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(Chest. 2005;127:266-274.)
© 2005 American College of Chest Physicians

Expression of Apoptotic and Antiapoptotic Markers in Epithelial Cells in Idiopathic Pulmonary Fibrosis*

Maria Plataki, MD; Anastassios V. Koutsopoulos, MD; Katherine Darivianaki, BS; George Delides, MD, PhD; Nikolaos M. Siafakas, MD, PhD, FCCP and Demosthenes Bouros, MD, FCCP

* From the Departments of Pneumonology (Drs. Plataki and Siafakas) and Pathology (Drs. Koutsopoulos and Delides, and Ms. Darivianaki), Medical School, University of Crete, Heraklion, Crete, Greece; and the Department of Pneumonology (Dr. Bouros), Medical School, University of Thrace, Alexandroupolis, Thrace, Greece.

Correspondence to: Demosthenes Bouros, MD, FCCP, Professor of Pneumonology, Medical School University of Thrace, University General Hospital, Alexandroupolis 68100, Greece; e-mail: bouros{at}med.duth.gr


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objective: Idiopathic pulmonary fibrosis (IPF) is a chronic, usually fatal lung disease of unknown etiology. A common feature is the presence of microscopic areas of epithelial cell dropout. Increased apoptosis of these cells could elucidate the speculative pathogenesis of the disease. Therefore, the aim of our study was to examine the expression of p53, p21, bcl-2, bax, and caspase-3 in association with DNA strand breaks in bronchial and alveolar epithelial cells in lung specimens from IPF patients and control subjects.

Patients and methods: We examined by immunohistochemistry the expression of p53, p21, bax, bcl-2, and caspase-3 in association with DNA strand breaks detected by terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) in bronchial and alveolar epithelial cells in lung specimens taken by biopsy in 12 IPF patients and 10 control subjects. An independent tissue evaluation by two pathologists graded semiquantatively the degree of staining present.

Results: TUNEL was positive in epithelial cells in all IPF patients and only in one control subject. The expression of p53, p21, bax, and caspase-3 was up-regulated in IPF patients compared to control subjects. Bcl-2 was expressed less in IPF patients than in control subjects.

Conclusions: These results confirm that apoptotic hyperplastic epithelial cells are present in patients with IPF and that the expression of p53, p21, bax, and caspase-3 appears to be up-regulated and that of bcl-2 down-regulated in these cells. The increased expression of proapoptotic molecules in epithelial cells in IPF may be involved in the inadequate and delayed reepithelialization, which in turn contributes to fibroblast proliferation.

Key Words: apoptosis • epithelial cells • idiopathic pulmonary fibrosis • pathogenesis


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease of unknown etiology. The diagnosis of IPF requires compatible clinical history, and spirometric and roentgenographic findings, and the exclusion of other known causes of interstitial lung disease.1 Usual interstitial pneumonia (UIP) is the histopathologic pattern that identifies patients with IPF in lung biopsy specimens.1 UIP is characterized by alternating areas of normal lung, interstitial inflammation, fibroblastic foci, dense fibrosis, and honeycomb changes.12 It has been widely held that pulmonary fibrosis begins with alveolar inflammation and that chronic inflammation injures the lung and modulates fibrogenesis, leading to fibrosis.23 However, inflammation is not a prominent histopathologic finding, and epithelial injury in the absence of ongoing inflammation is sufficient to stimulate the development of fibrosis.2 A new theory suggests that IPF involves abnormal wound healing in response to multiple, microscopic sites of ongoing alveolar epithelial injury and activation that are associated with the formation of fibroblast-myofibroblast foci, which evolve into fibrosis.2 A common feature is the presence of microscopic areas of epithelial cell dropout.4567 The explanation, however, for the loss of the alveolar epithelial cells remains speculative. Increased apoptosis of the pulmonary epithelial cells could contribute to the loss of balance in cell turnover and the abnormal reepithelialization implicated in the pathogenesis of the disease.

