Idiopathic Pulmonary Fibrosis: Prevailing and Evolving Hypotheses ...

Review Idiopathic Pulmonary Fibrosis: Prevailing and Evolving Hypotheses about Its Pathogenesis and Implications for Therapy Moise´s Selman, MD; Talmadge E. King Jr., MD; and Annie Pardo, PhD

Idiopathic pulmonary fibrosis is a progressive and usually fatal lung disease characterized by fibroblast proliferation and extracellular matrix remodeling, which result in irreversible distortion of the lung’s architecture. Although the pathogenetic mechanisms remain to be determined, the prevailing hypothesis holds that fibrosis is preceded and provoked by a chronic inflammatory process that injures the lung and modulates lung fibrogenesis, leading to the end-stage fibrotic scar. However, there is little evidence that inflammation is prominent in early disease, and it is unclear whether inflammation is relevant to the development of the fibrotic process. Evidence suggests that inflammation does not play a pivotal role. Inflammation is not a prominent histopathologic finding, and epithelial injury in the absence of ongoing inflammation is sufficient to stimulate the development of fibrosis. In

addition, the inflammatory response to a lung fibrogenic insult is not necessarily related to the fibrotic response. Clinical measurements of inflammation fail to correlate with stage or outcome, and potent anti-inflammatory therapy does not improve outcome. This review presents a growing body of evidence suggesting that idiopathic pulmonary fibrosis involves abnormal wound healing in response to multiple, microscopic sites of ongoing alveolar epithelial injury and activation associated with the formation of patchy fibroblast–myofibroblast foci, which evolve to fibrosis. Progress in understanding the fibrogenic mechanisms in the lung is likely to yield more effective therapies.

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computed tomography, HRCT, bronchoalveolar lavage, prognosis, prognostic factors, pathogenesis, fibroblasts, myofibroblasts, epithelial cells, macrophages, neutrophils, eosinophils, coagulation, collagen, collagenase, metalloproteinases, TIMP, basement membrane, therapy, treatment, review. Pulmonary fibrosis was used alone, with the same mentioned key words, and with transgenic mice, experimental models, bleomycin, and silica. Other terms were usual interstitial pneumonia, nonspecific interstitial pneumonia, BOOP, organizing pneumonia, desquamative interstitial pneumonia, and idiopathic interstitial pneumonias. The reference lists of identified articles and additional sources, such as textbook chapters and meeting abstracts, were reviewed for relevant publications. All of the included papers were peer-reviewed. Data were also extracted from results of unpublished studies conducted by the National Institute of Respiratory Diseases, Mexico; the Faculty of Sciences, National Autonomous University of Mexico; and the National Jewish Medical and Research Center, Denver, Colorado.

diopathic pulmonary fibrosis is a specific form of chronic fibrosing interstitial pneumonia limited to the lung. Although its cause remains unknown, advances in cellular and molecular biology have greatly expanded our understanding of the biological processes involved in its initiation and progression. Recently, an international consensus statement defining the diagnosis, evaluation, and treatment of patients with idiopathic pulmonary fibrosis was produced as a collaborative effort by the American Thoracic Society, the European Respiratory Society, and the American College of Chest Physicians (1). The purpose of the consensus statement was to assist clinicians in the diagnosis and management of idiopathic pulmonary fibrosis. We provide a focused update of the pathogenesis of this disease and discuss the therapeutic implications of these findings. In particular, we examine the hypothesis that idiopathic pulmonary fibrosis is a fibrotic rather than an inflammatory disease.

METHODS We reviewed the epidemiologic characteristics, clinical behavior, pathogenesis, experimental models, and treatment of idiopathic pulmonary fibrosis. By searching MEDLINE, we identified articles published from 1965 to 2000. The following search terms were used: idiopathic pulmonary fibrosis, alone and with epidemiology, 136 © 2001 American College of Physicians–American Society of Internal Medicine

Ann Intern Med. 2001;134:136-151. For author affiliations and current addresses, see end of text.

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EPIDEMIOLOGIC AND CLINICAL CHARACTERISTICS Patients with idiopathic pulmonary fibrosis are usually between 50 to 70 years of age at presentation; two thirds are older than 60 years of age (2). The estimated annual incidence is 7 cases per 100 000 for women and

