those in close proximity to the airway epithelium, are likely regulators of local responses. In a recent commentary, Smith and colleagues discussed the possibility that resident fibroblasts may act as sentinel cells for these responses (2). In addition to their role as connective tissue cells, fibroblasts also produce cytokines and chemokines in response to various stimuli. Their fixed position in the tissue suggests that they can respond in a local manner to bacterial products, tissue injury, or other environmental factors. The relationship between cytokines and inflammatory cells in asthmatic airways also indicates a similar role for fibroblasts. In the asthmatic lung, the fibroblast plays a key role as a resident mesenchymal cell beneath the epithelium, receiving and sending information to epithelial and inflammatory cells (3, 4). Additionally, these fibroblasts are thought to be responsible for the subepithelial fibrosis associated with asthma. The significance of resident fibroblasts in the airway during inflamation has been described; however, the concept of an anatomically distinct group of fibroblasts associated with airway epithelium has not been explored.

In 1990, Brewster and associates described a layer of subepithelial fibroblasts in the bronchi of normal and asthmatic human subjects that were positioned to allow close interaction with the epithelium, neural tissues, and extracellular matrix (5). The population of cells was shown to comprise fibroblasts and myofibroblasts, and individual cells were reported to be as large as 100 µm in diameter. A detailed description of the subepithelial layer of resident fibroblasts in the rat trachea was reported by Evans and coworkers (6). In tissue sections, the cells appear as a layer of attenuated cell processes closely opposed to the lamina reticularis of the basement membrane zone, about 1.9 µm beneath the epithelial basal lamina. The cells are intermeshed with each other, about 40 µm in diameter with the attenuated portions about 0.55 µm in thickness. The cell is thicker near the nucleus and contains abundant rough endoplasmic reticulum near the nucleus. There were no apparent bundles of microfilaments in the thin or thick portions of the cell as there are in myofibroblasts. The cells were determined to be stellate in shape. They exist as a layer of large, flat cells covering about 70% of the interstitial surface of the lamina reticularis, and make numerous contacts with the lamina densa of the basement membrane zone (approximately 7,000 times per mm²). This layer of thin mesenchymal cells was named the attenuated fibroblast sheath (6), with properties of a layer of similar cells in the gut identified as the pericryptal fibroblast sheath (7). On the basis of the data from this previous paper (6), we constructed a three-dimensional model of the attenuated fibroblast sheath (Figure 1). The total attenuated fibroblast sheath, with its large surface area and close proximity to the epithelial/environmental interface, defines an anatomic unit of resident fibroblasts that could respond in a local manner to various stimuli. In this role, the attenuated fibroblast sheath represents the mesenchymal component of interactions with the epithelium, extracellular matrix, neural tissues, and migratory cells of the inflammatory response. The anatomic and functional relationship between the attenuated fibroblast sheath, epithelial and neural tissue, and also the extracellular matrix appears to serve as an epithelial-mesenchymal trophic unit. The epithelial-mesenchymal trophic unit would allow local exchange of information between the different tissue elements in response to various stimuli. This concept is similar to that of other investigators, with the exception that it recognizes that the subepithelial fibroblasts exist as a specific layer of resident fibroblasts beneath the epithelium instead of being randomly distributed in the lamina propria (2).

View larger version (107K): [in this window] [in a new window]   Figure 1.   The attenuated fibroblast sheath consists of a layer of mesenchymal cells (Mesenchymal Cell Layer) opposing a layer of epithelial cells (Epithelial Cell Layer). The area between the cell layers is termed the Basement Membrane Zone. Nerves and migratory inflammatory cells may interact with both the epithelial and mesenchymal cell layers. The large surface area occupied by the attenuated fibroblast sheath (approximately 70%), and its close proximity to the epithelium, indicate that it represents the mesenchymal component of epithelial-mesenchymal interactions in the airways. This illustration was constructed from morphometric data obtained from transverse and longitudinally sectioned rat trachea described previously (6).

Of the tissues in the epithelial-mesenchymal trophic unit, the least is known about the attenuated fibroblast sheath. Examples of the attenuated fibroblast sheath can be seen in most published electron microscope studies of the upper respiratory tract as a thin layer of attenuated cell processes immediately beneath the epithelium. Present in all animal species examined to date, the attenuated fibroblast sheath extends from the proximal to the distal regions of the conducting airways (5, 6, 8). Conceptually, it continues into the gas exchange region as interstitial fibroblasts in the alveolar walls (9). The main difference between fibroblasts of the attenuated fibroblast sheath and other fibroblasts is that those in the attenuated fibroblast sheath are flat, lying against the lamina reticularis of the basement membrane, as opposed to the more fusiform shape of fibroblasts in the lamina propria.

Most morphologic studies recognize fibroblasts and myofibroblasts surrounding the airways. However, there is considerable evidence indicating that fibroblasts in the lung are a heterogeneous population of cells, capable of changing phenotype much as lymphocytes can change phenotype. For example, there is evidence that both inflammatory responses and lung development are regulated by fibroblast subpopulations that are both functionally and spatially distinct. The onset of lung fibrosis is controlled by two subsets of fibroblasts. One subset controls the inflammatory response that precedes lung fibrosis, whereas the other subset controls hyperplasia and the production of extracellular matrix (10). Lung development is also controlled by at least two subsets of fibroblasts. Fibroblasts that are spatially close to the epithelium produce differentiation factors, whereas more distant fibroblasts produce factors that stimulate proliferation (11). The fibroblasts of the attenuated fibroblast sheath lie flat against the basement membrane zone, creating a layer of cells beneath the epithelium. They represent a morphologically distinct population of fibroblasts compared with those fibroblasts deeper in the extracellular matrix.