p53 is a tumor suppressor gene that appears to be a critical mediator of the following two different cellular responses following exposure to DNA damage: growth arrest in the G1 phase of the cell cycle while the cell attempts DNA repair; or apoptosis in the case of irreparable damage.48 p21, variously known as Cip1, Sdi1, and Waf1, is a 21-kd protein that is induced in wild- type p53-containing cells following exposure to DNA-damaging agents, but not in mutant p53-containing cells.489 Transcriptional activation of the p21 gene by p53 results in the inhibition of G1 cyclin-dependent kinases through the increased binding of p21 to these cell-cycle regulatory complexes, leading to G1 arrest.9

The bcl-2 family of proteins regulates cell death.101112 Bcl-2 is an intracellular membrane-associated oncogene, which functions by extending cellular survival via inhibiting a variety of apoptotic deaths, including those that have been shown to be both p53-dependent and p53-independent. Bax, a member of the same family, was first identified based on its ability to associate with bcl-2 and has been shown to antagonize the ability of bcl-2 to suppress apoptosis.811 Bax is an early-response apoptosis-promoting gene of p53.1213 The relative levels of bcl-2 and bax may dictate whether a cell is susceptible to apoptosis.11

Caspases are a family of proteolytic enzymes, which play an essential role in the execution of apoptosis.14 During apoptosis, a cascade of proteolysis beginning with the activation of initiator caspases, like caspase-8, causes the activation of effector caspases, such as caspase-3, leading to the cleavage of several substrates that result in the morphologic changes observed in apoptosis.

In this study, we examined the expression of p53, p21, bcl-2, bax, and caspase-3 in association with DNA strand breaks in bronchial and alveolar epithelial cells in lung specimens in 12 patients with IPF and 10 control subjects.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
This study was performed on lung specimens from 12 patients with IPF. Samples were obtained by thoracoscopic lung biopsy. The study was approved by the institutional review board for human studies. All patients included in the study were male, except one, and were 42 to 77 years old (median age, 65.3 years old). Six patients were ex-smokers, and six patients were nonsmokers. The diagnosis of IPF was based on strict criteria (ie, clinical history, physical examination, roentgenographic findings, laboratory tests, pulmonary function tests, histologic findings, and the exclusion of other known causes of interstitial lung disease), according to the latest American Thoracic Society/European Respiratory Society criteria.1 The histologic diagnosis in all specimens was compatible with that of UIP. All patients were treatment naïve when included in the study. Normal lung parenchyma specimens that were obtained from 10 patients undergoing thoracotomy for bullectomy, during the same time period as the IPF patients, were used as control subjects. Patients in the control group had no other underlying lung pathology, apart from bullas. The control group was composed of eight male subjects and two female subjects (age range, 15 to 66 years; median age, 35.6 years), five smokers, two ex-smokers, and three nonsmokers.

Tissue Preparation
IPF and control specimens underwent the same procedures throughout the study and were stained simultaneously. Tissue samples were fixed in 10% formalin overnight and embedded in paraffin. A 4-µm-thick paraffin section was adhered to slides (SuperFrost/Plus slides; O. Kindler GmbH; Freiburg, Germany). Deparaffinization was accomplished by heating the sections for 1 h at 60°C. These sections were dewaxed by washing three times for 5 min each in xylene, then were dehydrated in 100% and 95% ethanol three times for 5 min each, and 80% and 70% ethanol for 5 min each, and finally were rinsed with distilled water.