Idiopathic Pulmonary Fibrosis

10 cases per 100 000 for men (3). The incidence, prevalence, and death rate increase with age (3–5). Cigarette smoking has been identified as a potential risk factor for this disease (6). Although other risk factors have been suggested, the studies attempting to define them have often examined unconfirmed cases of idiopathic pulmonary fibrosis (7–13). Several viruses have been implicated in pathogenesis, but no clear evidence points to a viral cause (14 –24). In general, evidence of predisposing or etiologic factors is weak, and most patients do not have any obvious risk factors. Familial cases of pulmonary fibrosis provide compelling evidence for participation of genetic factors, but no specific genetic abnormality has been identified (25–38). Most patients have symptoms for more than 6 months before diagnosis (average duration, 24 months). The clinical manifestations include dyspnea on exertion, nonproductive cough, and inspiratory crackles, with or without digital clubbing noted on physical examination. Chest radiography and high-resolution computed tomography typically show patchy, predominantly peripheral, subpleural, lower lung zone reticular opacities (1, 39). High-resolution computed tomography also shows variable but limited ground-glass opacity (usually associated with traction bronchiectasis) and subpleural honeycombing. Confluent alveolar opacities, evidence of pleural disease, or lymphadenopathy suggest another diagnosis (39 – 43). Pulmonary function tests reveal restrictive impairment, reduced diffusing capacity for carbon monoxide, and arterial hypoxemia exaggerated or elicited by exercise (44 – 49). The definite diagnosis of idiopathic pulmonary fibrosis requires a compatible clinical history, the exclusion of other known causes of interstitial lung disease (such as drug injuries, environmental exposures, or collagen vascular disease), and a surgical lung biopsy showing usual interstitial pneumonia (1). Previously, pathologists included several histopathologic patterns among the cases labeled as usual interstitial pneumonia, especially desquamative interstitial pneumonia, nonspecific interstitial pneumonia, and bronchiolitis obliterans organizing pneumonia. However, the histopathologic definition for usual interstitial pneumonia has narrowed and excludes these patterns (1, 50, 51). The histologic hallmark of usual interstitial pneumonia is a heterogeneous appearance at low magnification with alternating areas of normal lung, interstitial inflammation, fibrowww.annals.org

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blastic foci, dense fibrosis, and honeycomb change. These histopathologic changes affect the peripheral subpleural parenchyma most severely.

PREVAILING HYPOTHESIS: INFLAMMATION LEADS TO FIBROSIS It has been widely held that a common pathogenetic sequence underlies all fibrotic lung diseases, regardless of cause. This “inflammatory fibrosis” hypothesis asserts that chronic inflammation injures the lung and modulates fibrogenesis, leading to the end-stage fibrotic scar (52). Several of the key concepts that formed the basis for this hypothesis no longer seem valid. Inflammation Is Not a Prominent Histopathologic Finding in Usual Interstitial Pneumonia

A major driving force sustaining the “inflammatory fibrosis” theory for usual interstitial pneumonia was the commonly held view that desquamative interstitial pneumonia, a predominately intra-alveolar macrophage accumulation (that is, “alveolitis”), was the earliest lesion in this disorder (52, 53). However, desquamative interstitial pneumonia is a nonspecific response to cigarette smoke (54, 55). Alveolar septa may be thickened by a sparse inflammatory infiltrate; however, this lesion rarely contains fibroblastic foci or the extensive fibrotic pattern common in usual interstitial pneumonia. Consequently, given that most patients with idiopathic pulmonary fibrosis are current or former cigarette smokers, intra-alveolar macrophage accumulation is to be expected and probably does not play a key role in pathogenesis. Most important, little evidence supports the concept that inflammation is more prominent in early stages of usual interstitial pneumonia. Careful review of larger numbers of better defined cases shows that the inflammatory component is usually mild, occurs mainly in areas of collagen deposition or honeycomb change, and rarely involves otherwise unaltered alveolar septa (50). Finally, interstitial lung diseases in which inflammation is a prominent feature of early disease—for example, hypersensitivity pneumonitis— often do not progress to end-stage fibrosis. Inflammation Is Not Required for the Development of a Fibrotic Response

Some evidence, primarily from transgenic animals, shows that it is possible to dissociate the inflammatory response from the fibrotic response. Interleukin-10 – 16 January 2001 Annals of Internal Medicine Volume 134 • Number 2 137

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deficient mice instilled with silica showed greater lung inflammation but less fibrotic response than wild-type mice (56). Munger and colleagues (57) demonstrated in bleomycin lung injury that mice deficient in the integrin ␣v␤6 (␤6⫺/⫺), a ligand that binds and activates transforming growth factor-␤, developed exaggerated inflammation but were protected from pulmonary fibrosis. Both the ␤6⫺/⫺ and ␤6⫹/⫹ mice showed similar levels of total transforming growth factor-␤ protein. Sime and coworkers (58), using two replication-deficient adenoviruses expressing active and latent transforming growth factor-␤ 1, showed that overexpression of both transgenes provoked transient inflammation. However, only the active form induced fibroblast proliferation and progressive extracellular matrix accumulation (58). Adamson and colleagues (59) were able to induce a lung fibrotic response in a blood-free environment. Mice were exposed to 95% hyperoxia, and lung explants were cultured at various stages of hyperoxic injury. As epithelial alveolar damage increased, epithelial cell proliferation in the explants was retarded while fibroblast growth became predominant mainly at areas of epithelial necrosis. Collagen production also increased. It is important to note that these explants had few macrophages and no blood components. Thus, epithelial injury in the absence of ongoing inflammation is adequate to stimulate the development of fibrosis. Clinical Measurements of Inflammation Fail To Correlate with Stage or Outcome in Idiopathic Pulmonary Fibrosis

