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Roles for Insulin-Like Growth Factor I and Transforming Growth Factor-ß in Fibrotic Lung Disease^*

^* From the Immunology Research Group, University of Calgary, Calgary, AB, Canada.

Correspondence to: Brent W. Winston, MD, FCCP, Departments of Medicine, Biochemistry and Molecular Biology, and Critical Care Medicine, University of Calgary, Health Sciences Center, Room 1843, 3330 Hospital Dr NW, Calgary, AB, Canada, T2N 4N1; e-mail: bwinston{at}ucalgary.ca

	Abstract

TOP Abstract Introduction IGF-I TGF-ß Divergent Roles of TGF... Conclusion References

 
Idiopathic pulmonary fibrosis (IPF) is a lung disease that is characterized by epithelial cell damage and areas of denuded basement membrane resulting in inflammation, fibroblast proliferation, excessive extracellular matrix (ECM) deposition, and remodeling of alveolar gas exchange units. The progressive loss of lung gas exchange units in patients with IPF leads to respiratory failure and eventually to death. While the etiology of this disease is unknown, for many years studies suggested that chronic inflammation was the underlying factor that caused fibroproliferation and structural alterations of the lung. Recent data show that fibroproliferation and fibrosis can occur independently of inflammation, suggesting that IPF is a disease caused by a mesenchymal, rather than an immune disorder. Mesenchymal growth factors, including transforming growth factor (TGF)-ß, insulin-like growth factor (IGF)-I, platelet-derived growth factor, connective tissue growth factor, fibroblast growth factors, and keratinocyte growth factors, as well as proinflammatory cytokines such as tumor necrosis factor- and interleukin-1ß, have been shown to be exaggerated in several fibrotic lung disorders including IPF, ARDS, sarcoidosis, and bronchopulmonary dysplasia, as well as pulmonary manifestations of systemic diseases such as rheumatoid arthritis or progressive systemic sclerosis (scleroderma). We argue that inflammation is required to initiate growth factor production and repair of the damaged alveolar epithelial lining in fibrotic lung diseases and that exaggerated TGF-ß production may be responsible for the fibrotic response seen in diseases such as IPF. We recognize the potential role of several growth factors in the fibroproliferative process in the lung, and in this brief report we focus on the possible roles of the growth factors IGF-I and TGF-ß in cell migration, proliferation, and ECM synthesis in patients with IPF.

Key Words: fibroproliferation • insulin-like growth factor-I • pulmonary fibrosis • transforming growth factor-ß

	Introduction

TOP Abstract Introduction IGF-I TGF-ß Divergent Roles of TGF... Conclusion References

 
Idiopathic pulmonary fibrosis (IPF) is one of a number of fibrotic lung diseases that is characterized by dyspnea, nonproductive cough, and interstitial infiltrates on radiographic or CT scan examination. Patients usually present in the fourth or fifth decade of life and show a steady decline in lung function that ultimately results in death. The etiology of IPF is unknown, although many studies have attempted to link risk factors such as smoking, viral infections, and genetic factors to the development of the disease.¹ IPF is characterized at the pathologic level with areas of denuded lung epithelium and exposed basement membrane overlying fibroblast foci with exaggerated extracellular matrix (ECM) deposition. The progressive proliferation of fibroblasts, and the exaggerated deposition and remodeling of ECM proteins leads ultimately to fibrosis and the failure of the alveolar units. Several growth factors have been implicated in the pathogenesis of IPF. These factors have been identified by virtue of their ability to stimulate fibroblast division and ECM production, as well as their presence in the lungs and lung fluids of patients or animals with fibrotic lung disease. These growth factors include transforming growth factor (TGF)-ß, insulin-like growth factor (IGF)-I, platelet-derived growth factor, connective tissue growth factor, and members of the fibroblast growth factor and keratinocyte growth factor families.^{2 3}