DNA Nick End-labeling of Tissue Sections
The method of terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) was performed using an in situ cell death detection kit (Boehringer Mannheim; Mannheim, Germany). The nuclei of tissue sections were stripped of proteins by incubation with 20 µg/mL proteinase K for 30 min at 37°C. The TUNEL reaction mixture was prepared by adding 50 µL terminal deoxynucleotidyl transferase from a calf thymus to 450 µL nucleotide mixture in a reaction buffer. A total of 50 µL TUNEL reaction mixture was added to cover each section. The slides were then incubated in a humidified chamber for 60 min at 37°C while covered with coverslip to prevent drying of sections. After washing three times with phosphate-buffered saline solution (PBS), the slides were incubated with 50 µL Converter-alkaline phosphatase (AP) (antifluorescein antibody, a Fab fragment from sheep conjugated with AP) for 30 min at 37°C in a humidified chamber. The slides were rinsed three times with PBS and were incubated with 50 µL substrate buffer with Fast Red for 10 min at room temperature (RT). The slides were washed with PBS, counterstained with methyl green, and covered using an aqueous mounting medium. Then they were examined under a light microscope.

Immunohistochemistry
Immunohistochemistry (IHC) for p53 (mouse monoclonal antibody [MoAb], clone DO-7; DAKO PATTS; Glostrup, Denmark), p21 (mouse MoAb, clone EA10; Oncogene Science; Cambridge, MA), bax (mouse MoAb, clone 4F11; Immunotech; Westbrook, ME), bcl-2 (mouse MoAb, clone 124; DAKO PATTS), and caspase-3 (mouse MoAb, clone 3CSP03; NeoMarkers; Fremont, CA) was performed. The monoclonal mouse antihuman p53 protein recognizes both wild-type and mutant forms of the p53 protein.

Slides were placed in a plastic jar filled with 0.01 mol/L (pH 6.0) citrate buffer, as an unmasking fluid. We treated the slides three times for 5 min at 500 W in a household microwave oven. After that, we allowed them to cool at RT, and washed them with distilled water and Tris-buffered saline solution (pH 7.6).

Immunohistochemical staining was carried out manually according to the AP-anti-AP (APAAP) complex technique (DAKO PATTS), except for bax, in which we used the secondary antibody D314 (DAKO PATTS) conjugated with AP, and D314 (rabbit antimouse Ig-AP). Briefly, the slides were treated with normal rabbit serum for 20 to 30 min at RT and were incubated for 60 min at RT, for p53, p21, and bax antibodies. The incubation of caspase-3 took place overnight at 4°C, and that for bcl-2 was performed for 120 min at RT. For the APAAP complex technique, after rinsing with Tris-buffered saline solution the sections were incubated with rabbit antimouse Ig (Z259) for 30 min and then with APAAP mouse MoAb (D651) for 30 min at RT. The same procedure was repeated with a 15-min incubation time. The substrate chromogen used was K699, a Fast-Red system (DAKO PATTS). Slides were counterstained with hematoxylin and subsequently were mounted with glycergel. An independent tissue evaluation by two pathologists (K.A. and D.G.) graded semiquantatively the degree of staining that was present, as follows: grade 0, no staining present; grade 1, < 10% of the cells are positive; grade 2, > 10% but < 50% of the cells are positive; and grade 3, > 50% of cells are positive.

Statistical Analysis
The percentage of IPF patients and control subjects with positive immunohistochemical signals were compared using the {chi}2 test (Microsoft Excel 2000; Microsoft Corp; Bellevue, WA). The statistical correlation between smoking status and TUNEL results and the expression of these markers, in patients and control subjects, was evaluated with the Spearman test using a statistical software package (SPSS, version 11; SPSS Inc; Chicago, IL). A p value of < 0.05 was considered to be significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Due to the heterogeneity of UIP, the specimens included areas of relatively normal lung parenchyma, as well as areas of distorted lung architecture and honeycomb lesions. The normal epithelium was examined separately from the hyperplastic epithelium. Reactive large, cuboidal cells, which protrude into the alveolar lumen and have replaced the thin, flat, elongated normal type I cells, were considered to be hyperplastic type II pneumocytes, which are easily observed in routine sections. The percentage of epithelial cells that are positive in each IPF patient and control subject is shown in Tables 1 and 2 .


