Transforming growth factor-{beta} induces fibrosis in immune cell-depleted lungs

Mitchell A. Olman1,2 and Michael A. Matthay3

Departments of 1Medicine and 2Pathology, Division of Pulmonary and Critical Care Medicine, University of Alabama, Birmingham, Alabama 35294; and the 3Cardiovascular Research Institute, University of California at San Francisco, San Francisco, California 94145

THE STUDY by Xu and colleagues, one of the current articles in focus (Ref. 30a, see p. L527 in this issue), addresses the potential role of epithelial cell production of transforming growth factor (TGF)-{beta} on fibrosis using immune cell-depleted lung tissue slices. This question is important because activation of TGF-{beta} through interaction with the protease cleavage, binding to the matricellular protein thrombospondin-cell surface receptor CD36 complex, or the epithelial-specific integrin {alpha}v{beta}6 may result in temporally and spatially restricted TGF-{beta} to sites of injury. If epithelial-specific activation pathways are found to be dominant, they could be modulated with therapeutic intent.

Fibroblast epithelial cell interactions in fibrosis. The in vivo and in vitro studies of Adamson et al. (2, 3) and others reveal a complex interdependence of fibroblast and epithelial cells during lung injury and repair. Epithelial cells can produce soluble factors that modulate fibroblast proliferation, migration, apoptosis, and matrix synthesis, including TGF-{beta} and platelet-derived growth factor (PDGF). In turn, fibroblast production of soluble factors, such a keratinocyte growth factor (KGF), hepatocyte growth factor, and epidermal growth factor, will mediate alveolar type II cell migration, proliferation, and phenotype. Direct fibroblast-epithelial cell-cell contacts occur in the lung, and these contacts may modulate the effects of soluble growth factors. For example, fibroblasts produce a soluble factor(s) that stimulates epithelial cell proliferation, KGF, but direct epithelial cell-fibroblast contacts, as noted in vivo, abrogates this effect (2, 33). The physical and biochemical interactions between epithelial cells and subjacent mesenchymal cells, whether they are fibroblasts or myofibroblasts, are bidirectional, dynamic, and reciprocal. The end result of their interaction depends on the coordinated signals of both soluble and matrix-bound factors as well as their individual and changing profile of cell surface receptors. These interactions have been aptly described as an "epithelial-mesenchymal trophic unit" (12). Resident and recruited cells, including dendritic cells, inflammatory cells, and endothelial cells, and the matrix itself, participate in the injury-repair process through modulation of dynamic epithelial-mesenchymal cell interactions in vivo.

Studies generally support a net inhibitory role for epithelial cells on fibroblast proliferation and collagen synthesis. For example, exposure of epithelial cells to silica particles in vitro induces the production of soluble mitogenic inhibitors of fibroblasts and inhibitor of collagen synthesis that is indomethacin inhibitable (14). Recent work reveals that an aspirin-sensitive (perhaps PGE2), fibroblast antiproliferative factor is produced in greater quantities in alveolar epithelial cells from fibrosis-resistant mice deficient in chemokine receptor CCR2 (19). Furthermore, epithelial cell injury/activation modifies their effects on fibroblasts. For example, the antiproliferative activity of freshly isolated epithelial cells is not observed when epithelial cells from late stage fibrotic lungs were utilized (33). The mechanism of the reciprocal and dynamic interactions of epithelial and fibroblastic cells remains incompletely defined but clearly involves both soluble cell membrane and matrix-derived signals.

Model systems to study pulmonary fibrosis. A number of progressively more complex systems have been used in an attempt to mechanistically address the fibroproliferative effects of epithelial-fibroblast interactions. Transfer of conditioned media or fibroblast-epithelial cell monolayer coculture systems demonstrate that soluble factor(s) are produced by epithelial cells in response to cytokines/chemokines elevated in lung injury or physical factors such as changes in cell shape (19, 23). Evidence for the matrix-induced modulation of this interdependence is available. For example, alveolar type II cell proliferation was increased, and the phenotype switch was greater when cells were cultured on an endothelial cell-derived matrix compared with other substrates. This effect was enhanced as a consequence of fibroblast KGF production in a two-chamber coculture system (3). Acknowledgment of the importance of cell-matrix interactions and epithelial cell-fibroblast cell-cell contact has prompted studies using three-dimensional systems, including an epithelial monolayer grown on a three-dimensional layer of collagen-embedded fibroblasts. This system can provide biophysical cues to the fibroblast in a manner known to modulate its response to growth factors. However, the biochemical and physical organization of lung tissue-derived matrix, especially during lung injury/repair, is difficult to model in vitro.

