PERSPECTIVE
Mesenchymal Regulation of Alveolar Repair in Pulmonary Fibrosis

Kenneth C. Fang

Cardiovascular Research Institute and Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, San Francisco

Induction of alveolar epithelial cell phenotypes via mesenchymal signaling regulates pneumocyte differentiation not only during lung development (1, 2) but also during the response to a myriad of injuries inciting fibrogenesis (3). Signals generated in the epithelium and the mesenchyme regulate critical functions of cells in both compartments reciprocally, with transduction mediated by soluble growth factors, membrane-anchored receptors, and extracellular matrix proteins (4). Such epithelial-mesenchymal interactions characterize remodeling in a variety of tissues, including airways and lung parenchyma (3, 5, 6). Transition from injury to the repair phase in alveolar remodeling requires epithelialization to regenerate pneumocyte populations and the epithelial component of the alveolar barrier. Ordered layering of type I and type II pneumocytes, basement membranes, and capillary endothelial cells establishes compartmentalization that normalizes communication among cells and extracellular matrix components in distinct epithelial and mesenchymal layers. Restoration of alveolar quiescence reverses ventilation-perfusion defects to equilibrate gas exchange. By contrast, unrelenting signaling between the compartments leads to continued invasion of fibroblasts and excessive deposition of matrix proteins, causing intra-alveolar fibrosis and the loss of surface area, which defines the pathophysiology of pulmonary fibrosis (3).

Epithelial cells differentiate into subpopulations, which reconstruct a protective barrier and regulate clearance of alveolar exudates. Type II cells differentiate from cuboidal progenitor cells at septal intersections into thin type I cells that line approximately 90% of the alveolar unit. Subpopulations of type II pneumocytes synthesize surfactants needed to reduce alveolar surface tension and also produce matrix constituents such as fibronectin, collagen IV, and proteoglycan, essential for remodeling. Other type II cells help resorb proteinaceous exudates by releasing fibrinolytic proteases or by upregulating expression of water and ion channels. Some type II pneumocytes also serve as stem cells to replenish the type II epithelial cell population itself (3). Failure of the alveolar epithelium to perform these functions results both in the unchecked migration of fibroblasts onto the provisional matrix and the continued matrix protein deposition, which propagate fibrogenesis. Premature demise of the epithelial cell population may result from apoptosis due to incessant injury or from apoptotic signals arising from the mesenchyme (7). Thus, epithelial dysfunction permits sequential flooding and increased surface tension in the alveolar space, apposition and fusion of exposed basement membranes, and irreversible collapse and fibrosisall of which diminish effective alveolar surface area (3).

Bleomycin induction of lung fibrosis in rodents is a preferred model because of a similarity of the resulting pathology with that in lung biopsy specimens from patients with interstitial pulmonary fibrosis (IPF) (8). In rodents, parenchymal inflammation and repair accelerates after intratracheal administration of bleomycin, which induces well characterized spatial and temporal responses. Necrosis of type I cells precedes acute alveolitis by two to three days, with intense interstitial inflammation evident by four to 12 days. Alveolar re-epithelialization, fibroblast proliferation, and matrix protein deposition follows, with a two-fold increase in collagen content after 21 days (8). Applications of this model to mice engineered to express loss- or gain-of-function phenotypes are providing new insights into the mechanisms of alveolar fibrogenesis. Use of lpr and gld mice lacking Fas and Fas ligand, respectively, demonstrated that the Fas pathway regulates alveolar epithelial cell apoptosis in pathways favoring fibrogenesis (9). Evidence of neovascularization in fibrotic lungs encouraged investigations of proangiogenic pathways. These studies identified regulatory roles for chemokines, such as interferon (IFN)--inducible protein (IP)-10 and macrophage inflammatory protein (MIP)-2 (10, 11). Other work reveals a critical relationship between integrins and growth factors, demonstrating interactions between integrin v6 and transforming growth factor (TGF)-1 latency-associated peptide in the activation of TGF-1; these studies have established that a null mutation for the integrin 6 subunit protects mice against bleomycin-induced lung fibrosis (12). Antagonization of signaling by TGF- occurs after expression of Smad 7, a member of the Smad family of proteins that transduces TGF- signals to the nucleus and attenuates lung fibrosis (13). Use of 6^/ mice and oligonucleotide arrays has also enabled characterization of the temporal expression of individual and clustered genes in the regulation of pulmonary inflammation and fibrosis induced by bleomycin (14).

These novel molecular and cellular interactions contribute to our understanding of epithelial and mesenchymal communication, essential for tissue remodeling during both development and wound repair. Reciprocal signaling, which governs differentiation of progenitor cells and morphogenesis of tissues during organ development, is also thought to play a role in processes regulating repair and remodeling of injured tissues (1, 2, 5). Epithelial and mesenchymal cells signal each other bidirectionally and dynamically, either by the release of soluble mediators in a paracrine fashion or by direct cellular contact. Epithelial cells release growth factors such as TGF- and platelet-derived growth factor (PDGF), both of which regulate fibroblast proliferation, migration, apoptosis, and matrix protein deposition. Fibroblasts influence epithelial cell function by releasing epidermal growth factor (EGF), keratinocyte growth factor (KGF), or hepatocyte growth factor (HGF), all of which regulate type II cell migration, proliferation, differentiation, deposition of matrix proteins, and production of urokinase-type plasminogen activator (uPA) (3, 4).