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  		Table 1. Percentage of Positive Alveolar Cells in Each IPF Patient and Control Subject*

 

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  		Table 2. Percentage of Positive Bronchial Epithelial Cells and Epithelial Cells Lining Honeycomb Lesions in Each IPF Patient and Control Subject*

 
Positive signals by TUNEL were detected in all IPF patients (12 of 12 patients; 100%), especially in hyperplastic alveolar and bronchial epithelial cells, whereas only a few alveolar epithelial cells stained positive in one control subject (1 of 10 subjects; 10%; p < 0.0001) [Fig 1 , top left, A, and top inset, B]. Few alveolar macrophages, vascular endothelial cells, and fibroblasts also stained positive in IPF.



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  		Figure 1. Top: representative staining results of TUNEL in paraffin-embedded lung tissue from patients with IPF (top, A, original magnification x200; top inset, B, original magnification x400). The TUNEL method stained red the nucleus of hyperplastic alveolar epithelial cells (arrows). Few alveolar macrophages, vascular endothelial cells, and fibroblasts also stained positive. Bottom: immunohistochemical localization of caspase-3 in IPF patients (bottom, C, original magnification x200; bottom inset, D, original magnification x400). The expression of caspase-3 was detected both in the cytoplasm (bottom, C) and the nucleus (bottom inset, D) of alveolar epithelial cells (arrows). The significance of the different cellular distributions of caspase remains unclear.

 
Caspase-3 stained positive in hyperplastic alveolar and bronchial cells in all IPF patients (12 of 12 patients; 100%) and in few epithelial cells in 5 of 10 control subjects (50%; p < 0.0001). Caspase-3 stained not only the cytoplasm (Fig 1, bottom left, C) but also the nucleus in some cells (Fig 1, bottom inset, D). Immunoreactivity for caspase-3 was not detected in macrophages, whereas some lymphocytes stained positive.

IHC for p53 was positive in hyperplastic alveolar or bronchial epithelial cells in 10 of 12 IPF patients (83.4%). In contrast, it was negative in all but one control subject (1 of 10 control subjects; 10%; p < 0.0001) [Fig 2 , top, A].



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  		Figure 2. Top, A: expression of p53 in IPF patients (original magnification x400). Proapoptotic p53 stained red the nucleus of hyperplastic alveolar epithelial cells (arrows). Bottom: expression of p21 in IPF patients (bottom, B, original magnification x100; bottom inset, C, original magnification x400). Hyperplastic alveolar epithelial cells showed strong expression of p21 (red nuclei) in IPF patients (arrows). p21 stained a greater percentage of epithelial cells than p53.

 
p21 was strongly expressed in all patients with IPF (12 of 12 patients; 100%), especially in hyperplastic alveolar epithelial cells, while only 3 of 10 control subjects (30%) showed a few bronchial cells that were positive for p21 (p < 0.0001) [Fig 2, bottom left, B, and bottom inset, C]. p21 was expressed in a greater number of epithelial cells than p53. As in TUNEL, few alveolar macrophages stained positive for p53 and p21 in IPF patients.

By IHC for bax, all IPF patients showed positive signals (12 of 12 patients; 100%) in a small percentage of hyperplastic epithelial and bronchial cells, and 2 of 10 control subjects (20%) had a few epithelial cells that stained positive (p < 0.0001; Fig 3 , top, A).



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  		Figure 3. Top, A: immunohistochemical localization of proapoptotic bax in IPF patients (original magnification x200). Bax was strongly expressed in the cytoplasm of alveolar epithelial cells (arrows). Some lymphocytes also stained positive for bax in IPF. Middle and bottom: expression of antiapoptotic bcl-2 protein in IPF patients (middle, B) and control subjects (bottom, C, and bottom inset, D) [middle, B, and bottom, C, original magnification x200; bottom inset, D, original magnification x400]. In IPF patients (middle, B) bcl-2 stained the lymphocytes (white arrow), which served as an internal control, and not the epithelial cells (black arrows). In control subjects (bottom, C, and bottom inset, D), bcl-2 stained the cytoplasm of some alveolar epithelial cells (arrows) and lymphocytes. The relative levels of bax and bcl-2 dictate whether a cell is susceptible to apoptosis. Weakstaining of bcl-2 was detected in the smooth muscle cells of vascular and small airway walls in IPF patients and control subjects.

