Introduction

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Interstitial pulmonary fibrosis (IPF), which is characterized by an excess deposition of extracellular matrix (ECM) in the interstitial spaces of the lung, is the end stage of many lung disorders (Crouch, 1990). The bleomycin (BL) model of lung fibrosis involves an initial injury to the lung, followed by an influx of inflammatory cells that release cytokines that increase the synthesis of collagen by the mesenchymal cells, resulting in an increased connective tissue deposition (Bitterman et al., 1983). The inflammatory response includes an initial phase involving an increase in the number of neutrophils followed by a sustained increase in the number of macrophages and lymphocytes at the later phases in the lungs (Chandler et al., 1983). The alveolar macrophages are known to be the predominant cell type in the alveolar space and are known to play a crucial role in inflammation and wound repair. In the BL-induced lung injury, there is an increase in the influx of macrophages into the lung, and these activated macrophages produce a variety of cytokines such as interleukin (IL)-1, tumor necrosis factor-, platelet-derived growth factor (PDGF), and transforming growth factor (TGF)- (Martinent et al., 1987; Jordana et al., 1988; Piguet et al., 1989; Khalil et al., 1989) in the lung. TGF- is known to play a central role in modulating the inflammatory response and in regulating the pathogenesis of pulmonary fibrosis (Khalil et al., 1989, 1991).

TGF- is known to exist in five different isoforms, and types I, II, and III have been identified in mammals (Roberts and Sporn, 1990). Type I is the isoform known to be most implicated in fibrosis (Border and Noble, 1994). Numerous resident and recruited cell types, including activated macrophages, platelets, lymphocytes, epithelial cells, and fibroblasts, are known to release TGF- by paracrine or autocrine mechanisms at the site of lung inflammation and injury (Gauldie et al., 1993). Three different receptor isoforms of TGF- have been identified on a variety of cell types. TGF- mediates most of its actions via type I and type II receptors and it plays a regulatory role in initiating, maintaining, and amplifying the effects of other chemoattractants and cytokines involved in the pathogenesis of lung injury. TGF- is known to influence the metabolism and turnover of ECM by increasing its deposition and inhibiting its degradation (Roberts and Sporn, 1990). In the process of wound healing, TGF- elevation often leads to an exuberant deposition of ECM. An increased level of TGF- is known to precede increases in collagen, fibronectin, and proteoglycan deposition (Westergren et al., 1993).The elevated levels of TGF- in animal models of lung fibrosis, and in the bronchoalveolar lavage fluid (BALF) and lung tissues of patients suffering from pulmonary fibrosis, are well documented by various investigators (Brokelman et al., 1991; Gurujeyalakshmi et al., 1998).

Treatment for IPF continues to remain a challenge for all physicians and researchers. The currently available therapeutic measures for treatment of IPF are inadequate. In addition, these measures are associated with severe side effects (Giri, 1990). Because TGF- is central to the molecular mechanism for an excess deposition of ECM, suppressing its overproduction constitutes a rational therapeutic approach in the management of pulmonary fibrosis.

Our laboratory has previously demonstrated the antifibrotic and therapeutic potential of pirfenidone (PD) in a BL hamster model of lung fibrosis (Iyer et al., 1995, 1998). We have also demonstrated that PD down-regulates the BL-induced overexpression of procollagen I and III gene expression. (Iyer et al., 1999) There is evidence that TGF- stimulates procollagen gene expression (Raghow et al., 1985, 1987; Khalil et al., 1989; Breen et al., 1992). We tested the hypothesis that PD perhaps acts more proximally and initially down-regulates the BL-induced overexpression of TGF- gene, followed by a subsequent down-regulation of BL-induced overexpression of lung procollagen genes, as demonstrated in our earlier study (Iyer et al., 1999). To test this hypothesis, we have evaluated the effects of PD treatment on the influx of inflammatory cells and TGF- in the BALF and expression of TGF- message in the lungs at various times during the course of BL-induced lung fibrosis in hamsters.