Because alveolitis was presumed to have a central role in the pathogenesis of fibrotic lung diseases, it was believed that methods to determine its character and intensity had to be identified in order to stage and treat patients. However, most markers of inflammation failed to correlate with disease stage or outcome in idiopathic pulmonary fibrosis. Thus, it has been proven that cellular constituents of bronchoalveolar lavage, gallium-67 lung scan and tissue, and circulating immune complexes have rather limited clinical value in staging or monitoring idiopathic pulmonary fibrosis (60 –73). High-resolution computed tomography lung scanning has been proposed as a technique to determine the “activity” of idiopathic pulmonary fibrosis, since it has been shown that the extent and severity of ground-glass opacities correlate with alveolitis in several diffuse infiltrative lung disorders (74). However, in idiopathic pul138 16 January 2001 Annals of Internal Medicine Volume 134 • Number 2

monary fibrosis, ground-glass opacities do not show a relationship with the inflammatory response. In fact, they are often associated with reticular opacities and traction bronchiectasis, findings usually related to alveolar septal fibrosis and granulation tissue within the alveoli (75, 76). Moreover, in idiopathic pulmonary fibrosis, the areas of ground-glass opacities typically progress and are replaced by evidence of lung fibrosis (77–79). By contrast, in desquamative interstitial pneumonia, hypersensitivity pneumonitis, nonspecific interstitial pneumonia, or systemic sclerosis, the ground-glass opacity usually regresses or stays stable in response to treatment (79 – 83).

Anti-Inflammatory Therapy Does Not Improve Disease Outcome

Idiopathic pulmonary fibrosis is almost always progressive and leads to death, despite potent and long-term anti-inflammatory therapy. On the basis of the presumption that the disease is an inflammatory process with concurrent remodeling of the lung by fibrosis, the natural choice for treatment has been oral glucocorticoids (84). Several early clinical series reported few, usually short-term, steroid responders among patients with presumed idiopathic pulmonary fibrosis (63, 85). This finding has been difficult to confirm because controlled studies with adequate numbers of participants have not been performed. More important, in most studies, not all patients had biopsy-proven disease and some patients had other lung disorders, especially desquamative interstitial pneumonia, nonspecific interstitial pneumonia, or even collagen vascular disease. For example, a retrospective review of biopsy specimens confirmed the diagnosis of idiopathic pulmonary fibrosis in only 62% of patients who originally received it (51). Remaining patients displayed nonspecific interstitial pneumonia, bronchiolitis obliterans organizing pneumonia, and other diseases. Median survival of the group with usual interstitial pneumonia was 2.8 years, which was significantly worse than for the other groups. Currently, a growing body of evidence supports the notion that high doses of glucocorticoids are not useful in patients with idiopathic pulmonary fibrosis (usual interstitial pneumonia) because marginal or no response has been seen, even when glucocorticoids are administered with potent immunosuppressive drugs (84, 86 –92). www.annals.org


By contrast, other potentially fibrotic lung disorders that have an inflammatory process during early stages often respond to glucocorticosteroid therapy, particularly if therapy is begun during the inflammatory phase. This favorable response has been widely documented in sarcoidosis, hypersensitivity pneumonitis, nonspecific interstitial pneumonia, and desquamative interstitial pneumonia (93–96).

EVOLVING HYPOTHESIS: FIBROSIS RESULTS FROM EPITHELIAL INJURY AND ABNORMAL WOUND HEALING In addition to emerging evidence that inflammation is not more prominent in early stages of usual interstitial pneumonia (50), it is becoming clear that the primary sites of ongoing injury and repair are the regions of fibroblastic proliferation, so-called fibroblast foci (50, 97). These small aggregates of actively proliferating myofibroblasts and fibroblasts constitute many microscopic sites of ongoing alveolar epithelial injury and activation associated with evolving fibrosis (Figure 1) (50, 97, 98). We hypothesize that usual interstitial pneumonia represents a model of abnormal wound healing in the lung. It is important to note that this process differs from the normal wound healing model because of the absence of adequate reepithelialization and abnormalities in myofibroblast behavior (Figures 2 and 3).

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Figure 1. Fibroblast foci.

Reepithelialization: An Essential Step in Normal Repair

After injury, the alveolar epithelium must initiate a wound healing process to restore its barrier integrity. One important step is the rapid reepithelialization of the denuded area through epithelial cell migration, proliferation, and differentiation. In idiopathic pulmonary fibrosis, this response seems slow and inadequate. The alveolar epithelium shows a marked loss of or damage to type I cells, hyperplasia of type II cells, and altered ex-

Figure 2. The normal wound healing model.

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Figure 3. Hypothetical scheme of the abnormal wound healing model for idiopathic pulmonary fibrosis.