Direct communication between epithelial and mesenchymal cells is achieved via intercellular contacts between type II cells and fibroblasts. Type II cells may inhibit fibroblast growth by this direct physical interaction, and fibroblast proliferation may occur when epithelial injury interferes with cellular contact (15). This apparent requirement for an anatomical association of epithelial cells with fibroblasts has engendered the concept of an "attenuated fibroblast sheath," within which such contacts occur. These contacts are a subset of interactions taking place within the so-called "epithelial-mesenchymal trophic unit" (5, 6). Complex interactions in the contact region of the basement membrane flanked by epithelium and mesenchyme involve epithelial cells, fibroblasts, recruited inflammatory cells, neural tissues, and extracellular matrix functioning as a coordinated unit. The attenuated fibroblast sheath is a thin layer of a subpopulation of fibroblasts, under and in proximity to epithelial cells, which facilitate the exchange of signals to enable an appropriate response to various stimuli. While the in vivo role of the sheath is unclear, subpopulations of fibroblasts, such as myofibroblasts, are considered likely candidates to implement critical functions such as signaling and deposition of matrix (5, 6).

Recognizing that specialized epithelial-mesenchymal interactions may also direct alveolar repair during fibrogenesis, Terasaki and colleagues report in this issue of the AJRCMB that mesenchymal cell expression of epimorphin, a key regulator of lung epithelial cell morphogenesis (16), coincides with the process of re-epithelialization during the response to bleomycin-induced injury. Epimorphin is a member of a gene family, localized to human chromosome 7 (17), and also encodes cytoplasmic trafficking proteins known as syntaxins (16). During development, mesenchymal cells express epimorphin in different isoforms, with some anchored to the cell surface by a putative membrane-spanning region and others localized to the cytoplasm. Immunohistochemical studies using monoclonal antibody MC-1 localize epimorphin to stromal proteins and to mesenchymal cells adjacent to epithelial cells, suggesting the potential for interactions involving soluble factors or cell contacts. MC-1 antibody blocks embryonic tubular formation, ductal branching, and lumen formation, indicating that a specific epimorphin epitope is essential for epithelial morphogenesis (16). Parallel requirements for alveolar epithelialization in lung morphogenesis and parenchymal lung fibrosis provide the rationale for exploring epimorphin expression in murine models of bleomycin-induced fibrosis (18).

Bleomycin administration induces expression of MC-1 immunoreactive epimorphin protein in a temporal and spatial pattern that appears to coincide with the process of re-epithelialization in remodeling alveoli. Lung tissue of adult mice demonstrate constitutive, low-level expression of epimorphin restricted to the bronchiolar, alveolar, and vascular walls and remains unchanged in control animals. Thus, although initially reported to be a morphogen critical in development, epimorphin may also play a role in homeostasis in mature lungs. By contrast, expression increases in the same areas after bleomycin treatment, with increased detection of epimorphin in mesenchymal cells and regions demonstrating intra-alveolar fibrosis. Specifically, epimorphin expression increases in fibrotic regions and is localizable to vimentin-positive mesenchymal cells at three weeks, in the absence of the appearance of keratin-positive epithelial cells. By four weeks, expression of epimorphin protein and mRNA peaks, corresponding to invasion of intra-alveolar fibrotic regions by epithelial cells. In situ hyridization and immunohistochemical studies demonstrate restriction of increases in epimorphin mRNA and protein signals to mesenchymal cells in fibrotic lesions. Immunoelectron microscopy localizes epimorphin protein to the endoplasmic reticulum of mesenchymal cells, but also to basement membranes and collagen fibrils in fibrotic regions. Thus, Terasaki and coworkers suggest that expression of epimorphin may facilitate migration or proliferation of epithelial cells during the process of re-epithelialization in profibrotic lung injury.

