help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on January 13, 2006, doi:10.1164/rccm.200505-717OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
200505-717OCv1
173/7/769    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chaudhary, N. I.
Right arrow Articles by Park, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chaudhary, N. I.
Right arrow Articles by Park, J. E.
American Journal of Respiratory and Critical Care Medicine Vol 173. pp. 769-776, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200505-717OC

Original Article

Pharmacologic Differentiation of Inflammation and Fibrosis in the Rat Bleomycin Model

Nveed I. Chaudhary, Andreas Schnapp and John E. Park

Department of Pulmonary Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany

Correspondence and requests for reprints should be addressed to Dr. John Park, Ph.D., Department of Pulmonary Research, Boehringer Ingelheim Pharma GmbH & Co., KG Birkendorferstrabetae 65, Biberach an der Riss D-88937, Germany. E-mail: john.park{at}bc.boehringer-ingelheim.com


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: The model most often used to study the pathogenesis of pulmonary fibroses is the bleomycin (BLM)-induced lung fibrosis model. Several treatments have been efficacious in this model, but not in the clinic.

Objectives: To describe the time course of inflammation and fibrosis in the BLM model and to study the effect of timing of antiinflammatory and antifibrotic treatments on efficacy.

Methods and Measurements: Rats were given single intratracheal injections of BLM on Day 0. At specified time points, 10 rats were killed and their lungs studied for proinflammatory cytokines and for profibrotic growth factor mRNA. After a single intratracheal injection of BLM on Day 0, rats were treated from Day 1 or 10 daily with oral prednisolone (10 mg/kg) or oral imatinib mesylate (50 mg/kg) for 21 d.

Results: After BLM administration, the expression of inflammatory cytokines was elevated and returned to background levels at later time points. Profibrotic gene expression peaked between Days 9 and 14 and remained elevated till the end of the experiment, suggesting a "switch" between inflammation and fibrosis in this interval. Antiinflammatory treatment (oral prednisolone) was beneficial when commenced at Day 1, but had no effect if administered from Day 10 onward. However, imatinib mesylate was effective independently of the dosing regime.

Conclusions: The response of the BLM model to antifibrotic or antiinflammatory interventions is critically dependent on timing after the initial injury.

Key Words: bleomycin • fibrosis • imatinib mesylate • lung • prednisolone

Fibrosis can occur in all tissues, but is especially prevalent in organs with frequent exposure to chemical and biologic insults including the lung, skin, digestive tract, kidney, and liver (1–3). Fibrosis often severely compromises the normal functions of the organ, and many fibrotic diseases are life-threatening or severely debilitating (4). Treatment options for these diseases are often limited to organ transplantation, a risky and expensive procedure.

Pulmonary fibrosis is a pathologic condition which accompanies a wide range of inflammatory conditions of the airways. In patients with chronic obstructive pulmonary disease, a patchy alveolar wall fibrosis with peribronchiolar distribution is observed, whereas in patients with chronic asthma, a fibrotic response predominantly in the lamina reticularis, leading to a thickening of the basement membrane has been demonstrated (5, 6). In both diseases, an ongoing inflammation-repair cycle in the airways leads to permanent structural changes in the airway wall (remodeling), of which fibrosis is a major constituent (7, 8). Furthermore, severe and lethal pulmonary fibrosis is prevalent in patients with diseases such as idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome, and unusual interstitial pneumonitis. In these diseases, fibrosis is associated with extreme morbidity and patients have a very poor survival prognosis, half of whom die within 30 mo of diagnosis because of the absence of efficacious treatment options (4). Although the degree of pulmonary fibrosis can occur with different severities, there is much evidence to suggest that the pathophysiologic mechanisms underlying the development of pulmonary fibrosis are similar across diseases.

In all forms of pulmonary fibrosis, the predominant cell types are the fibroblasts and myofibroblasts (9, 10), and there is increased evidence to suggest the activation of an epithelial-mesenchymal trophic unit (a phenomenon in which the damaged epithelium can activate fibroblasts in the underlying mesenchyme through the secretion of cytokines and growth factors) (7, 11). Inflammatory damage to the effected epithelium results in the secretion of growth factors (12), which stimulate the underlying fibroblasts in the mesenchymal compartment of the airway. Depending on the presence of the correct stimulatory milieu, the stimulation of the fibroblast may lead to one of two main events, namely the transformation of fibroblasts to myofibroblasts, or fibroblast proliferation (13–16). Myofibroblasts secrete a wide array of growth factors and extracellular matrix components, all of which contribute to increased collagen, fibronectin, and extracellular matrix deposition, which is the hallmark of fibrosis (17). The number of myofibroblasts in the submucosa has been reported to correlate with subepithelial collagen deposition in asthma (5). The increased smooth muscle mass in asthma is also thought to be due to increased activity of the fibroblasts, which, when activated by factors secreted by the damaged epithelium, transform to myofibroblasts (18). It has been suggested that repeated stimulation of the myofibroblasts can result in further differentiation to smooth muscle cells (19). It is believed that the myofibroblasts do not proliferate per se. Rather the increase in their number is believed to result from the increased proliferative activities of the fibroblasts before transformation. Recent evidence also shows that the increased number of fibroblasts present at sites of lung injury could be especially recruited in from the blood. Moore and coworkers have shown that bone marrow–derived fibrocytes are found in the alveolar space after fluorescein isothiocyanate–induced lung fibrosis in mice, and that increased expression of CCR2 maybe responsible for this recruitment (20).