 
Bcl-2 protein was detected in bronchial and alveolar epithelial cells in all control subjects (10 of 10 subjects; 100%) [Fig 3, bottom left, C, and bottom inset, D]. Little positive staining (ie, < 10% of the cells) for bcl-2 was seen in epithelial cells, hyperplastic and normal, in 3 of 12 IPF subjects (25%; p < 0.0001) [Fig 3, middle, B]. Weak staining of bcl-2 was detected in the smooth muscle cells of vascular and small airway walls in IPF patients and control subjects. Some lymphocytes also stained positive for bax and bcl-2, in both IPF patients and control subjects. There was no statistical correlation of TUNEL results and the expression of p53, p21, bcl-2, bax, and caspase-3 with the smoking status of either IPF patients or control subjects.


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we found increased apoptosis of hyperplastic epithelial cells and the overexpression of all proapoptotic molecules studied in IPF patients compared to control subjects. We also found reduced expression of the antiapoptotic bcl-2 protein in epithelial cells in IPF patients compared to control subjects, underlining the potential contribution of the imbalance between apoptosis-inducible and apoptosis-inhibitory proteins in apoptosis in epithelial cells in IPF patients. Our study is the first to examine the expression of bax in combination with bcl-2, and the second to examine the expression of p21 and caspase-3 in epithelial cells in IPF patients. All of these apoptosis-regulatory molecules have not been studied in combination before. These findings support the notion that apoptosis could be a key element in the pathogenesis of IPF and indicate possible pathways implicated in this disease as future targets of therapeutic attempts.

Apoptosis is programmed cell death, a highly ordered physiologic process by which useless, irreparably damaged, or unwanted cells are eliminated during development and normal biological processes. Fewer articles appear each year investigating apoptosis in the lungs than investigating other major organs.1516 Elucidating the regulating mechanisms of apoptosis in specific lung cell types would contribute to the understanding of normal lung function as well as the pathophysiology of many lung disorders, and also would allow for specific therapeutic targets.16

IPF is a poorly understood disease with fatal consequences to those persons with IPF. The concept about its pathogenesis that dominated in the 1970s and 1980s has been described as the "inflammatory" concept of pulmonary fibrosis.17 This hypothesis supports a sequence of events that is initiated by a lung injury from an exogenous insult, which is followed by an inflammatory process, and then by fibroproliferation and fibrosis. However, clinical measurements of inflammation fail to correlate with stage or outcome, and potent antiinflammatory therapy does not improve the outcome.2 Another theory has suggested that IPF represents a model of abnormal wound healing in the lung in response to multiple, microscopic sites of ongoing alveolar epithelial injury and activation associated with fibroblast-myofibroblast foci, which evolve into fibrosis.2 Some evidence has shown that it is possible to dissociate the inflammatory response from the fibrotic response and that inflammation is not required for the development of a fibrotic response.2

In this study, we examined the expression of p53, p21, bcl-2, bax, and caspase-3 in association with DNA strand breaks in bronchiolar and alveolar epithelial cells in patients with IPF and control subjects in order to assess the role of apoptosis in the pathogenesis of IPF. Bcl-2 was selected in order to examine the antiapoptotic side. It is the balance between bax and bcl-2 that dictates whether a cell will undergo apoptosis or not. Increased apoptosis could also be due to a decreased antiapoptosis balance.

A very important step for the preservation of normal alveolar epithelial function after injury, which seems to be impaired in IPF, is the rapid reepithelialization, through the migration, proliferation and differentiation of cells.2 In UIP, the alveolar epithelial cells show ultrastructural alterations, hypertrophy, and hyperplasia, and a modulation of a series of structural and membrane proteins.18 The damaged epithelial type I cells are replaced by hyperplastic type II cells. The proliferative capacity of type II cells to restore damaged type I cells seems to be seriously affected in patients with fibrosis, resulting in epithelial cuboidalization and transitional phenotypes between type II and type I cells. For this reason, we decided to examine separately the relatively normal epithelium and the hyperplastic epithelium. Our results confirmed that apoptotic hyperplastic epithelial cells are present in patients with IPF, and show that the expression of p53, p21, bax, and caspase-3 appears to be up-regulated and that of bcl-2 appears to be down-regulated in these cells in IPF patients.