Experimental evidence suggests that fibrotic lung diseases such as IPF are inflammatory disorders at their inception. This evidence includes information from a transgenic mouse model of fibrosis, which develops spontaneously when tumor necrosis factor (TNF)-, for example, is overexpressed in the lungs. Histologically, the fibrotic disease produced in this mouse model is closely related to that of IPF.⁴ Additionally, in a bleomycin model of pulmonary fibrosis in animals, the fibrosis is preceded by profound inflammation, including the production of high levels of TNF-, and the fibrosis can be blocked by the simultaneous administration of an anti-TNF antibody. Importantly, TNF--deficient or TNF- receptor-deficient animals do not develop fibrotic lung disease.^{3 5} Despite this and other experimental evidence that fibrosis is related to inflammation, anti-TNF- therapy and antiinflammatory corticosteroid treatments in animals and humans have shown little or no reproducible improvement of lung function in most studies.

Some studies have attempted to show that fibrotic lung disease is not an inflammatory disorder. For example, Sime et al⁶ demonstrated this by using an adenoviral transfer of active TGF-ß to the lungs of animals, which resulted in sustained fibrosis with only a transient inflammatory response. Further data using the adenoviral transfer of TNF- to the lungs of animals showed fibrotic changes present only in areas of TNF--induced TGF-ß expression.⁷ These studies have led authors to conclude that TGF-ß is sufficient to induce fibrotic changes in the lung in the absence of inflammation. To disregard inflammation based on the transient expression of TNF- in the lung using the adenoviral expression system may not be accurate, as human IPF likely involves chronic rather than transient inflammation. Support for this idea comes from studies of animals that develop lung fibrosis when TNF- production in the lung is sustained for longer periods of time.⁴

In an ex vivo experimental system, Adamson et al⁸ showed that lung explants developed fibroproliferation and excessive collagen synthesis in the absence of blood cells or serum factors, concluding that damaged epithelial cells secrete signals that induce a fibroblast response. This study, however, did not rule out the presence and role of interstitial macrophages and other immune cells.

Fibroblasts isolated from the lungs of individuals with fibrotic lung disease have been shown to secrete enhanced levels of growth factors when compared to the levels from normal lung fibroblasts, further supporting the theory that a mesenchymal disorder is the cause of fibrosis.⁹ Further evidence suggesting that inflammation is not the underlying cause of fibrosis is that the presence of multiple markers of inflammation do not correlate with disease progression or outcome in patients with IPF¹ ; however, many of these studies used alveolitis as a marker of inflammation, whereas the documentation of interstitial inflammation may have been more accurate.

In a general sense, fibrosis is wound healing gone awry. As in other organ systems, inflammation is the immediate response following injury to the lung vasculature or epithelium. The recruited and activated immune cells secrete cytokines and growth factors that mediate the recruitment and proliferation of mesenchymal cells to replace the cells of the lung and to repair damaged stromal structures. In the normal process of healing, the increased cellularity resolves, leaving alveolar structures essentially intact. In fibrosing lung diseases, the repair stage persists, leading to areas of fibroblast foci, ECM deposition, and largely remodeled alveoli. While the cause is unclear, many believe the inability of the epithelium to fully cover the alveolar walls leads to a chronic reparative condition and fibrosis.¹

	IGF-I

TOP Abstract Introduction IGF-I TGF-ß Divergent Roles of TGF... Conclusion References

 
IGF-I is a 70-amino acid, 7.6-kd, single-chain nonglycosylated polypeptide with structural similarity to insulin. IGF-I has a broad range of physiologic functions including the stimulation of cell division, differentiation, migration, growth, inhibition of apoptosis, and the regulation of gene transcription. The actions of IGF-I occur primarily via the IGF-I receptor (IGF-IR), a tetrameric, type-1 surface receptor that is composed of two -chains and two ß-chains that are joined by disulfide linkages. The half-life of IGF-I is extended and its availability is modulated by binding to any of six IGF-binding proteins, the actions of which in turn are modulated by IGF-binding protein proteases, leading to a complex system of IGF-I availability to the cell surface receptor.¹⁰ IGF-I binds to cysteine-rich regions of the IGF-IR -subunit and induces a conformational change resulting in autophosphorylation of the intracellular portion of the ß-chains. This phosphorylation of tyrosine residues in turn enhances the activity of the tyrosine kinase domain of the ß-chains. Subsequently, adaptor proteins, such as members of the IRS, Grb, and SHC families, bind the ß-chain and initiate intracellular signal transduction cascades including the Ras-mitogen-activated protein kinase and phosphoinositol 3-kinase-Akt/protein kinase B pathways involved in cell proliferation, and inhibition of apoptosis respectively.¹⁰