As mentioned, the cellular milieu can drastically affect the fate of the stimulated fibroblast. The most potent factor able to transform fibroblasts to myofibroblasts is transforming growth factor beta (TGF-beta) (21). Furthermore, there has been in vitro evidence suggesting that at low concentrations, TGF-beta also stimulates the proliferation of fibroblasts (15). Besides TGF-beta, there are other growth factors present in the milieu of the inflamed lung that are much more effective at stimulating the proliferation of fibroblasts. Such factors include endothelin 1, insulin-like growth factor 1, and platelet-derived growth factor (PDGF), which has very potent promitogenic effects on these cells (12, 14).

The animal model most often used to study the pathogenesis and treatment of IPF and related pulmonary fibroses is the bleomycin-induced lung fibrosis model in rodents (22). In this model, bleomycin is administered intratracheally directly into the lungs, which induces lung injury from bleomycin-mediated cleavage of DNA. The resulting inflammatory response causes damage to the airway epithelium, activation of fibroblasts, and subsequent fibrosis. Numerous agents targeting such diverse signaling pathways as EGFR and PDGFR signaling (21), antiinflammatory agents such as nonsteroidal antiinflammatory drugs and corticosteroids (23), cytokines, antioxidants, anticoagulants (24), various gene therapies, and angiotensin II antagonists (25), inhibit fibrosis very effectively in this model. Almost invariably, the effectiveness of the pharmacologic intervention was determined by beginning treatment 1 d after administration of bleomycin and addressing efficacy of treatment at the end of the experiment. Because none of these molecules has demonstrated clear efficacy in the treatment of IPF, some doubt occurred as to the relevance of the bleomycin model to assess the value of potential IPF therapies.

In this study, we used a prototypic antiinflammatory agent, prednisolone, and a potential antifibrotic agent, imatinib mesylate (21), a PDGFR/cAbl/cKit kinase inhibitor, to determine the relative contributions of inflammation and fibrosis in the rat bleomycin model and demonstrate that one can distinguish agents with antifibrotic and antiinflammatory modes of action. These findings might offer a preclinical model to ultimately more accurately judge the therapeutic potential of such compounds for the treatment of IPF.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Compounds
Imatinib mesylate (Novartis, Basel, Switzerland) and bleomycin sulfate (Bleomycin; Hexal AG, Holzkirchen, Germany) were purchased from a local pharmacy. Prednisolone was obtained from Sigma-Aldrich.

Bleomycin Administration and Treatment Protocols
All experiments were performed in accordance with German guidelines for animal welfare and were approved by the responsible authorities.

To determine the optimal dose of bleomycin sulfate, 10 male Wistar rats per group were intratracheally injected with bleomycin sulphate from 0.05 to 2.75 mg/kg in 300 µl saline using a catheter (0.5-mm internal diameter, 1.0-mm external diameter) through the nasal passage. After 21 d, animals were killed with a lethal intraperitoneal injection of Narcoren (pentobarbital sodium; Rhone Merieux, Athens, GA); collagen staining and the expression of profibrotic markers were analyzed as described in the following section. The 2.2 mg/kg dose was determined to result in a strong and reproducible pulmonary fibrosis with no effect on animal survival or body weight.

The fully effective doses of prednisolone and imatinib mesylate were determined performing dose–response experiments in rats treated with bleomycin (2.2 mg/kg). Both compounds were administered orally in 1 ml 0.1% Natrosol (Hercules Inc., Wilmington, DE). Again, fibrosis was analyzed at Day 21. For imatinib mesylate three doses (10, 30, and 50 mg/kg; 10 animals/group) were used. The dose of 50 mg/kg was observed to be the most efficacious dose consistent to previously published data (26). Prednisolone was tested (5, 10, and 30 mg/kg) and the dose of 10 mg/kg was seen to be adequately efficacious to the 30 mg/kg dose at inhibiting inflammation in this model.