Genetically altered mice offer an experimentally robust system to examine mechanisms of fibrosis in vivo through their inclusion of inflammatory cell components and their interaction with endothelium and plasma-derived factors (16). Work on the effect of overproduction or deletion of a given factor in epithelial cells, during a specific time of injury in vivo is progressing, but issues of redundant pathways may cloud the picture (27). In addition, deletion of lung injury-related growth factors may confer a embryonic lethal phenotype and/or have development-specific effects that may not be recapitulated in adults during injury and repair. In regard to TGF-{beta}, the TGF-{beta} and/or receptor/signaling molecule-deleted mice have severe phenotypic alterations and die in utero or shortly after birth. The study of lung injury and repair in conditional, cell type-specific, genetically altered mice will likely greatly contribute to our understanding of these processes.

Tissue explant models, such as those reported by Xu et al. (30a), place the epithelium and fibroblasts in their natural orientation, surrounded by their natural complement of matrix proteins in their native conformations along with matrix-bound regulatory molecules such as proteases and growth factors. Xu et al. cleverly utilize a rat lung tissue explant model that is depleted, but not devoid, of inflammatory cells. This model develops histopathological changes similar to human idiopathic pulmonary fibrosis when the epithelial cells are induced to overexpress a constitutively active form of TGF-{beta}. Histopathological alterations included not only type II cell hyperplasia but fibroblastic bud-like lesions along with an increase in interstitial collagens.

The solid rationale for this work is based on the appearance of epithelial cell and subepithelial matrix-localized TGF-{beta} in areas of advanced fibrosis and in the presence of increased inactive and biologically active TGF-{beta} in the bronchoalveolar lavage of patients with pulmonary fibrosis. Furthermore, both abrogation of fibrosis through administration of TGF-{beta}-neutralizing antibodies to bleomycin-injured animals and the induction of fibrosis in mice using adenovirally mediated overexpression of TGF-{beta} demonstrate the critical importance of active TGF-{beta} in the fibrotic process (7, 11, 28). What is uncertain, however, is the relative contribution of individual cellular components to the total active TGF-{beta}, the mechanism of its activation in vivo, and the mechanism(s) of its in vivo profibrotic effect. The ability to both measure and control conditions over time with the pulmonary cells and matrix in their natural configurations is a substantial strength of this model system.

Rat lung tissue slice explants were partially depleted of inflammatory cells and cultured on agarose. The slices were treated with KGF, KGF/empty retroviral vector, or KGF/retroviral vector expressing a constitutively active form of TGF-{beta}. As expected, all KGF-treated explants demonstrated hyperplasia of epithelial cells, confirming previously described in vivo findings. In explants treated with KGF and the TGF-{beta}-expressing vector, several key features of fibrosis were noted. These include histopathological areas of fibrosis and increases in matrix proteins, including collagen (types I, II, IV, and V) and fibronectin, as well as evidence of an increase in TGF-{beta} cellular responses, including phosphorylation of Smad2, increases in connective tissue growth factor protein, and an increased number of myofibroblasts. Several of these changes were partly abrogated with antibodies to TGF-{beta} or a TGF-{beta} receptor II-competitive inhibitor, fetuin, demonstrating their specificity. Of further significance, the total TGF-{beta} in the overlying media was equally elevated in all groups; however, active TGF-{beta} (3% of the total) was only detected in the explants treated with KGF/active TGF-{beta}-expressing vector.