	Materials and Methods

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Treatment of Animals. Male Golden Syrian hamsters weighing 90 to 110 g were purchased from Simonsens, Inc. (Gilroy, CA). Hamsters were housed in groups of four, in facilities with filtered air and constant temperature and humidity. All care was in accordance with the National Institutes of Health Guide for Animal Welfare Act. The hamsters were allowed to acclimate in facilities for 1 week before any treatments. A 12-h light/dark cycle was maintained. The hamsters had access to water and either pulverized Rodent Laboratory Chow 5001 (Purina Mills, St. Louis, MO) or the same pulverized chow containing 0.5% PD (w/w). The animals were fed these diets starting 2 days before intratracheal (IT) instillation and continuing throughout the course of the experiment. Under pentobarbital anesthesia, hamsters were IT instilled with saline (SA; 4 ml/kg) or BL (6.5 U/4 ml/kg). Animals were randomly divided into four experimental groups: SA-instilled with a control diet (CD; SA + CD); SA-instilled with the PD diet (SA + PD); BL-instilled with the control diet (BL + CD); and BL-instilled with the PD diet (BL + PD).

Total and Differential Cell Counts in BALF. Total cell number in BALF was estimated directly by Coulter Counter (Model F; Coulter Electronics Inc., Hialeah, FL) and macrophage cell numbers were obtained from differential cell counts. For differential cell counts, the slides were prepared according to the method of Wilcox et al. (1988) and a total of 500 cells were counted.

TGF-₁ Level in BALF. The BALF was obtained from five hamsters in each group at various times after SA or BL instillation. The TGF- levels in the BALF were assayed using the commercially available Predicta TGF- enzyme-linked immunosorbent assay kit (GENZYME Diagnostics, Cambridge, MA). The enzyme-linked immunosorbent assay kit contained a 96-well microtiter plate with immunomobilized mouse monoclonal antibody to TGF- with a reported sensitivity of 0.05 ng/ml. The activation of the samples to release the active TGF-, and the rest of the assay were carried out as described per manufacturer recommendations. The standard curve was generated using the TGF- standard provided with the kit. The TGF- assays were carried out in duplicate and the results were reported as the mean of five samples in picogram per lung of total BALF recovered.

Total RNA Isolation and Hybridization Analysis. The animals were sacrificed by decapitation at various times and their lungs were quickly dissected out after SA or BL instillation. They were freeze clamped and dropped in liquid nitrogen and stored at 80°C. The total RNA from the whole lung was isolated using RNeasy total RNA extraction kit, according to the manufacture's instructions (Qiagen, Chatsworth, CA). After isolation, the RNA was quantitated and checked for integrity. Northern blot experiments were performed as described previously (Gurujeyalakshmi et al., 1996). Briefly, 5 µg of RNA was electrophoresed through 1% agarose in 2.2 M formaldehyde gels and transferred to a nylon membrane. The membrane was air dried for 20 min, UV cross-linked, and prehybridized at 42°C for 2 h in a solution containing 50% formamide, 5× sodium chloride sodium phosphate ethylenediamine tetraacetic acid, 0.3% sodium dodecyl sulfate, and 200 µg/ml sheared salmon sperm DNA. Radiolabeled probe was prepared by random primer method (Bio-Rad, Richmond, CA). The membranes were hybridized either with TGF-₁ cDNA, or 18S rRNA cDNA probe at 42°C for 16 h. RNA hybridization and washings were done as described previously (Gurujeyalakshmi et al., 1996). Relative intensities of either TGF- or 18S band were determined using a dual-wavelength flying spot-scanning densitometer (model CS-9301PC; Shimadzu, Columbia, MD). The results were expressed as the ratio of the signal intensity for TGF-₁ mRNA/18S rRNA bands in the same lane to limit variations in the quantity of RNA loaded in each lane.

Nuclear Runoff Studies. The nuclei from the whole lung were isolated from hamsters in all the groups, and the transcription assay was carried out by the method of Gurujeyalakshmi and coworkers (1998). Plasmids containing cDNA inserts of TGF-₁ and 18S rRNA were isolated and linearized with the appropriate restriction enzymes. Twenty micrograms of plasmid with cDNA inserts were slot blotted onto nylon membrane strips, air dried, and UV cross-linked. Plasmid pBR322 with no cDNA insert was included as a control for nonspecific binding. The membrane strips were prehybridized and then hybridized with buffer containing ³²P-labeled transcripts (4-6 × 10⁶ cpm/assay). The RNA-DNA binding was evaluated by autoradiography and densitometry.

