Pulmonary Fibrosis
Pathways Are Slowly Coming into Light

J.Allen D. Cooper Jr.

Pulmonary Sections, Birmingham V.A.M.C.; and the University of Alabama at Birmingham, Birmingham, Alabama

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Pulmonary fibrosis is a devastating disorder that is resistant to treatment. Patients with idiopathic pulmonary fibrosis (IPF) have a median survival of 4 to 5 yr after onset of symptoms (1). Although corticosteroids continue to be the primary mode of treatment for this disorder, they improve lung function in less than 30 percent of treated patients. Pulmonary fibrosis can also arise from known precipitating factors, including lung exposure to exogenous agents by inhalation, as with pneumoconioses or hypersensitivity pneumonitis, or systemically, as with drug-induced pulmonary fibrosis. One drug, bleomycin, is associated with significant pulmonary side effects, including fibrosis, that can limit its use. Bleomycin was first noted to cause pulmonary fibrosis in initial trials using the drug (2). Since that time, risk factors and incidence of bleomycin-induced pneumonitis/fibrosis have been elucidated (3). Overall, approximately four percent of patients treated with the drug develop pneumonitis and fibrosis. Although prognosis of bleomycin pneumonitis/fibrosis is difficult to quantitate because of the affected patients' underlying illnesses, it is most likely much better than for patients with IPF. Bleomycin also has well documented subcellular effects on normal and malignant tissues, including the generation of reactive oxygen species (4) and induction of apoptosis (5).

Shortly after pulmonary fibrosis was noted in humans receiving bleomycin, an animal model was developed (6). Subsequently, there has been a wealth of studies employing bleomycin in mice, rats, hamsters, dogs, and other species (5). Although there are some limitations in these animal models, they have generally been helpful in directing research toward the understanding of the mechanisms of pulmonary fibrosis in humans. The limitations of extrapolating animal models of bleomycin-induced pulmonary fibrosis to IPF or bleomycin toxicity in humans include (1) the natural resolution of fibrosis induced by intratracheal bleomycin instillation in animals compared with the common progression of IPF in humans; (2) the favorable response of bleomycin-induced pneumonitis/fibrosis in animals or humans to agents, such as corticosteroids (3), that are less effective in IPF, and (3) the common use of intratracheal bleomycin to induce fibrosis in animal models, which may change the dynamics of the process in comparison with the intravenous route of administration given humans. However, as noted, the use of these animal models has been helpful in partly establishing pathways of lung damage leading to fibrosis, and comparison studies of patients with IPF have validated many of these animal studies. This Perspective article discusses some of these studies and attempts to compare the data between each study to help uncover pathways of pneumonitis/fibrosis, pathways already being used to design treatment strategies.

The process leading to pulmonary fibrosis originates in the alveolus (6). The earliest event in lungs of patients developing bleomycin-induced pneumonitis/fibrosis is probably endothelial damage possibly manifested by a transient reduction in diffusing capacity for carbon monoxide (3). However, epithelial injury and alveolar inflammation caused by plasma exudation and increased macrophage numbers is probably the initial event resulting in clinical disease (6) (Figure 1). Pulmonary fibrosis resulting from chemotherapeutic agents has been particularly associated with dysplastic alveolar epithelial cells, but studies have suggested that this finding is nonspecific (3). In virtually all cases of pneumonitis/fibrosis, regardless of precipitating stimuli, there are detectable alterations in alveolar epithelial cells. After the initial alveolitis has occurred, there is organization of alveolar exudate and incorporation of the fibroproliferative process into alveolar walls, resulting in interstitial fibrosis with scarring and altered function of the alveolar unit (6). Factors modulating the initial alveolar epithelial cell alterations are not totally defined, but knowledge of some pathways is beginning to emerge.

View larger version (39K): [in this window] [in a new window]   Figure 1.   Early events in the pathogenesis of pneumonitis/fibrosis (borrowed with permission from an article by Kuhn and colleagues [6]).