Retroviral vector integration efficiency is greatly enhanced in proliferating cells. The potent epithelial cell mitogen KGF was added to obtain impressive retroviral epithelial cell infection rates (%15). KGF levels are increased in patients with lung injury, and KGF administration induces alveolar cell hyperplasia but protects animals from the mortal and fibrotic effects of lung injury in response to a panoply of agents (30). A number of mechanisms have been proposed to explain KGF's beneficial in vivo effect that directly impacts on injury-repair processes involved in fibrogenesis. These include mitogenic effects on alveolar type II cells, induction of surfactant protein production, enhancement of cell spreading and motility, and alteration of protease release (including urokinase and matrix metalloproteases). KGF may also effect injury/repair through its ability to increase the epithelial cell's resistance to mechanical-, radiation-, and oxidant-induced injury, including resistance to apoptosis and enhanced DNA repair (30). Last, KGF modulates important autocrine/paracrine profibrotic cytokine expression, including PDGF-BB and TGF-{beta}, after bleomycin-induced lung injury. In the Xu et al. (30a) explant model, it is notable that KGF administration alone produced epithelial hyperplasia but not fibrosis. Perhaps more importantly, KGF administration did not abrogate the TGF-{beta} overexpression-induced fibrosis. This key finding does not identify precisely what types of epithelial cell-fibroblast interactions support or abrogate fibrogenesis. Because adenoviral transfection rates are high in nonproliferating epithelial cells, a similar study with adenovirally mediated overexpression of TGF-{beta} may help to sort out the potentially confounding effects of KGF on TGF-{beta}-induced lung injury/repair.

TGF-{beta} action in fibrogenesis. TGF-{beta} is a member of the superfamily of genes that also includes bone morphogenetic protein 2/4, activin, and several Drosophila and Caenorhabditis elegans homologs that play important roles in embryonic patterning, organogenesis, immune system regulation, and tissue homeostasis (36, 38). The three isoforms of TGF-{beta} have largely overlapping functions. TGF-{beta} is produced in latent form by virtue of its noncovalent association with latency-associated peptide (LAP). Its secretion is enhanced though covalent association of latent TGF-{beta} with a latency TGF-{beta}-binding protein (LTBP) family member. LTBP is directly relevant to matrix remodeling since LTBP will target latent TGF-{beta} to the extracellular matrix of fibroblasts (29). Furthermore, release of matrix-bound latent TGF-{beta} is accomplished through proteolytic cleavage by proteases involved in lung injury, including elastase and plasmin. In fact, mice lacking neutrophil elastase have a reduced fibrotic response to bleomycin, possibly due to cleavage of LTBP and loss of matrix-bound TGF-{beta} (9).

Cell types of all lineages express TGF-{beta} and its signaling receptors, but the response to TGF-{beta} varies with the cell type, its state of differentiation, and the presence or absence of other growth factors and cytokines. TGF-{beta} will inhibit growth of endothelial and epithelial cells while promoting growth and differentiation of connective tissue cells. Fibroblasts respond to TGF-{beta} through induction of genes encoding for matrix proteins, protease inhibitors that block matrix-degrading proteases, and by differentiation into contractile, smooth muscle actin-rich myofibroblasts, while its effects on proliferation are variable (34).

Binding of TGF-{beta} to its type II receptor induces phosphorylation of the type I receptor by type II receptors, resulting in the initiation of downstream signaling. Both receptors are expressed on many cell types present in normal and diseased human lungs. Type I receptors phosphorylate the R-Smads (largely Smads 2 and 3), which then form a heteromeric complex with a co-Smad (common Smad4) in the cytoplasm, and the complex translocates into the nucleus (5, 36). Smads 6 and 7 are inhibitory. The role of modulation of TGF-{beta} signaling at the receptor and Smad levels in patients is largely unknown. However, areas of chronic fibrosis in patients with idiopathic pulmonary fibrosis contained cells with relatively fewer type I receptors (13a). Furthermore, adenovirally mediated overexpression of Smad7 will ameliorate bleomycin-induced pulmonary fibrosis in mice (22).

The transcription factor activator protein-1 (AP-1) is thought to play an important role in controlling the expression of many TGF-{beta}-responsive genes, including matrix proteins and proteases/protease inhibitors. Cooperation between Smads and AP-1 may participate in fibrogenic regulation through cellular signals generated on ligation of growth factor receptors and integrins. A number of other transcription factors have similarly been shown to affect TGF-{beta} signaling of transcriptional activity, including cAMP response element binding protein, SP-1, AP-1, TFE3, and p300/CBP, as well as the TGF-{beta}-inducible interaction of Smad2 with the homeobox protein TGIF (36, 38).