Multiple microinjuries damage and activate alveolar epithelial cells (top left), which in turn induce an antifibrinolytic environment in the alveolar spaces, enhancing wound clot formation. Alveolar epithelial cells secrete growth factors and induce migration and proliferation of fibroblasts and differentiation into myofibroblasts (bottom left). Subepithelial myofibroblasts and alveolar epithelial cells produce gelatinases that may increase basement membrane disruption and allow fibroblast–myofibroblast migration (bottom right). Angiogenic factors induce neovascularization. Both intra-alveolar and interstitial myofibroblasts secrete extracellular matrix proteins, mainly collagens. An imbalance between interstitial collagenases and tissue inhibitors of metalloproteinases provokes the progressive deposit of extracellular matrix (top right). Signals responsible for myofibroblast apoptosis seem to be absent or delayed in usual interstitial pneumonia, increasing cell survival. Myofibroblasts produce angiotensinogen that as angiotensin II provokes alveolar epithelial cell death, further impairing reepithelialization. FGF-2 ⫽ fibroblast growth factor-2; MMP ⫽ metalloproteinase; PAI-1, PAI-2 ⫽ plasminogen activator inhibitor-1, -2; PDGF ⫽ platelet-derived growth factor; TGF-␤ ⫽ transforming growth factor-␤; TIMP ⫽ tissue inhibitors of metalloproteinases; TNF␣ ⫽ tumor necrosis factor-␣; VEGF ⫽ vascular endothelial growth factor.

pression of adhesion molecules and MHC antigens (99 – 102). Where the basement membrane remains intact, type II cells attempt to recover the epithelial surface; these cells express several enzymes, cytokines, and growth factors (99). In usual interstitial pneumonia, the capacity of type II alveolar cells to restore damaged type I cells is seriously altered, resulting in epithelial cuboidalization and the presence of transitional reactive phenotypes (99), abnormalities in pulmonary surfactant (103, 104), and alveolar collapse (105). Potential Fibrogenic Roles of Alveolar Epithelial Cells Epithelial Cells and the Coagulation System

During wound healing, tissue injury causes disruption of blood vessels and extravasation of blood constituents into the wound (106). This step reestablishes hemostasis and provides a provisional extracellular matrix 140 16 January 2001 Annals of Internal Medicine Volume 134 • Number 2

in which the repair process can begin. The actions of initiators and inhibitors of the coagulation cascade tightly regulate the amount of fibrin present at sites of injury. Usually, the fibrinolytic system, required to cleave a path for cell migration, is active during repair processes that restore injured tissues to normal (107). During normal wound healing, epithelial cells have to dissolve the fibrin barrier to migrate throughout the denuded wound surface (107). By contrast, increased local procoagulant activity has been found in fibrotic lung disorders, including idiopathic pulmonary fibrosis (108 –110). Most important, alveolar epithelial cells seem to contribute to the increased procoagulant and antifibrinolytic activities in this disorder. Tissue factor (a potent procoagulant factor) and plasminogen activator inhibitor-1 and -2 are strongly expressed by alveolar epithelial cells (109, 110). www.annals.org


These findings suggest that cell movement in this extracellular matrix may be impaired, thereby impeding the repair process. A study by Eitzman and coworkers (111) supports the importance of this process in the development of lung fibrosis. In their study, transgenic mice that overexpressed plasminogen activator inhibitor-1 developed significantly more fibrosis than littermate controls after bleomycin injury. By contrast, mice deficient in plasminogen activator inhibitor-1 behaved normally (111). In addition, when the gene encoding plasminogen has been knocked out in transgenic mice, wound reepithelialization is almost completely abrogated (112). Alveolar Epithelial Cells as a Primary Source of Profibrotic Cytokines

In idiopathic pulmonary fibrosis, epithelial cells express several cytokines and growth factors that may promote fibroblast migration and proliferation and extracellular matrix accumulation. Using immunofluorescence and in situ hybridization, we demonstrated that in idiopathic pulmonary fibrosis, alveolar epithelial cells expressed platelet-derived growth factor (113). Several studies have shown that in this condition, hyperplastic type II pneumocytes constitute the main site of synthesis of transforming growth factor-␤1 and tumor necrosis factor-␣ (114 –116). Khalil and colleagues (117), studying several different interstitial lung disorders, demonstrated that in early fibrotic lungs (with inflammation and minimal fibrosis), transforming growth factor-␤1 was found mainly in alveolar macrophages. By contrast, in advanced fibrotic honeycomb lesions primarily seen in usual interstitial pneumonia, the growth factor was mainly present in epithelial cells. These findings could be interpreted in several ways, but we can speculate that fibrosis occurs where there is epithelial expression of transforming growth factor-␤1. Fibroblastic Foci: Site of Ongoing Lung Injury and Repair

Alveolar epithelial cells may initiate the pathologic process, producing most of the factors inducing the phenotypic changes seen among fibroblasts during the progression to end-stage fibrosis. Present evidence suggests that the earliest, and possibly the only, morphologic change associated with subsequent progression to dense fibrosis is the presence and extent of fibroblastic foci in www.annals.org