Molecular Probes. TGF-₁ cDNA, containing a 1.05-kb EcoRI fragment was obtained from R. Derynk (Genentech, South San Francisco, CA). The 18S ribosomal RNA clone PN29III, with a 0.752-kb BamHI and SphI cDNA insert were obtained from the American Type Culture Collection (Rockville, MD). The plasmid pBR322 containing no cDNA insert was purchased from Pharmacia (Piscataway, NJ). The plasmids were isolated and purified using the Qiagen gel extraction kit and treated to complete restricted endonuclease digestion before use (Qiagen, Chatsworth, CA).

Statistical Analysis. Data were expressed as the mean ± S.E. Statistical differences among SA + CD, SA + PD, BL + CD, and BL + PD groups at the corresponding times were analyzed using two-way ANOVA, and a value of P  .05 was considered to be the minimum level of statistical significance. The t test was applied where two groups were involved, and a value of P  .05 was considered to be the minimum level of statistical significance.

	Results

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Influx of Inflammatory Cells in BALF. The effects of SA or BL instillation, with or without PD in diet, on the influx of inflammatory cells in BALF are summarized in Fig. 1. The total number of cells recovered in BALF from hamsters in the SA + CD group was almost the same as in SA + PD groups. IT instillation of BL significantly increased the total cell counts in BALF from BL + CD groups by 223, 310, 473, and 293% at 3, 7, 14, and 21days, when compared with the corresponding SA + CD control groups, respectively. Treatment with PD significantly inhibited the influx of total cells in BALF from hamsters in the BL + PD group at day 7, when compared with the BL + CD group at the same day.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Effect of IT instillation of SA (control) or BL with or without dietary intake of PD on total cell count in the BALF. The total cell count was determined as described in Materials and Methods. Each value represents the mean ± S.E. of five animals. Treatment groups are indicated along the x-axis. SA + CD = SA-instilled, fed CD; SA + PD = SA-instilled, fed the same diet containing PD; BL + CD = BL-instilled, fed CD; BL + PD = BL-instilled, fed the same diet containing PD. *, significantly higher (P  .05) than other groups at the corresponding times, except BL + PD at 3, 14, and 21 days. +, significantly lower (P  .05) than BL + CD group at the corresponding time.

Effects of PF on Macrophages in BALF. Figure 2 demonstrates the effect of PD on the number of macrophages in BALF at various times after SA or BL instillation. There were no statistically significant differences in the number of macrophages in BALF from SA + CD and SA + PD groups at 3, 7, 14, and 21 days. Treatment with BL significantly elevated the number of macrophages in BL + CD groups by 3-, 4.3-, and 3.2-fold of the corresponding SA + CD controls at 7, 14, and 21 days, respectively. However, treatment with PD statistically reduced the number of macrophages in the BL + PD group by 58% at day 7, when compared with the BL + CD group at the same time point.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Effect of dietary intake of PD on BL-induced increases on the number of macrophages in the BALF from hamster lungs at different time points after IT instillation of SA or BL. The number of macrophages was determined by differential cell analysis as described in Materials and Methods. Each value represents the mean ± S.E. of five animals. See the legend to Fig. 1 for treatment details and explanations for abbreviations. *, significantly higher (P  .05) than SA + CD and SA + PD groups at the corresponding times and BL + PD group at 7 days. +, significantly lower (P  .05) than BL + CD group at the corresponding time.

Effects of PF on TGF- Levels in BALF. The effects of PF on the TGF- protein levels in BALF at various time points after SA or BL instillation are shown in Fig. 3. The TGF- levels in SA + CD and SA + PD groups were similar at all time points. The IT instillation of BL caused significant increases in the TGF- levels in BALF from hamsters in BL + CD groups by 255, 284, 290, and 452%, when compared with the corresponding SA + CD controls at 3, 7, 14, and 21 days, respectively. Treatment with PD suppressed the BL-induced increases in the TGF- levels significantly in BL + PD groups by 78 and 47% at 14 and 21 days, respectively, when compared with the corresponding BL + CD groups.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effect of IT instillation of SA (control) or BL with or without dietary intake of PD on TGF- in the BALF from hamster lungs at different times. The TGF- protein levels were measured as described in Materials and Methods. Each value represents the mean ± S.E. of five animals. See the legend to Fig. 1 for treatment details and explanations for abbreviations. *, significantly higher (P  .05) than SA + CD and SA + PD groups at the corresponding times, and BL + PD groups at 14 and 21 days. +, significantly lower (P  .05) than BL + CD groups at the corresponding times.