Immunolocalization of epimorphin expression to vimentin-positive cells suggests that contacts between mesenchymal cells and pneumocytes may orchestrate the process of re-epithelialization. Immunodetection of epimorphin using MC-1 antibody requires recognition of a specific epitope exposed only in complexes of epimorphin protein. Whereas epimorphin cDNA predicts a 34-kD protein (16), analysis of native or recombinant protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) reveals an electrophoretic profile with products at 34, 70, and 150 kD. Polyclonal antibodies detect all forms, but MC-1 detects only the 150-kD form whose susceptibility to trypsin digestion prior to isolation from cells suggests its localization to the membrane. Thus, cells may express monomeric 34-kD epimorphin, which is detectable in the cytoplasm, or SDS-resistant complexes with only the cell surface-anchored 150-kD form being available to contact with other cells (22). The critical regulatory epitope of epithelial morphogenesis is a 19 residue (NGNRTSVDLRIRRTQHSVL) NL-peptide located in the central portion of 150-kD epimorphin (23). Thus, these data presented by Terasaki and colleagues suggest that cells expressing membrane-anchored epimorphin may establish contacts with epithelial cells during repair after bleomycin-induced lung fibrosis. The results emphasize the potential importance of cellular contacts in epithelial-mesenchymal interactions during fibrogenesis. However, the identity of the cells expressing epimorphin remains unclear. Vimentin is a component of intermediate filaments, which, together with actin microfilaments and microtubules, forms the multicomponent scaffold of intracellular architecture (24). A candidate epimorphin-expressing cell is the myofibroblast, a subtype of fibroblast with unclear origins, that is widely believed to play a role in fibrosis in many organs (25). Lung myofibroblasts express vimentin and -smooth muscle actin, but not desmin, and are thus classified as the VA phenotype, and lung myofibroblasts can also be detected by PR2D3 antibody in the subepithelium (25, 26). Thus, the authors' findings suggest the intriguing possibility that mesenchymal myofibroblasts express a membrane-anchored 150-kD complex of epimorphin, via which they establish direct contacts with pneumocytes during re-epithelialization associated with alveolar repair.

Molecular and cellular interactions regulating epimorphin expression and activity remain to be deciphered. Specificity of MC-1 antibody for the 150-kD surface isoform emphasizes that characterization of biochemical pathways that regulate complex formation from a 34-kD monomer and guarantee surface accessibility of the NL-peptide epitope is critical to understanding the regulation of epimorphin-dependent cell-cell interactions. Localization of MC-1 antibody immunoreactive protein to the extracellular matrix suggests that cell surface processing of the 150-kD isoform, perhaps via sheddases, may be an important regulatory pathway because the secreted 34-kD monomer or 70-kD complex would not be identified by MC-1 antibody. Investigations of mammary gland morphogenesis suggest that epimorphin provides directional information to cells, guiding the development of lumens, branches, or spheres, depending upon the extent of cellular exposure to epimorphin. Putative molecular mechanisms regulating epimorphin signaling include ligation of an as-yet-unidentified epimorphin receptor and binding to membrane-anchored or extracellular matrix-associated molecules (27). Epimorphin regulation of mammary epithelial cell morphogenesis demonstrates a potential adjunctive role for soluble mediators such as EGF, KGF, FGF, and HGF, which are already implicated in epithelial-mesenchymal interactions in pulmonary fibrosis and promote proliferation of epimorphin-expressing cells, but do not themselves function as morphogens (27). Epimorphin may also indirectly regulate matrix remodeling via induction of uPA or gelatinase A (matrix metalloproteinase [MMP]-2) (27). Further characterization of the mesenchymal cells responsible for epimorphin expression would provide more specific targets for additional study. In addition to VA myofibroblasts, vimentin-expressing mesenchymal cells include non-VA myofibroblasts and fibroblasts, dendritic cells, and mast cells (25, 28). As residents of the mesenchyme, mast cells establish close contacts and interact synergistically with fibroblasts and may act in lung fibrosis by contributing degranulated mediators (29, 30). Although deemed unnecessary for the propagation of fibrosis in previous studies using bleomycin in murine models (31), mast cells and their secreted products participate in physiologic processes such as angiogenesis, apoptosis, collagen synthesis, and MMP zymogen activation (32), thus suggesting the alternative possibility that they participate in normal tissue remodeling and perhaps block fibrogenesis. Thus, while bidirectional communication between the epithelium and mesenchyme may determine the global response of the alveolar unit to profibrogenic injuries, local cell-cell and cell-matrix interactions mediated by epimorphin may be similar to those governing lung morphogenesis and may perhaps ultimately determine whether injured lungs heal or scar.

View larger version (18K): [in this window] [in a new window]   Figure 1.   Epithelialization in alveolar repair. Proliferation and phenotypic differentiation accompany migration of type II epithelial cells to repair breaches in the alveolar barrier and establish contacts with mesenchymal cells. Bidirectional signaling between epithelial and mesenchymal cells involves cell-cell and cell-matrix interactions with the release of soluble mediators in a contact region known as the "epithelial-mesenchymal trophic unit." Subpopulations of fibroblasts in an "attenuated fibroblast sheath" may establish contacts with epithelial cells (5, 6). Local cell-cell interactions may also involve contacts between fibroblasts, myofibroblasts, dendritic cells, and mast cells resident in the mesenchyme.

	Footnotes

Abbreviations: idiopathic pulmonary fibrosis, IPF; transforming growth factor , TGF-; urinary-type plasminogen activator, uPA; vimentin -smooth muscle actin myofibroblasts, VA.

(Received in original form June 7, 2000).

Acknowledgments: This work is supported in part by Mentored Clinical Scientist Development Award HL-03345 from the National Institutes of Health and the Dalsemer Research Scholar Award from the American Lung Association. The author thanks George Caughey, Harold Chapman, and Paul Wolters for helpful comments regarding the manuscript.

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