The mechanism through which epithelial cell apoptosis and denudation could lead to fibrosis remains unknown, however, a dysfunctional epithelial repair process with a delay in appropriate reepithelialization seems to be a key feature. Adamson et al,19 in a study of mouse lung explants after exposure to hyperoxia, found that epithelial injury alone could stimulate fibrosis in a blood-free environment. Witschi,20 in an analysis of toxic lung damage focusing on the biological response, suggested that delayed reepithelialization of the alveolar surface may lead to pulmonary fibrosis. It also has been demonstrated that the mechanical injury of epithelial cells induces myofibroblast differentiation when coculturing airway epithelial cells and fibroblasts.21 Moreover, studies on the repopulation of denuded tracheal explants by epithelial cells have shown that the denuded tracheal implants fill rapidly with fibroblasts, unless enough epithelial cells are introduced into the lumen to control fibroblast proliferation.22 The intact alveolar epithelium produces prostaglandins of the E series, which are known inhibitors of fibroblast proliferation.523 Furthermore, it protects underlying interstitial cells from exposure to growth factors that are released by intraalveolar macrophages and other inflammatory cells.23 Apoptotic cells are cleared in vivo with minimal local reaction, and it has been suggested that this results from an active production of antiinflammatory mediators, particularly transforming growth factor (TGF)-ß.2425 TGF-ß, however, is a known profibrotic agent. A repetitive or continuous supply of apoptotic cells could lead to an increasing supply of TGF-ß by neighboring cells, contributing to the fibrotic response.24 The epithelium may be implicated in IPF at the following two levels: as an initial target for injury/turnover; and as a normally regulating agent to stop ongoing fibrosis or even to induce its resolution. Alveolar epithelial cell hyperplasia may represent an attempt to resolve the fibrosis.24

A gradually growing body of literature confirms the presence of apoptosis in the alveolar and bronchial epithelial cells as an early feature in IPF. In a study by Uhal et al,5 both electron microscopy and picrosirius red staining confirmed epithelial cell apoptosis adjacent to foci of collagen accumulation surrounding fibroblast-like cells. Moreover, earlier work from the same laboratory26 showed that abnormal fibroblast phenotypes isolated from fibrotic human lung produce factors that are capable of inducing apoptosis of alveolar epithelial cells in vitro. Barbas-Filho et al27 also have reported that numerous epithelial cells were found to undergo apoptosis in otherwise normal areas of lung parenchyma in patients with IPF/UIP. The presence of apoptotic epithelial cells in patients with IPF detected by the TUNEL method was confirmed by Kuwano et al4 and Maeyama et al.6