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the injured lung (50, 97, 98) (Figure 1). Fibroblasts within these foci continually modify their interactions with the microenvironment. Fibroblasts assume first a migratory phenotype, second a proliferative phenotype, and finally a profibrotic phenotype, during which they produce abundant extracellular matrix components. Most cells in the fibroblast foci are myofibroblasts aligned parallel to one another and thus probably contribute to active contraction and distorted architecture (98). It is not known whether fibroblasts from patients with idiopathic pulmonary fibrosis are genetically susceptible to abnormal response after stimulation. It is possible that some inciting factor or factors activate epithelial cells and induce secretion of profibrotic molecules, which in turn act on genetically defective fibroblasts and provoke a continuous fibrotic response. Of interest, lung fibroblasts isolated from these patients have a defect in cyclooxygenase-2 expression and a failure in their capacity to synthesize prostaglandin E2 that has, among other functions, an antifibrogenic effect (118).

Failure of Reepithelialization: The Role of Fibroblast-Mediated Alveolar Epithelial Cell Death

A plausible explanation for the absence of appropriate reepithelialization in idiopathic pulmonary fibrosis remains unclear. A common feature in this disease is the presence of microscopic areas of epithelial cell dropout. In other areas, hyperplasia of type II pneumocytes occurs while cystic fibrotic air spaces (frequently lined by bronchiolar epithelium and filled with mucin) are present in areas of honeycomb change (100). Other mechanisms of epithelial repair may also be involved. In bleomycin-induced injury in mice, a subpopulation of epithelial cells that seemed to express components of alveolar and Clara cells—probably representing a multipotential stem-cell population— has been described (119). Some evidence suggests that apoptosis may play an important causal role in microscopic areas of epithelial cell dropout. Fibroblasts and myofibroblasts from lungs with idiopathic pulmonary fibrosis induced alveolar epithelial cell death in vitro (120). In addition, numerous apoptotic alveolar epithelial cells have been demonstrated in vivo by in situ end labeling and electron microscopy. These apoptotic alveolar epithelial cells are de16 January 2001 Annals of Internal Medicine Volume 134 • Number 2 141

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tected primarily in areas immediately adjacent to underlying foci of myofibroblasts (121). More recently, it was found that angiotensinogen is the factor responsible for myofibroblast apoptotic activity (122). Likewise, patients with idiopathic pulmonary fibrosis exhibit an upregulation of the tumor suppressor proteins p53 and p21Waf1/Cip1/Sdi1, primarily in hyperplastic alveolar epithelial cells, that may also result in apoptosis (123). Basement Membrane Disruption

The basement membrane is a complex structure that includes type IV collagen, laminin, entactin, fibronectin, and heparin sulfate–chondroitin proteoglycans (124). It plays a dynamic role in maintaining the integrity and differentiation of the alveolar epithelium, and its disruption is important in the pathogenesis of lung fibrosis (125–127). Migration of fibroblasts and myofibroblasts into the alveolar spaces occurs through partially disrupted and denuded epithelial basement membranes (97, 98, 128). The disrupted basement membrane may also contribute to the failure of an orderly repair of the damaged alveolar type I epithelial cells. The mechanisms involved in the disruption of the basement membrane remain unknown. Also, biochemical information about basement membrane turnover in pulmonary fibrosis is limited. Gelatinases A and B, two members of the matrix metalloproteinase family that are produced by several types of lung cells, have been shown to degrade different components of the basement membrane, primarily type IV collagen (129 –132). In addition, in idiopathic pulmonary fibrosis, gelatinases are synthesized by subepithelial myofibroblasts, coinciding in some areas with denuded alveolar basement membranes (133, 134). This finding suggests that myofibroblasts play a role in the degradation of the basement membrane, facilitating their migration into the alveolar spaces. Likewise, in bleomycin-induced pulmonary fibrosis, increased gelatinase B activity and disruption of the alveolar epithelial basement membrane have been found (135). Extracellular Matrix Accumulation and Remodeling

Under physiologic conditions, an exquisite coordination between extracellular matrix, integrins, growth factor signaling, and parenchymal cells integrates the normal lung structure and function. Fibroblasts and 142 16 January 2001 Annals of Internal Medicine Volume 134 • Number 2