In the case of the time course studies, the rats were treated with bleomycin at Days 0 and 10 rats were killed at specified time points (i.e., Days 0, 3, 6, 9, 14, and 21).

After the rats were killed, their lungs were removed, blotted dry, and half were snap frozen in liquid nitrogen and stored at –80°C. The other half was fixed in 4% formalin for subsequent paraffin embedding and histology. Another lobe of the lungs was cut into approximate 3-mm cubes and snap frozen in liquid nitrogen for subsequent RNA and protein analysis.

Histology
The collapsed lung tissues fixed in 4% formalin were embedded into paraffin using a Tissue-Tek VIP (Sakura, Zoeterwoude, The Netherlands) embedding machine. Five-micrometer sections were cut using a microtome (SM200R; Leica, Wetzlar, Germany) and placed on poly-L-lysine–coated slides. The sections were then dried onto the slides (60°C, 2 h) and then left to cool at room temperature. Collagen deposition was assessed using Masson's Trichrome staining as previously described (27, 28). The sections were then cover-slipped and observed under a light microscope.

Total RNA Extraction and Synthesis of cDNA
One part of the frozen lung tissue dedicated to investigation of gene expression was cut into small pieces using a sterile scalpel blade. Approximately 100 mg of tissue was then placed into a 2-ml Eppendorf tube and 1.5 ml of Trizol was added. A sterile Tungsten carbide bead (Qiagen) was then added to the tube and the tube was placed in a Retsch MM300 Tissue disruptor (Qiagen, Düsseldorf, Germany) at a frequency of 30.0 Hz for 8 min. After this time, the bead was removed and the sample centrifuged at 12,000 rpm for 10 min to remove tissue debris. The RNA was extracted using a modified version of the manufacturer's protocol supplied with Trizol. Briefly, 0.3 ml chloroform was added to the tube and the tube shaken vigorously and then left to incubate at room temperature for 5 min, after which the tube was centrifuged for 15 min at 12,000 rpm at 4°C. The upper colorless aqueous phase was then collected and added to 750 µl isopropanol. This was then shaken vigorously and stored at –80°C overnight. The samples were then incubated at room temperature for 15 min, after which they were centrifuged for 40 min at 12,000 rpm at 4 °C. The supernatant was then removed and 500 µl of 70% ethanol was added to wash the pellet then the sample was centrifuged for 10 min at 12,000 rpm an 4°C, this wash step was repeated twice, after which the pellet was left to dry for 10–15 min. Finally the pellet was resuspended in 2.0 µl RNase free water and stored at –80°C. The concentration of each sample was then measured using a spectrophotometer.

Using the Superscript III (Invitrogen, Paisley, UK) reverse transcriptase first-strand synthesis kit, 2 µg of each mRNA sample was reversed transcribed using a modified version of the manufacturer's protocol. Briefly, a mixture of 2 µg RNA, 1 µl random hexamer primers (50 ng/µl), and 1 µl deoxyribonucleotide triphosphate mix (10 mM) was made up to 10 µl with diethylpyrocarbonate-treated water and incubated at 65°C for 5 min, after which it was placed on ice for 5 min. After this, to each reaction, 2 µl reverse transcriptase buffer (10X), 4 µl MgCl2 (25 mM), 2 µl dithiothreitol (0.1 M), 1 µl RNaseOUT (40 U/µl), and 1 µl SuperScript III enzyme (200 U/µl) was added and the mixture placed in a thermal cycler (Applied Biosystems, Foster City, CA) under the following conditions: 25°C for 10 min, 50°C for 50 min, and 85°C for 5 min, after which 1 µl of RNase H was added and incubated at 37°C for 20 min. The synthesized cDNA was diluted to 5 ng/µl under the assumption that the reverse transcriptase reaction fully transcribed all of the mRNA to cDNA and had a concentration of 100 ng/µl.

Investigation of Gene Expression Using Real-Time Polymerase Chain Reaction
Gene expression was investigated in each of the samples using the Applied Biosystems 7700 sequence detection system. Primers for the 18S endogenous control and TGF-beta1 were purchased as predeveloped assay reagent kits (PDAR; Applied Biosystems), whereas primers and probes (Table 1) for pro-collagen I and fibronectin were designed using PrimerExpress (Applied Biosystems), ensuring that at least one of the primers or probes in each set overlapped an intron/exon junction, thus eliminating the possibility of amplifying any contaminating genomic DNA in the cDNA sample. The purchased PDARs also amplified only cDNA.