Effects of PF on TGF- Gene Expression in Lung. The effects of dietary intake of PF on the lung TGF- gene expression in hamsters receiving SA or BL IT are shown in Fig. 4. The Northern blot technique was used in the gene expression studies. The levels of lung TGF- mRNA in groups SA + CD and SA + PD were similar to each other at all time points and remained unchanged throughout the study period. The IT instillation of BL significantly up-regulated the TGF- gene expression at 7, 14, and 21 days by 270, 425, and 301% in the BL + CD groups, respectively, when compared with the corresponding SA + CD groups. Treatment with PF down-regulated the BL-induced overexpression of TGF- mRNA at all time points in the BL + PD groups, and significant reductions occurred at 7, 14, and 21 days by 60, 75, and 62%, respectively, compared with the corresponding BL + CD groups. Figure 5 represents the Northern blot and densitometric reading showing the overexpression of the TGF- gene in the BL+ CD group and its down-regulation by PD treatment in the BL+ PD group.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of IT instillation of SA (control) or BL with or without dietary intake of PD on the levels of TGF- mRNA in hamster lungs at different time points after IT instillation. TGF- mRNA was quantitated as described in Materials and Methods. Each value represents the mean ± S.E. of four animals. See the legend to Fig. 1 for treatment details and explanations for abbreviations. *, significantly higher (P  .05) than other groups at the corresponding times. +, significantly lower (P  .05) than BL + CD groups at the corresponding times.

View larger version (65K): [in this window] [in a new window]   Fig. 5.   Demonstration of the temporal effect of PD on TGF- gene expression in BL-instilled hamster lungs. Total RNA was extracted and TGF-mRNA (A) was quantitated as described in Materials and Methods. The membrane shown in (A) was hybridized with TGF- cDNA and then rehybridized with a cDNA probe for 18S rRNA as shown in (B). C, densitometric analysis of the autoradiogram shown in (A).

Effects of PF on TGF- gene Expression in Lung at the Transcriptional Level. We carried out the nuclear run-off transcription assay to determine whether the down-regulation of TGF- gene in the BL + PD groups occurred at the transcriptional level. The assay was performed as described previously by Gurujeyalakshmi and coworkers (1996). The nuclei from the lungs of hamsters in SA + CD, SA + PD, BL + CD, and BL + PD groups were isolated at 14 days. The nascent transcripts were labeled using ³²P-UTP and hybridized with the probes as described in Materials and Methods. The labeled transcripts that hybridized to the cDNA of TGF-₁ were significantly reduced by 33% in the BL + PD group when compared with the BL + CD group, as shown in Table 1 and Fig. 6. The transcription of TGF- in the SA + CD and SA + PD was extremely low (data not shown), when compared with the BL + CD and BL + PD groups. We observed that the transcription of the gene encoding 18S rRNA in lung nuclei of BL + CD and BL + PD groups was almost identical. Our results demonstrate that PF down-regulates the expression of TGF- gene at the transcriptional level. This would partly explain the subsequently observed down-regulation in TGF- gene expression and TGF- protein production.