myofibroblasts play a central role in synthesis, deposition, and remodeling of the extracellular matrix. Increased deposition of extracellular matrix, including fibrillar collagens, fibronectin, elastic fibers, and proteoglycans, is the hallmark of the aberrant tissue remodeling in idiopathic pulmonary fibrosis (136, 137). The final result is an extensive structural disorganization in the lung microenvironment, with spatial–temporal and quantitative– qualitative changes in the normal interactions and consequent progressive loss of the alveolar– capillary units. The molecular mechanisms behind the aberrant tissue remodeling that results in exaggerated extracellular matrix accumulation, cell migration, and angiogenesis involve proteolysis by matrix metalloproteinases (138 – 140). Matrix metalloproteinases, a family of highly regulated zinc-dependent peptidases, have been implicated in the remodeling of extracellular matrix and in the events underlying cell migration (138 –140). Extracellular control of matrix metalloproteinases activity is achieved by members of a specific family of inhibitors known as tissue inhibitors of metalloproteinases (TIMPs). There are currently four members of this family, which have in common matrix metalloproteinase inhibitory action. However, they differ in expression patterns and other properties, such as association with latent gelatinases, cell growth–promoting activity, cell survival–promoting activity, and apoptosis (141–143). We recently demonstrated a higher interstitial expression of the four TIMPs compared with interstitial collagenases, suggesting that a nondegrading fibrillar collagen microenvironment is present in idiopathic pulmonary fibrosis (133). Collagenase-1 was expressed by alveolar macrophages, alveolar epithelial cells, and bronchiolar epithelial cells lining honeycomb cystic spaces, but it was virtually absent in the interstitial compartment. Collagenase-2 was revealed in neutrophils, and collagenase-3 was not detected (133). In contrast, TIMP-1 was present in interstitial cells associated with fibrous tissue, TIMP-2 was present in myofibroblasts within fibroblast foci, TIMP-3 strongly stained the elastic lamina of vessels, and TIMP-4 was present in interstitial macrophages and plasma cells. Therefore, an imbalance between collagenases and TIMPs seems to play a role in fibrogenesis. Previous studies have also shown increased staining for TIMP-1 and TIMP-2, mainly in alveolar epithelial cells and myofibroblasts (128, 134). It www.annals.org


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Table 1. Morphologic Differences between Masson Bodies (Organizing Pneumonia) and Fibroblast Foci (Usual Interstitial Pneumonia)* Process

Masson Bodies

Fibroblast Foci

Reference

Inflammation

Mixed inflammatory cell population seen in the central region Fibroblasts undergo specific changes (i.e., express desmin) Early apoptosis and disappearance occur; predominant localization of matrix metalloproteinases occurs Loose mode of organization causes susceptibility to degradation; myofibroblasts phagocytose collagen fibrils Occurs rapidly; basement membrane is not severely impaired; regenerated type I alveolar epithelial cells surround the residual collagen globule Newly formed vessels with well-developed basement membranes are seen

Minimal or no inflammation seen

98, 154

Myofibroblasts do not express desmin

154

Survival is prolonged; predominant localization of TIMP-2 occurs Imbalance occurs between matrix metalloproteinases and TIMPs; dense collagen is progressively deposited Is delayed or absent because of apoptosis and disruption of basement membrane

128, 133, 155

New vessels are usually not found

128

Fibroblasts cytoskeleton Myofibroblasts Extracellular matrix

Reepithelialization

Neovascularization

98, 128, 133, 134

120, 121, 125

* TIMP ⫽ tissue inhibitors of metalloproteinases.

is important to emphasize that increased TIMP-1 and TIMP-2 expression may induce mesenchymal cell proliferation, while TIMP-3 may induce apoptosis (141– 143). In this context, myofibroblast expression of TIMP-2, specifically observed in the fibroblast foci, could be related (in addition to its enzymatic inhibitory effect) to the stimulation of fibroblast proliferation and activation of latent gelatinase A. Association of TIMP-2 with the expansion of the myofibroblast cell population was assumed because in these foci, the inhibitor co-localized with nuclear markers of cell proliferation (133). This process may explain, at least in part, the survival of mesenchymal cell populations in the fibroblast foci, in contrast with the expected cell death as it is observed in a normal wound healing model (144). Supporting this point of view, rats showing spontaneous recovery from liver fibrosis exhibit a rapid decrease in TIMP-1 and TIMP-2, increased collagenase activity, and apoptosis of hepatic stellate cells (145). Angiogenesis

The formation of new blood vessels requires coordinated regulation of matrix proteolysis and endothelial cell migration. Studies of angiogenesis in idiopathic pulmonary fibrosis are few, but this process has been demonstrated in the disease as well as in experimental models of fibrosis, and it is probably implicated in anastomoses between systemic and pulmonary microvasculature found in fibrotic regions (146, 147). Collagenase-1 and gelatinases A and B, through the degradation www.annals.org

of extracellular matrix, may initiate and promote angiogenesis, whereas TIMPs inhibit neovascularization (148, 149). The angiogenic molecules involved in this process (vascular endothelial cell growth factor, angiopoietins, fibroblast growth factor-2) and the contribution of neovascularization to the progression of fibrosis are largely unknown (150, 151). At least theoretically, angiogenesis may have a deleterious role (as suggested in cancer), an irrelevant role (as suggested in early cardiac infarction), or even an antifibrogenic role during the development of idiopathic pulmonary fibrosis. Recent studies suggest that neovascularization enhances fibrogenesis (152, 153). In bleomycin-induced lung fibrosis, neutralization of an angiogenic chemokine or administration of an angiostatic chemokine reduced the fibrotic response, which was paralleled by a reduction in the level of angiogenesis (152, 153). However, neovascularization is a prominent feature in organizing pneumonia, a usually reversible fibrogenic process (128).