Several pieces of information suggest that transforming growth factor  (TGF-) is important in the process of bleomycin-induced pulmonary fibrosis in animals (7), as well as IPF in humans (11, 12). TGF- pulmonary levels are elevated in mice (7) and rats (8) after intratracheal bleomycin instillation, during the development of pneumonitis/fibrosis. Mice strains sensitive to the effects of bleomycin instillation exhibit a greater degree of TGF- stimulation with the agent than do strains that are insensitive (7). In addition, the antibody to TGF- (9) and a receptor antagonist for this cytokine (10) attenuate pulmonary collagen accumulation induced by bleomycin. In humans with IPF, evidence also shows that TGF- is increased (11, 12), and treatment with gamma interferon decreases levels of this cytokine in conjunction with slowing progression of the disease (12). These studies provide evidence that TGF- is at least one important mediator of pulmonary fibrosis in both animals and humans.

The specific cellular source of TGF- production in the setting of pulmonary fibrosis is not entirely clear. Bleomycin stimulates endothelial TGF- production in vitro through its effects on gene transcription (13). Because endothelial damage is probably an early event in bleomycin toxicity, this may be the initial source of this cytokine. However, other studies suggest pulmonary macrophages and epithelial cells produce increased amounts of TGF- in animals exposed to bleomycin (14) and patients with IPF (11). One study (14) on lungs from rats exposed to intratracheal bleomycin showed a variation in the location of immunohistochemical staining for TGF- according to the time after drug exposure. Between two hours and four days after exposure, TGF- was primarily localized to the bronchial epithelium. Between four and seven days after exposure, alveolar macrophages showed increased TGF- concentrations. After seven days TGF- was localized to areas of pneumonitis and matrix deposition. In a study of patients with pulmonary fibrosis due to IPF (11), as well as other causes, TGF-₁ staining was noted in epithelial cells and macro- phages in patients with endstage fibrosis, whereas lungs from patients with early disease demonstrated localization of TGF- primarily in alveolar macrophages. However, it appears that there are multiple cellular sources of TGF- during the process of pneumonitis developing into fibrosis. The relative order in which specific cells demonstrate increased TGF- expression should be interpreted with caution in an animal model using intratracheal bleomycin because of the difference in site of exposure compared with patients receiving intravenous bleomycin. Multiple cellular sources of TGF- appear to be activated during development of pulmonary fibrosis, with macrophages and epithelial cells being the primary sources.

What effects of TGF- mediate pulmonary fibrosis? There are multiple effects of this cytokine that can explain amplification of this process. TGF- is chemotactic for fibroblasts (15) and polymorphonuclear neutrophils (PMN) (16). Fibroblasts have obvious importance in the fibrotic process. PMN are also present in increased numbers in the lung in the setting of bleomycin injury (17) and IPF (18), although the role of these cells in pneumonitis/fibrosis remains somewhat controversial. In addition, a potentially important effect of TGF- is induction of programmed cell death (apoptosis) in epithelial cells (19). There is increasing interest in the importance of epithelial cell apoptosis during development of pneumonitis/fibrosis. A recent study (20) demonstrated that mice deficient in Fas or Fas ligand, a molecular system important for cellular apoptosis, develop less fibrosis after exposure to intratracheal bleomycin when compared with wild-type mice. This protection from fibrosis occurs in conjunction with a reduction in epithelial cell apoptosis. Because research over the past 10 to 15 years has suggested that pulmonary fibrosis is initiated in the alveolus, with epithelial injury being an early event, the link between apoptosis of these cells due to Fas activation and progression of pneumonitis/fibrosis is intriguing. In addition, another study (21) has demonstrated that soluble Fas ligand is potently chemotactic for PMN, suggesting another potential role for the Fas/Fas ligand system in inflammation associated with pneumonitis/fibrosis.

In this issue, Mishra and colleagues demonstrate alveolar epithelial damage in mice exposed to intratracheal bleomycin in conjunction with increased expression of several proteins that affect cell growth and proliferation (22). They find that bleomycin exposure induces increased concentrations of p53 tumor-suppressor protein, a DNA damage-inducible protein, as well as p21^WAF1/PiC1 and proliferating cell nuclear antigen (PCNA), two proteins involved in DNA replication and repair. Immunohistochemical studies demonstrate that p53 concentrations are localized to a number of cell types, including epithelial cells present in fibrotic regions, whereas unaffected regions of lung do not show a significant increase in staining for this protein. Double immunostaining suggests type II alveolar epithelial cells overexpress all of these proteins. In general, levels of all of these proteins peak seven to nine days after exposure to the drug, then start to fall.