An important aspect of the attenuated fibroblast sheath is that the fibroblasts maintain a constant area relationship with the basement membrane zone. How the attenuated fibroblasts determine their position near the basement membrane zone is not known, but neuropeptides, epithelial cells, and extracellular matrix proteins can affect the position of fibroblasts in vitro and possibly also in vivo (12). Maintenance of a flattened or spreading morphology, as well as a defined spatial relationship with each other, suggests the cells are anchored in place. The spatial relationship that exists between cells of the attenuated fibroblast sheath also implies there is a mechanism for communication between them. Presumably there is some local communication by way of soluble mediators. In addition, attachment of cells to the matrix and to each other is also a means of cellular communication. Adhesion plaque formation with the matrix serves as a means for signal transduction between the matrix and the cell, and adhesion plaques between cells are a means of cell-cell signal transduction (15, 16). It is also possible that there is direct signaling between attenuated fibroblasts via gap junctions, as there is between pericryptal fibroblasts in the gut (17). Adhesion plaques and gap junctions may prove to be very significant means of communication because conceptually, the attenuated fibroblast sheath is an anatomic unit of mesenchymal cells that is continuous throughout the interstitial space of the entire lung (9).

Very little is known about how the attenuated fibroblast sheath functions in vivo. One key role for the peribronchiolar fibroblast is its transformation into myofibroblasts as well as proliferation in the lungs of asthmatics. In asthmatic subjects there is an increase in the number of subepithelial myofibroblasts compared with nonasthmatic control subjects (5). Subepithelial myofibroblasts have been shown to be increased in response to allergen (18). Myofibroblasts differ primarily from fibroblasts by containing -smooth-muscle actin microfilaments arranged in bundles and having an indented nucleus. It is thought that most new myofibroblasts are derived from fibroblasts; however, some could be derived from smooth-muscle cells (19). The attenuated fibroblast sheath is clearly involved in the airway remodeling of the entire epithelial-mesenchymal trophic unit that is characteristic of asthma, although its role in this process in vivo remains to be elucidated.

In vitro studies of fibroblasts isolated from the attenuated fibroblast sheath were carried out by Zhang and colleagues (20). Using special techniques, they isolated and cultured cells, which they considered to be myofibroblasts, from the basement membrane region (lamina reticularis) of human bronchial biopsy samples. These cells produced granulocyte macrophage colony-stimulating factor, interleukin-8, and stem-cell factor in response to tumor necrosis factor-. These investigators concluded that subepithelial myofibroblasts were involved in the regulation of inflammatory cell recruitment and activation by interaction with the cytokine network in the bronchial mucosa. Similar fibroblast cell cultures also enhanced the survival of eosinophils by inhibiting apoptosis (21). In further studies, cells isolated in the same manner were grown on collagen gels (22), and a human bronchial epithelial cell line was plated on the surface of the gel, forming a coculture of epithelial cells and fibroblasts. These three-dimensional cocultures of epithelial and mesenchymal cells in collagen gels resemble portions of the epithelial-mesenchymal trophic unit that we describe here for the airways in vivo. After injury to the epithelial cells in vitro, it was found that growth factors were secreted by bronchial epithelial cells that controlled myofibroblast proliferation. Other studies using bronchiolar explants from mice have shown that when the airways are cultured as a tube, keeping the microenvironment intact, the epithelium can recapitulate bronchiolar repair in vitro (23). These studies underscore the close interaction that exists between the components of the epithelial-mesenchymal trophic unit.

The attenuated fibroblasts in the sheath may also be members of a functionally related family of subepithelial mesenchymal cells termed juxtaparenchymal cells (17). A similar layer of subepithelial mesenchymal cells is present in the gut between the enterocytes and the lamina propria. This layer of cells was originally named the pericryptal fibroblast sheath (7, 24). These cells have since been shown to be subepithelial myofibroblasts. Other members of the juxtaparenchymal family of cells identified by Valentich and Powell include renal and pulmonary alveolar interstitial cells, vascular pericytes, breast and endometrial stromal cells, hepatic Ito cells, and orbital, synovial, and granuloma fibroblasts (17). Little is known about how these different cells function as juxtaparenchymal cells; however, Valentich and Powell speculated that they may mediate interactions between parenchymal cells and soluble mediators of the immune, endocrine, and neural responses of the various tissue compartments in which they reside (17).

In summary, the epithelial-mesenchymal trophic unit consists of opposing layers of epithelial and mesenchymal cells. The area between these two cell layers, the basement membrane zone, contains extracellular matrix and a network of nerve fibers. Recognition of the attenuated fibroblast sheath as a distinct layer of resident fibroblasts is key to the concept of an epithelial-mesenchymal trophic unit. The fixed position of the attenuated fibroblast sheath beneath the epithelium indicates that it responds in a local manner to bacterial products, tissue injury, or other environmental factors as they impinge on the epithelium and mediate the response of other tissues to these stimuli. However, the mechanisms by which the epithelial-mesenchymal trophic unit actually functions in vivo have only recently begun to be studied.

	Footnotes

Address correspondence to: Michael J. Evans, Ph.D., Dept. of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616.

(Received in original form May 14, 1999 and in revised form July 9, 1999).

Acknowledgments: This work was supported by NIH grants ES00628, ES04311, ES06700, RR00169, and ES05707; the American Lung Association; and funds from the State of California Tobacco-Related Disease Research Program (6KT-0306) and the UCD Health System Research Program.

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