View this table: [in this window] [in a new window]   TABLE 1 The effect of pirfenidone on the transcription of TGF- and 18S rRNA at 14 days after BL instillation The nuclear run-off experiment was carried out as described in Materials and Methods. Each value represents mean ± S.E. of five animals in duplicate experiments.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   A representative Northern blot of nuclear runoff study, showing the inhibitory effect of PD in BL + PD group on BL-induced overexpression of TGF- transcripts in the BL + CD group at 14 days. The transcription assays were performed as described in Materials and Methods. After the isolation of nuclei from all lung lobes of each animal in BL + CD and BL + PD groups, 32P-labeled RNAs (4  6 × 106 cpm) in each assay were hybridized to 20 µg of TGF- and 18S rRNA containing the cDNA inserts, which had been linearized, denatured, and immobilized on Nytran membranes. Insert-free plasmid pBR322 was included as the control for nonspecific binding. See Table 1 for mean ± S.E. of five animals for nuclear runoff experiment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, a variety of experiments were performed to determine the mechanisms by which PD treatment inhibits the BL-induced increased production of TGF-. Our investigation focused on this potent cytokine, which is in part derived from alveolar macrophages. The macrophages are known to play a critical role in the pathogenesis of BL-induced pulmonary damage, including the orchestration of both inflammatory and fibroproliferative events associated with the fibrotic processes of the lung. The alveolar macrophages regulate the inflammatory phase by releasing a variety of proinflammatory cytokines such as IL-1, tumor necrosis factor-, macrophage inflammatory protein-1, and chemotactic cytokines such as IL-8. They also influence the fibrotic phase by releasing growth factors such as PDGF, fibroblast growth factor, and insulin growth factor-1, which are mitogenic to mesenchymal cells and release cytokines that are directly fibrogenic like those of TGF- (Shaw and Kelly, 1995). Previous studies have demonstrated the influx of macrophages and their activation during the course of BL-induced lung fibrosis (Chandler et al., 1983; Khalil et al., 1989). It is well documented that TGF- is involved in BL-induced lung fibrosis and its level in the BALF peaks to about 30-fold of the controls in BL-treated animals (Khalil et al., 1989). TGF- functions as an amplifier of inflammatory response by acting as a potent chemoattractant to the monocytes and macrophages, and it also autoinduces its own production (Wahl et al., 1987). After macrophages are activated to secrete TGF-, the process results in continued activation, recruitment, and secretion of TGF-, which remains elevated throughout the entire course of the fibroproliferative process. It seems that there is a positive feedback loop that facilitates an increased production of TGF- by activated cells. (Wahl, 1994). Our findings that BL-treated hamsters in BL + CD groups had significantly greater influx of total cells and macrophages, and consequently greater amounts of TGF- in the BALF at almost all time points of the study than the hamsters in SA + CD groups, lend credence to the proposed concept of a positive-feedback loop of a sustained production of increased amount of TGF- by the activated macrophages in the BL + CD groups. Treatment with PF generally suppressed the BL-induced increases in the TGF- levels of the BALF from hamsters in BL + PD groups, and significant decreases occurred in these groups, compared with BL+CD groups at 14 and 21 days. Treatment with PF had no effect on the BL-induced increases in the influx of total cells and macrophages in BL + PD groups, except on day 7. On this day, the influx of both total cells and macrophages was reduced by more than 50% in the BL + PD group, compared with the BL + CD group. The significance of this marked reduction in the influx of inflammatory cells on this day is not clear. It is possible that a marked reduction in the number of macrophages may contribute to decreased levels of TGF- at later time points. It is puzzling that the decreased influx of macrophages on day 7 did not correlate with the decreased levels of TGF- in the BALF from hamsters in the BL + PD group on this day. This would imply that there are other possibilities, besides macrophages, that may account for decreased levels of TGF- in the BALF from hamsters treated with PD in the BL + PD groups. These include: 1) PF may directly act not only on the macrophages, but also on the epithelial cells and fibroblasts and compromise their ability to synthesize and release TGF-; and 2) PF may be directly inhibiting the activity of serine proteases required to convert the latent form of TGF- into the active form in a manner similar to that of plasmin (Khalil et al., 1996). Regardless of the mechanisms, our data suggest that one of the mechanisms for anti-inflammatory and antifibrotic effects of PF is its ability to suppress the BL-induced increased production of biologically active form of TGF-.

In addition to inhibition of TGF-, PF treatment may elicit its anti-inflammatory and antifibrotic effects in BL + PD groups by other mechanisms. It has been previously shown that PD down-regulates the expression of the intercellular adhesion molecule (Kaneko et al., 1998), which is an adhesion molecule and necessary for infiltration of some inflammatory cells in lung (Von Andrian et al., 1991). It has also been shown that PD scavenges reactive oxygen species (ROS) in vivo (Iyer et al., 1995) and in vitro (Valleyathan et al., 1996) conditions. It is possible that PD could be exerting its anti-inflammatory and antifibrotic effects in other ways, including decreasing the expression of adhesion molecules, and being able to directly scavenge the ROS generated by the influx of inflammatory cells and/or by the redox cycling of BL/DNA/Fe²⁺ complex (Sugiura and Kikuchi, 1978). Thus, a diminution in the severity of ROS-induced lung damage by PF treatment in BL + PD groups may subsequently attenuate the ensuing cascade of events involved in the development of lung fibrosis.