This study featured in this issue (22) complements the other recent study, which has suggested that the Fas/Fas ligand system is important for induction of fibrosis by bleomycin (23) and the wealth of information that suggests TGF- is important in the pathogenesis of pneumonitis/ fibrosis. Previous evidence indicates that TGF- induces p53 expression, that, in turn, causes Fas-receptor clustering and caspase-8 activation with subsequent cellular apoptosis (24, 25). Clustering of Fas is the result of altered receptor dynamics by p53. Apoptosis of malignant cells induced by bleomycin is also dependent on p53 through enhanced Fas expression (5). In human vascular smooth-muscle cells, p53 increases Fas expression by enhancing transport from the Golgi complex and transiently sensitizing cells to Fas-induced apoptosis (26). A similar mechanism may be active in epithelial cells, as these cells express both of these proteins. As Mishra and colleagues note, p53 may also be working through transcriptional activation of p21^WAF1/PiC1 and PCNA (22). Because p21 blocks the effect of PCNA on DNA polymerase, this could result in cell-growth arrest and apoptosis. The current study also confirms previous reports of increased p53 and p21 expression in the lungs of patients with IPF (27), suggesting the authors' findings in the current study are clinically relevant.

Some questions still have not been answered by the current study and are always difficult to address because of the dynamic nature of pneumontis/fibrosis. The first question is whether increased DNA damage-inducible proteins noted by the authors are part of the injury or repair process. Previous studies have demonstrated that Fas antibodies, which trigger cellular apoptosis by cross-linking Fas, can cause pulmonary fibrosis (28), suggesting that programmed cell death by itself is sufficient to cause this process. However, as noted by Mishra and colleagues, p53 is expressed during normal wound healing (29). What differentiates normal lung repair from abnormal scar formation is still under debate. The second difficulty is the question of how the method of injury contributed to the authors' findings. Certainly, intratracheal administration of bleomycin would be expected to affect epithelial cells first. However, alveolar deposition is probably low, so direct effects on alveolar epithelium are probably insignificant. As with other studies using intratracheal bleomycin, previous confirmatory immunohistochemical studies in humans are helpful (27). The last concern is of how bleomycin increases expression of p53 and the other proteins noted in the study. In fact, there may be multiple pathways of activation either through activation of cytokines, particularly TGF-, or by producing DNA abnormalities through production of reactive oxygen species. Use of specific antibodies to cytokines, transgenic mice strains deficient in cytokines or delivered antioxidants could be used to address this issue.

Taking all of these findings into account, a scheme for one pathway leading to pneumonitis/fibrosis is proposed in Figure 2. This is a very simplified model for only one pathway leading to pulmonary fibrosis. Although TGF- certainly plays some role in pulmonary fibrosis due to bleomycin or IPF, there may also be other pathways that result in epithelial cell apoptosis or other cellular alterations that play a part in the process. In the presence of iron and oxygen, bleomycin is a powerful oxidizing agent, and oxidants generated by this process may induce DNA strand breaks, leading to cellular apoptosis. Tumor necrosis factor (30) and the fibrinolytic system (31, 32) also play important roles in bleomycin-induced pneumonitis/fibrosis. In one study (30), mice given intratracheal bleomycin developed increased lung tumor necrosis factor- (TNF-) messenger RNA (mRNA) levels, and antimouse TNF- prevented lung collagen accumulation due to bleomcyin. Another study (31) has demonstrated that the procoagulant tissue factor and fibrinolytic molecule plasminogen activator inhibitor (PAI-1) increase in lungs injured by bleomcyin. In addition, a link between PAI-1 lung concentrations and bleomycin-induced fibrosis in transgenic mice expressing variable levels of this fibrinolytic agent has also been demonstrated (32). These studies suggest that the process leading to pneumonitis/fibrosis is complex, and therapeutic strategies may be difficult to devise. However, clinical trials have already begun employing biologic agents that short-circuit this process (12). Hopefully, other agents can be designed using information from these studies that will be more effective than the current therapeutic options for patients with IPF and other forms of pulmonary fibrosis.

View larger version (12K): [in this window] [in a new window]   Figure 2.   Potential pathways of pulmonary injury occurring during the process of pneumonitis/fibrosis.

	Footnotes

Address correspondence to: J. Allen D. Cooper, Jr., M.D., Professor of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, University Station, Birmingham, AL 35294.

(Received in original form February 14, 2000).

Acknowledgments: This study was supported by VA Merit Review Research funds.

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