NORMAL COMPARED WITH ABNORMAL WOUND HEALING: LESSONS FROM ORGANIZING PNEUMONIA Our hypothesis emphasizes that idiopathic pulmonary fibrosis is not an inflammatory disorder but rather an “epithelial–fibroblastic disease.” Consequently, we should search for new therapeutic approaches aimed at targeting both cell types. We should also begin to consider whether therapies could be designed to “reverse” fibrosis, a process that we have long viewed as unidirec16 January 2001 Annals of Internal Medicine Volume 134 • Number 2 143

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tional, with the subsequent restoration of the normal lung architecture. In this context, it is clear that the fibroproliferative process in organizing pneumonia is usually self-limited or reversible despite the striking similarity between the Masson bodies found in this disorder and the intraalveolar fibroblastic foci observed in usual interstitial pneumonia (98). The reasons for the dramatic difference in clinical outcome are unknown but might be related to some of the morphologic and biochemical distinctions presented in Table 1 (98, 120, 121, 125, 128, 133, 134, 154, 155). Thus, organizing pneumonia may represent the normal wound healing model in the lung, with appropriate extracellular matrix remodeling, fibroblast–myofibroblast apoptosis, and alveolar reepithelialization. Conversely, usual interstitial pneumonia seems to represent the abnormal wound healing model, with progressive extracellular matrix accumulation, decreased fibroblast–myofibroblast cell death, con-

tinuous epithelial cell apoptosis, and abnormal reepithelialization.

PROMISING THERAPEUTIC APPROACHES Conventional management of idiopathic pulmonary fibrosis has been primarily based on the concept that suppressing inflammation would prevent progression to fibrosis. However, despite the use of aggressive immunosuppressive and cytotoxic treatment regimens, idiopathic pulmonary fibrosis remains a progressive, irreversible, and fatal disease. Current treatment approaches have been recently reviewed (1, 84, 156). Future therapies should aim at preventing or inhibiting the fibroproliferative response and enhancing normal alveolar reepithelialization. This dual cell targeting will be crucial in reducing the impact of this problem on the health of persons with idiopathic pulmonary fibrosis. An overview of the potential agents that may prove ef-

Table 2. Molecules or Drugs That Inhibit Fibroblast Proliferation or Induce Fibroblast Apoptosis* Agent or Approach

Mechanism of Action

Comments

Colchicine

Inhibits collagen formation from fibroblasts and may increase collagen degradation (157, 158) Suppresses the release of alveolar macrophage–derived growth factor and fibronectin by alveolar macrophages from patients with idiopathic pulmonary fibrosis (159) Inhibits collagen synthesis by interfering with collagen cross-linking (160) Suppresses T-cell function (161)

Clinical studies have not shown it to be more effective than glucocorticoids (87–89, 173)

D-Penicillamine

Pirfenidone (5-methyl-1-phenyl-2[1H]-pyridone)

Interferon-␥ Interferon-␤1a Lovastatin Antisense therapy

Beractant

Relaxin

In vitro, inhibits TGF-␤–stimulated collagen synthesis, decreases the extracellular matrix, and blocks the mitogenic effect of profibrotic cytokines in adult human lung fibroblasts derived from patients with idiopathic pulmonary fibrosis (162) In vivo, inhibits TGF-␤ and platelet-derived growth factor gene expression (163, 164) Regulates macrophage functions (165) Inhibits fibrogenesis (166, 167) In vitro, shown to reduce fibroblast migration–proliferation (168) Inhibits collagen production by fibroblasts (167) Blocks formation of granulation tissue by induction of fibroblast apoptosis in vitro and in vivo (169) Antisense gene-specific oligonucleotide against c-Ki-ras protein substantially inhibits the proliferation of diploid human fibroblasts (170) A natural bovine lung extract containing phospholipids, neutral lipids, fatty acids, and surfactant-associated surfactant proteins B and C; promotes apoptosis of normal human lung fibroblasts (171) Inhibits the TGF-␤–mediated overexpression of collagens and fibronectin (172) Stimulates the expression of collagenase-1 by human lung fibroblasts in vitro (172)

Reduced fibrosis induced by radiation and bleomycin (174, 175) Limited studies have not shown efficacy in idiopathic pulmonary fibrosis (88, 176, 177) Ameliorates pulmonary fibrosis in animal models of bleomycin-induced lung injury (163, 164) Is well tolerated; slows progression of disease and allows tapering of glucocorticoid and immunosuppressive therapy (162) Combination of interferon-␥1b and an oral glucocorticoid lead to improvement or stabilization of idiopathic pulmonary fibrosis (178) Results of an ongoing trial in the United States and Canada will be available in 2001 No clinical trials available No clinical trials available

No clinical trials available

No clinical trials available; has been shown to block bleomycin-induced fibrosis in mice (172)

* Numbers in parentheses are reference numbers. TGF ⫽ transforming growth factor.