TGF- is a chemoattractant for fibroblasts (Postlethwaite et al., 1987), and it induces proliferation of fibroblasts through the production of growth factors such as PDGF. PDGF is known to stimulate, activate, and differentiate the fibroblasts to a more aggressive phenotype fibroblast population that relentlessly synthesizes connective tissue proteins (Roberts and Sporn, 1990). Because TGF- is known to influence mesenchymal cell proliferation via stimulating PDGF -receptors (Yamakage et al., 1992), the suppression of TGF- by PD could effect the mitogenic activity of TGF- on mesenchymal cells directly or indirectly. Our laboratory has recently demonstrated that treatment with PD inhibited the BL-induced increases in the PDGF-AA and -BB levels in BALF from hamsters in BL + PD groups (Gurujeyalakshmi et al., 1999). The observed reduction in the lung collagen content in this group of hamsters could be due to reduced mesenchymal cell proliferation secondary to inhibitory effects of PD on TGF- and PDGF production.

TGF- is known to have a direct profibrotic action by virtue of its stimulatory effects on the synthesis of ECM proteins. For instance, it has been shown that TGF- increases expression of fibronectin and collagen genes at the transcriptional and post-transcriptional levels (Raghow et al., 1985, 1987) It also stabilizes the ECM by preventing its proteolytic degradation by decreasing the synthesis of matrix-degrading proteinases such as serine protease, metalloproteinase, and collagenase (Roberts and Sporn, 1990). TGF- also increases the synthesis of specific proteinase inhibitors such as tissue inhibitors of metalloproteinase and plasminogen activator inhibitor (Edwards et al., 1987). These effects of TGF- eventually lead to an aberrant and excess deposition of ECM (Sporn et al., 1987). Our results indicate that BL treatment significantly up-regulated the TGF- gene expression at all time points of the study. This could be caused by an increased transcription of TGF- gene, as demonstrated in this study. However, we cannot rule out the increased TGF- mRNA stability as another contributing factor to increased levels of TGF- message in the lung of hamsters in BL + CD groups. Our results also demonstrate that treatment with PD significantly down-regulated the BL-induced overexpression of TGF- in BL + PD groups at 7, 14, and 21 days. The reduction in the TGF- mRNA levels by PD treatment in BL + PD groups is being mediated at the transcriptional level because the nuclear runoff studies revealed that PF caused a significant reduction in the synthesis of TGF- transcripts in hamsters in this group compared with hamsters in the BL + CD group. These results explain our previous findings that reduced TGF- levels are associated with the down-regulation of the transcription of the procollagen genes and collagen content (Iyer et al., 1999). The data obtained in the present study indicate that treatment with PF suppressed the BL-induced overexpression of TGF- production and TGF- mRNA at the transcriptional level and subsequently suppressed the BL-induced overexpression of procollagen genes in the lungs. These effects finally lead to a reduction in the synthesis and deposition of collagen in the lung of hamsters in BL + PD group.

Other studies involving neutralizing TGF- by its antibody (Giri et al., 1993) and binding TGF- by decorin (Giri et al., 1997) demonstrated amelioration of BL-induced lung fibrosis. However, the stage at which TGF- can be suppressed is critical because corticosteroids are somewhat beneficial at the earlier stages of fibrosis, when TGF- is derived from macrophages. In the advanced stages of IPF, when TGF- production is associated with epithelial cells, corticosteroids fail to elicit any beneficial response (Carrington et al., 1978; Pierce et al., 1989; Khalil et al., 1993). Our results suggest that the beneficial effects of PF against BL-induced lung fibrosis involve the suppression of TGF- effects at the proinflammatory and profibrogenic phases of the fibrotic processes. Thus, treatment with PD starting at the inflammatory stages of fibrosis may prove to be more beneficial to patients with early diagnosis of IPF.