TGF-{beta} activation. TGF-{beta} action can be regulated at the level of its expression, its activation, its receptors, and its intracellular signaling in an interactive manner. Evidence for the importance of the activation step in pulmonary fibrosis is available. Adenovirally mediated overexpression of wild-type TGF-{beta} in rat lungs resulted in a transient mononuclear infiltrate (28). In contrast, transient overexpression, a constitutively active form of TGF-{beta} (cysteine 223/225 in LAP-mutated to serines), resulted in prolonged and severe interstitial fibrosis (28). The use of an identically mutated TGF-{beta} by Xu et al. (30a) is well conceived. Nonetheless, in this study, biologically active TGF-{beta} represented only 2-3% of the total TGF-{beta} in the media overlying the transfected explants of all conditions. This is roughly the same as measured with adenovirally mediated expression of constitutively active TGF-{beta} expression in murine lungs in vivo (28). Although it is possible that the media levels of active TGF-{beta} do not reflect that seen by the tissue cells as a consequence of binding to plastic, degradation, or cell surface-localized activation, these observations provide further support for the importance of the activation of TGF-{beta} to the biological response.

In vitro, the active conformation of TGF-{beta} is generated through exposure to acidic conditions, through proteolytic cleavage [e.g., by plasmin, calpain, transglutaminase, matrix metalloproteinases (MMP)-2 and -9], interaction with extracellular matrix proteins, including thrombospondin, acidification of cellular microenvironments, reactive oxygen species, and treatment with retinoids, glucocorticoids, and vitamin D (8, 13, 20, 21, 32, 36). Another modulatory step with potential significance to TGF-{beta} activity in human lung injury is its binding to other provisional matrix proteins and/or soluble proteins through LTBPs that concentrate at sites of lung inflammation. Whether LTBP-matrix interactions act to sequester TGF-{beta} or whether they act to form a potentially matrix-bound useable pool of TGF-{beta} in vivo during the repair process is an important question. The predominant mechanism of in vivo activation of TGF-{beta} during human lung injury remains unknown.

The epithelial cell integrin, {alpha}v{beta}6, is a ligand for latent TGF-{beta}, and this integrin can participate in epithelial cell-specific, spatially restricted TGF-{beta} activation (20). Furthermore, mice lacking the {beta}6-integrin demonstrate a reduced pulmonary fibrotic response to bleomycin (20). These mice are also protected from the TGF-{beta}-dependent early (5 days) pulmonary edema in bleomycin and bacterial endotoxin models of lung injury (25). The de-adhesive protein thrombospondin is a prominent component of remodeling granulation tissue matrix. Thrombospondin-1 (TSP-1) binds to latent TGF-{beta} and participates in plasmin and CD36-dependent TGF-{beta} activation on macrophage cell surfaces. Treatment of TSP-1 null mice with a TSP-derived peptide activates TGF-{beta} and normalizes the phenotype (8, 13, 21, 32). The role of TSP-1 in lung injury-related TGF-{beta} activation remains to be determined, although peptide homologs of the TSP-1 receptor CD36 will ameliorate bleomycin-induced pulmonary fibrosis in rats (31). In support of protease-dependent TGF-{beta} activation is the observation that IL-13-overexpressing mice develop pulmonary fibrosis with high levels of activated TGF-{beta} that is inhibitable with aprotinin (serine protease inhibitor) and reduced in MMP-9 knockout mice (16). TGF-{beta} is known to interact with matrix components, including the matrix proteoglycan decorin. Decorin is a 100-kDa proteoglycan with two binding sites for TGF-{beta} that localize to sites of inflammation and granulation tissue remodeling in lung injury (4). Adenovirally mediated decorin gene transfer, as well as twice weekly intratracheal instillations of decorin, ameliorates bleomycin-induced hydroxypro-line accumulation and neutrophil recruitment in mice and hamsters (10, 15).