IGF-I has been shown to be elevated in the lungs of patients with IPF, fibroproliferative ARDS, and other fibrotic lung disorders, as well as in animal models of pulmonary fibrosis such as bleomycin-induced fibrosis. Early reports suggesting the involvement of IGF-I in pulmonary fibrosis identified IGF-I (then deemed alveolar macrophage-derived growth factor or AMDGF) in the BAL fluid of patients with IPF. Additionally, these studies^{11 12} identified alveolar macrophages as being a source of this protein in patients with IPF. More recently, we and others have shown the presence of an enhanced IGF-I protein by immunohistochemical staining in macrophages and epithelial cells from patients with fibrotic lung diseases, including IPF and fibroproliferative ARDS.^{13 14 15} IGF-I has been shown to be partially responsible for the fibroblast proliferative response that is seen in lung fluid isolated from patients with fibrotic lung disorders and has been shown to stimulate fibroblast collagen synthesis in vitro. The contribution of IGF-I to collagen synthesis in fibrotic lung disorders is unclear, since, in at least one study, using IGF-I antibody to block IGF-I activity had no effect on the ability of the alveolar lavage fluids to stimulate collagen synthesis.^{16 17 18}

Studies in our laboratory and those of others have led us to believe that IGF-I may play a role other than in stimulating fibroblast proliferation and collagen synthesis following acute lung injury, namely, in stimulating reepithelialization. As described above, epithelial damage and death is widely considered to be one of the most important persistent insults that leads to inflammation and the development of fibrosis in patients with IPF. Critical to the resolution of this damage is the division and migration of intact epithelial cells to cover the denuded basement membrane. IGF-I is well-known to have mitogenic and antiapoptotic functions and also has been shown to play a role in altering focal adhesions and actin structures in mammary epithelial cells, indicating a potential role in epithelial cell migration.^{10 19} We have shown, using both immunohistochemical staining of human lung sections and RNase protection assay of human lung epithelial cell lines, that lung epithelial cells express the IGF-IR. Interestingly, platelet-derived growth factor and basic fibroblast growth factor, factors that have been shown to be enhanced in patients following lung injury and in patients with fibrotic lung diseases, have been shown to increase IGF-IR expression.¹⁰ Furthermore, as mentioned previously, the IGF-I protein has been detected in fibrotic but not in normal lung sections, and we and others have shown that macrophages are a potential source of IGF-I. Interestingly, bone marrow-derived macrophages stimulated with the proinflammatory cytokine TNF- produce IGF-I messenger RNA in a dose-dependent fashion (Fig 1 , and data not shown). We contend that following damage to the lung epithelium, inflammation, including the production of TNF-, results in the production of IGF-I by macrophages and, potentially, by epithelial cells. The IGF-I protein in turn may act primarily to increase epithelial cell proliferation and migration and secondarily to stimulate fibroproliferation. Our laboratory is currently testing this hypothesis. Why then in IPF do fibroblast proliferation and the presence of a denuded basement membrane persist?

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 1.. Murine bone marrow-derived macrophages stimulated over 6 h with the cytokines TNF- and/or TGF-ß. Messenger RNA was quantified by phosphorimage analysis of RNase protection assays. IGF-I was normalized to ß-actin level and is presented as a percentage of unstimulated cells (n = 5). The results are plotted ± SEM. * = levels that are significantly different (ie, p 0.05) than the control values, as determined by two-tailed t test. The inset is a representative RNase protection assay.