Several mechanisms have been implicated in pneumocyte apoptosis. There is only one previous study by Kuwano et al4 examining p21, and one by Maeyama et al6 examining caspase-3 in epithelial cells in patients with IPF. The patients included in our study, however, were selected according to the latest criteria, whereas at the time the studies by Kuwano et al4 and Maeyama et al6 were performed the American Thoracic Society/European Respiratory Society criteria were not available. As well, in the study by Kuwano et al4 the authors did not state whether the histologic diagnosis of IPF patients was compatible with UIP. In agreement with our study, Kuwano et al4 found increased expression of p53 and p21 in the epithelial cells of patients with IPF compared to those in control subjects.4 In our study, p21 was expressed in a greater percentage of cells than p53. It has been suggested that p21 can be also activated by a p53-independent pathway.28 It is possible that in IPF patients p21 is activated not only by a p53-dependent pathway, but also by a p53-independent pathway by fibroblast-regulating cytokines that are released by inflammatory cells.429 The increased expression of caspase-3 in the epithelial cells of IPF patients found by Maeyama et al6 was confirmed in our study. We also observed both nuclear and cytoplasmic staining for caspase-3 in IPF patients. Maeyama et al6 have suggested that caspase-3 and caspase-1 may translocate into the nucleus during the apoptotic process in a human lung epithelial cell line, and that the nuclear expression of caspases in lung epithelial cells of IPF patients may reflect the activation of caspases and the executional process of apoptosis. The cellular distribution of caspases and its meaning remain to be elucidated. In contrast to that study, however, we found no positive signal for caspase-3 in macrophages in IPF patients and little positive signal in lymphocytes in IPF patients and control subjects. The expression of bcl-2 in patients with IPF was also examined by Kazufumi et al.30 They found no staining for bcl-2 in the epithelial cells of IPF patients, whereas we found little positive staining, in < 10% of epithelial cells, in 25% of IPF patients. In agreement with their study, positive staining was observed in lymphocytes, and weak staining was observed in the smooth muscle cells of vessel and small airway walls in both IPF patients and control subjects. The expression of bax has not been studied before in epithelial cells of lung biopsy specimens in humans with IPF. In a study of bleomycin-induced pulmonary fibrosis in mice,8 bax protein was up-regulated in epithelial cells, whereas the expression level of bcl-2 protein was not changed. Mermigkis et al31 found overexpression of the bcl-2 protein on neutrophils and eosinophils in the BAL fluid of IPF patients. The Fas-Fas ligand pathway to apoptosis also was found to be up-regulated in IPF patients,632 and this pathway has been implicated in the pathogenesis of fibrosis through the induction of apoptosis of alveolar epithelial cells.33343536

The etiologic relationship between apoptosis and the pathogenesis of fibrosis has been extensively studied in vitro. Repeated inhalation of an anti-Fas antibody mimicking Fas-Fas ligand cross-linking induced excessive apoptosis, which resulted in pulmonary fibrosis in mice.33 Moreover, the administration of a soluble form of Fas antigen or anti-Fas ligand antibody prevented the development of fibrosis after bleomycin instillation in mice, and lpr and gld mice (loss of functional mutations of Fas and Fas ligand) were resistant to fibrosis after intratracheal bleomycin instillation.35 The administration of a broad-spectrum caspase inhibitor by aerosol decreased the caspase-1 and caspase-3 activity, the number of apoptotic cells, and the pathologic grade of fibrosis in a bleomycin fibrosis model in mice.37 A broad-spectrum inhibitor of caspases was found to be able to block alveolar epithelial cell apoptosis, the accumulation of lung collagens, and lung fibrosis in vivo after intratracheal administration of bleomycin in rats.38 The antifibrotic potential of this agent seems to be related to its ability to prevent the apoptotic death of the epithelial layer.16

In conclusion, we found increased expression of proapoptotic and decreased expression of antiapoptotic molecules in epithelial cells in IPF patients, which may be responsible for the inadequate and delayed reepithelialization, which in turn contributes to fibroblast proliferation. Discovery of the pathogenetic mechanisms leading to this condition will help to develop new therapeutic strategies. Future research must concentrate on identifying the critical proapoptotic or antiapoptotic proteins that account for the final picture in IPF patients and on the possible ways to effectively block their activation. It is possible that no single molecular target and no single therapeutic approach will be sufficient to control the fibrosis.


   Footnotes
 
Abbreviations: AP = alkaline phosphatase; APAAP = alkaline phosphatase-antialkaline phosphatase complex; IHC = immunohistochemistry; IPF = idiopathic pulmonary fibrosis; MoAb = monoclonal antibody; PBS = phosphate-buffered saline solution; RT = room temperature; TGF = transforming growth factor; TUNEL = terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling; UIP = usual interstitial pneumonia

This study was supported in part by a research grant from GlaxoSmithKline Greece.

Received for publication April 28, 2004. Accepted for publication August 26, 2004.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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