Last, it is notable that fibrosis develops in the relative absence of a number of plasma-derived molecules that can modulate TGF-{beta} action, including {alpha}2-macroglobulin, TGF-{beta} and PDGF from platelets, and plasminogen/plasminogen activator inhibitor-1 (6, 24). However, it is possible that some plasma-derived factors are replaced by lung cell-produced proteins in the explant.

TGF-{beta} effects on inflammatory cells. In the ex vivo explant model reported by Xu et al. (30a), it is clear that no additional inflammatory cells are recruited to the lung upon explant culturing. However, despite solid effort, it is likely that resident interstitial inflammatory cells, including T cells, dendritic cells, and macrophages, as well as a fraction of alveolar macrophages, remain in the explant. While Xu et al. show that these cells are unlikely to be transfected with retroviral vectors and, therefore, do not overexpress TGF-{beta}, it is possible that they contribute to the overall tissue response to TGF-{beta} through production of TGF-{beta}-responsive, fibrosis-modulating factors.

There is ample evidence that TGF-{beta} is an important immunomodulatory molecule. The effects of TGF-{beta} on immune cells are dependent on the cell type, its state of differentiation, and on other cytokines present in the microenvironment (17). Thus not only is the activation of TGF-{beta} critical but the response of the cells to active TGF-{beta} is highly dependent on the overall context. This concept underscores the importance of the model by Xu and colleagues (30a). Although no fibroproliferative changes were noted in the empty vector/insulin/KGF/hydrocortisone-treated lung slices, the issue of interactive effects of TGF-{beta} and the KGF/insulin/hydrocortisone used in the model is a difficult one to resolve experimentally. This model could be used to ferret out the interactive and contextual pathways in a system with a clinically and biologically relevant end point.

The cytokine milieu influences the response, as TGF-{beta} pathways cross talk with other cytokines/chemokines/growth factors relevant to the fibroproliferative response. For example, the T helper type 1 cytokine, interferon-{gamma} (IFN-{gamma}), is antifibrogenic in numerous experimental systems, and administration of IFN-{gamma} shows promise in early clinical trials in human chronic pulmonary fibrosis (35, 37). IFN-{gamma}, among other things, inhibits TGF-{beta} signaling through the STAT-induced transcription of the inhibitory Smad7, an inhibitor of R-Smad phosphorylation (34, 38). Both TNF-{alpha} and TGF-{beta} neutralization can ameliorate bleomycin-induced pulmonary fibrosis, and TNF-{alpha} is a prominent cytokine that is increased and partially active in early lung injury (26). Overexpression of active TGF-{beta} in mice lacking the TNF receptors leads to pulmonary fibrosis, indicating that TGF-{beta} is either redundant or downstream, but not upstream, of TNF-{alpha} in the fibrogenic process (18). The use of an in vitro model system from genetically altered mice, from mice with the full complement of inflammatory cells, and/or inflammatory cell-depleted or null mice will likely lead to the discovery of novel and unexpected mechanisms for modulating TGF-{beta} activity in vivo.

A number of unanswered questions remain. For example, What is the role of epithelial cell-fibroblast contacts and the role of the extracellular matrix on modulation of cell-cell cross talk during injury and repair? What is the relative significance of the many individual events involved in the activation of TGF-{beta}? Are the TGF-{beta} activation steps cell type dependent in vivo? How is TGF-{beta} activated in human acute lung injury or idiopathic pulmonary fibrosis? What aspects of the cytokine milieu condition the response to TGF-{beta} in vivo? The ability to both measure and control conditions over time with the pulmonary cells and matrix in their natural configurations is a strength of the explant model system. Future additional mechanistic studies using this model system should provide new insights.


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This work was supported by grants from the Veterans Administration MERIT Review and National Heart, Lung, and Blood Institute Grants HL-58655 (to M. A. Olman) and HL-51854 and HL-51856 (to M. A. Matthay).


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Address for reprint requests and other correspondence: M. A. Olman, Division of Pulmonary and Critical Care Medicine, Dept. of Medicine, Univ. of Alabama at Birmingham Medical Center, 1900 University Blvd., 215 THT, Birmingham, AL 35294 (E-mail: Olman{at}uab.edu).


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