	TGF-ß

TOP Abstract Introduction IGF-I TGF-ß Divergent Roles of TGF... Conclusion References

	Divergent Roles of TGF-ß and IGF-I

TOP Abstract Introduction IGF-I TGF-ß Divergent Roles of TGF... Conclusion References

 
Studies in our laboratory have shown that bone marrow-derived macrophages in the presence of TGF-ß have reduced basal IGF-I messenger RNA production and, when stimulated with TNF-, macrophages, have been unable to enhance IGF-I messenger RNA production (Fig 1 ). Therefore, TGF-ß inhibits the ability of the macrophage to express IGF-I directly, by repressing IGF-I messenger RNA expression, and indirectly, by reducing proinflammatory cytokine production. Interestingly, Homma et al¹³ showed large numbers of IGF-I immunoreactive cells in the early stages of IPF, while in later phases of the disease the presence of IGF-I-positive cells was significantly reduced. This supports our theory that IGF-I acts early following inflammation to promote cell proliferation and that in later phases of disease, when TGF-ß is present, IGF-I has been negatively regulated.

Interestingly, fibroblasts and myofibroblasts in fibrotic foci that are adjacent to sites in a denuded epithelium secrete factors that induce apoptosis in epithelial cells (angiotensin and tissue inhibitor of metalloproteinase-3).^{21 22} Taken together, the loss of the antiapoptotic and mitogenic factor IGF-I, and the presence of inducers of apoptosis at these fibrotic foci support the theory that the inability of epithelial cells to repopulate areas of damage may propagate fibrosis. We theorize that in areas of the lung with dense fibroblast foci and denuded epithelium the enhanced production of TGF-ß leads not only to fibroblast ECM synthesis and myofibroblast differentiation, but also to enhanced epithelial cell death via the reduced expression of IGF-I and the secretion of apoptotic factors. The net result is prolonged fibroblast repair with an inability of the epithelium to regenerate, which supports the presence of fibrotic scarring.

Finally, Bloor et al²³ have recently shown that cells recovered by BAL from individuals with IPF display reduced IGF-I expression when compared to healthy control subjects. Interestingly, fibroblasts derived from individuals with IPF displayed enhanced IGF-I expression that could be augmented when stimulated with TGF-ß. Taken together, these data imply that there is a differential regulation of IGF-I between cell types and that the temporal regulation and localization of IGF-I production may play a significant role in the progression of fibrotic lung disease.

	Conclusion

TOP Abstract Introduction IGF-I TGF-ß Divergent Roles of TGF... Conclusion References

 
Inflammation has been shown to occur in the early stages of IPF, presumably in response to damage to the respiratory epithelium and the subsequent recruitment of leukocytes. While arguments persist as to whether fibrosis is a result of inflammation or whether fibrosis occurs without significant inflammation, we argue that inflammation and the subsequent local production of IGF-I are required for the efficient repair of epithelial injury. In the absence of inflammation or with the premature expression of high levels of TGF-ß, IGF-I production may be absent, leading to enhanced epithelial death and to the presence of chronically denuded alveolar basement membranes, which together result in an ongoing repair process including ECM deposition mediated by fibroblasts and myofibroblasts. Clearly, a delicate temporal balance exists between fibrosis and normal healing as the proinflammatory cytokine TNF- appears to be responsible for the production of both IGF-I and TGF-ß. We are currently studying the temporal regulation of growth factor expression in the course of fibroproliferation in patients with ARDS. We hope to gather information on growth factor expression in the lungs of patients whose disease progresses vs growth factor expression in patients whose disease resolves.

	Footnotes

 
Dr. Winston was supported by a Canadian Institutes of Health Research (Medical Research Council of Canada) Scholarship, an Alberta Lung Association Operating Grant, and an Alberta Heritage Foundation for Medical Research Clinical Investigator Grant. Mr. Krein was supported by an Alberta Heritage Foundation for Medical Research Studentship Award.

Abbreviations: ECM = extracellular matrix; IGF = insulin-like growth factor; IGF-IR = insulin-like growth factor-I receptor; IPF = idiopathic pulmonary fibrosis; TGF-ß = transforming growth factor-ß1; TNF = tumor necrosis factor

	References

TOP Abstract Introduction IGF-I TGF-ß Divergent Roles of TGF... Conclusion References

 

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