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Table 3. Molecules or Drugs That Inhibit Epithelial Injury or Enhance Repair* Agent

Mechanism of Action

Comments

N-Acetylcysteine

Prevents epithelial cell injury mediated by oxygen radicals (179–183) Induces proliferation of type II pneumocytes (184, 185)

Clinical studies have not shown it to be more effective than glucocorticoids (179) Proliferation occurs when factor is used after the fibrotic insult has been unsuccessful in acute lung injury (185) Abnormally stimulated alveolar epithelial cells may play a deleterious role, increasing intra-alveolar antifibrinolytic activity and secreting fibrogenic cytokines Trial is ongoing at the National Institute of Respiratory Diseases, Mexico (189)

Keratinocyte growth factor

Captopril

Inhibits the angiotensin-converting enzyme, completely abrogates Fas-induced apoptosis in human alveolar epithelial cells (186) Inhibits fibroblast proliferation in vitro and reduces fibrotic lung response in vivo (187, 188)

* Numbers in parentheses are reference numbers.

fective as antifibrotic agents (87– 89, 157–178) is provided in Table 2. Table 3 lists agents that have putative protective effects on alveolar epithelial cells (179 –189). However, most of the mentioned agents have not been adequately studied in humans. A logical therapeutic approach would be the downregulation of myofibroblasts through induction of apoptosis, as occurs in normal wound healing (144, 190). Programmed cell death allows the elimination of specific populations of cells without additional tissue damage. No specific drug for that purpose is clinically available, but experimental results with lovastatin are encouraging (169). However, these findings should be interpreted carefully. For example, we need to be sure about cell specificity and the interdependence between epithelial cells and fibroblasts. In other words, although reduction of fibroblast growth or stimulation of fibroblast apoptosis is desirable, inhibition of alveolar epithelial cell proliferation or induction of epithelial apoptosis should be avoided to enhance reepithelialization. In this context, the experiments of Bowden and colleagues (191) suggest that inhibition of fibroblast growth does not necessarily promote normal lung repair. Use of a proline analogue that reduces fibroblast proliferation failed to inhibit fibrosis in an in vivo–ex vivo model because concentrations that reduced fibroblast growth also inhibited epithelial repair. Another exciting approach is related to the possibility of changing the phenotype of myofibroblasts. Myofibroblasts represent an aggressive profibrotic phenotype that also plays a deleterious role in increasing lung contractility and decreasing compliance. It has been shown that interferon-␥ decreases the expression of ␣-smooth www.annals.org

muscle actin and alters the spindle morphologic characteristics of fibroblasts pretreated with transforming growth factor-␤; these findings suggest that interferon-␥ is able to reduce the amount of myofibroblasts (192). In addition, evidence suggests that agents that directly induce type II pneumocytes to proliferate might ameliorate the fibrotic response to different injurious agents and reduce subsequent death. Keratinocyte growth factor seems to have this effect (179, 180). Similarly, therapy with repeated transfections of the human hepatocyte growth factor gene strongly inhibited fibrogenesis and hepatocyte apoptosis and completely resolved fibrosis in a rat model of liver cirrhosis (193).

CONCLUSION Over the past decade, it has been evident that idiopathic pulmonary fibrosis, a disease more common than previously believed, constitutes a challenge for clinicians and researchers. Although its pathogenesis remains incompletely understood, we propose that this disease represents a form of abnormal wound healing in the lung that is characterized by fibroblast–myofibroblast migration and proliferation, decreased myofibroblast apoptosis, and increased activity and responses to fibrogenic cytokines (transforming growth factor-␤1, tumor necrosis factor-␣, platelet-derived growth factor, and insulin-like growth factor). Likewise, an absence of appropriate reepithelialization and impaired extracellular matrix remodeling (including basement membrane disruption, angiogenesis, and fibrosis) has been proposed to help explain the abnormal repair process. An integral understanding of the sequence of the pathogenic mechanisms 16 January 2001 Annals of Internal Medicine Volume 134 • Number 2 145

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as well as the plethora of biological events that control the fibrotic response will improve therapeutic strategies—and ultimately outcome—in patients with this disease. From Instituto Nacional de Enfermedades Respiratorias and Universidad Nacional Autońoma de Me´xico, Mexico City, Mexico; and the University of California, San Francisco, San Francisco, California. Acknowledgments: The authors thank Dr. Iasha Sznajder for providing the impetus for the writing of this manuscript. Requests for Single Reprints: Moise´s Selman, MD, Instituto Nacional de Enfermedades Respiratorias, Tlalpan 4502, CP 14080, Mexico DF, Mexico; e-mail, [email protected]. Current Author Addresses: Dr. Selman: Instituto Nacional de Enfermedades Respiratorias, Tlalpan 4502, CP 14080, Mexico DF, Mexico. Dr. King: Department of Medicine, San Francisco General Hospital, University of California, San Francisco, 1001 Potrero Avenue, Room 5H22, San Francisco, CA 94110. Dr. Pardo: Facultad de Ciencias, UNAM, Apartado Postal 21-630, Coyoacan, Mexico DF 04000, Mexico.

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