View this table:
[in this window]
[in a new window]
  		TABLE 1. QUANTITATIVE PCR PRIMER AND PROBE SEQUENCES FOR FIBRONECTIN AND PRO-COLLAGEN I

 
Real-time polymerase chain reaction (PCR) was carried out in 25-µl reactions, using 25 ng (5 µl) of cDNA per reaction. A quantitative PCR core kit was purchased (Eurogentec, Seraing, Belgium) and a master mix was made up as follows for 100 reactions: 500 µl 10X reaction buffer, 500 µl MgCl2 (50 mM), 200 µl dNTP mix solution (5 mM), 25 µl Hot Goldstar enzyme, 75 µl 18S PDAR, 22.5 µl forward primer, 22.5 µl reverse primer, 15 µl probe, and 640 µl DPEC-treated water. Twenty microliters of this master mix was then added to 25 ng (5 µl) target cDNA. Each analysis was carried out in triplicate. To quantify the gene expression, a standard curve was constructed for each primer set and was included on each plate. The standards were made up of a mix of all the cDNAs under investigation; this mix of cDNAs was serially diluted 10, 20, 50, 100, and 1,000 times. A standard curve was constructed of the obtained CT (cycle at which amplification reaches a set threshold) against the log10 of the dilution factor. Curves were drawn for the target gene and the 18S rRNA endogenous control. The CT value for both targets for each of the samples was then converted to a fold dilution using the standard curve, and the target gene value was normalized to the 18S gene value.

Protein Extractions and Investigation of Protein Expression Using Multiplex Luminex Bead Technology
One part of the frozen lung tissue dedicated to investigation of protein expression was cut into small pieces using a sterile scalpel blade. Approximately 150 mg of tissue was placed in a tube and 3 ml of cold phosphate-buffered saline (containing protease cocktail inhibitor tablet [Roche, Mannheim, Germany]) was added. The sample was then disrupted using an Ultra-Torax (Ika-Werk, Staufen, Germany) for 3 min and then 2 ml of RIPA buffer was added. The pH of the solution was then adjusted to 7.2 using 1 M HCl.

The amount of protein in each sample was determined using Bio-Rad protein assay solution (Bio-Rad, Munich, Germany) following the manufacturer's protocol, using a spectrophotometer. A standard curve using serial dilutions of bovine serum albumin was constructed and the obtained absorbance from each sample was translated to protein concentration using the standard curve.

Multiplex cytokine detection was carried out using the Bioplex system (Bio-Rad) using a predeveloped cytokine assay (Rat 9-plex) that detected interleukin 1{alpha} (IL-1{alpha}), IL-1beta, IL-2, IL-4, IL-6, IL-10, granulocyte-macrophage colony–stimulating factor, IFN-{gamma}, and tumor necrosis factor-{alpha}. The assay was carried out using the manufacturer's protocol, and the results analyzed using Bio-Plex Managed V 3.0 software (Bio-Rad).

Statistics
All statistical analyses were carried out using GraphPad Prism V 4.02 software (GraphPad Software, San Diego, CA). Comparisons were made using a nonparametric t test (Mann-Whitney U test) and a significant value was considered to be p <= 0.05. On all graphs, one asterisk signifies a significance level of p <= 0.05, two asterisks signify a significance level of p <= 0.01, and three asterisks signify a significance level of p <= 0.0001.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Time Course for Development of Fibrosis in the Rat Bleomycin Model
To investigate the time course for the development of fibrosis in the rat bleomycin model rats were intratracheally instilled with bleomycin (2.2 mg/kg) and the mRNA expression profiles of fibrotic markers including TGF-beta1, fibronectin (an extracellular matrix protein found within fibrotic lesions), and procollagen I, were examined in lung tissue prepared at various time points during the course of the experiment. As shown in Figure 1, TGF-beta1 exerts a first peak of expression at Day 6, a decline on Day 9, and a second peak of expression at Day 21. The expression of fibronectin begins and peaks at Day 6, followed by a gradual decrease down to very low levels after Day 14, whereas expression of pro-collagen I remains at low levels until Day 9, shows a peak on Day 14 and a subsequent reduction of mRNA signal down to levels seen at the beginning of the experiment. To address the expression profile of collagens at the protein level, lung sections obtained at the various time points were stained with Masson's Trichrome. As shown in Figure 2, collagen deposition, as indicated by the blue staining, is weak at Days 0, 3, and 6. Starting with Day 9, an increased degree of collagen deposition is observed, which is even more pronounced on Days 14 and 21. At the later time points, the increase in collagen staining is distributed uniformly over the entire lung (data not shown). Furthermore, the increase in collagen deposition is associated with a loss of alveolar structures. On Day 3 and Day 6, an increased cell influx, possible from the recruitment of inflammatory cells, is seen.


Figure 1
View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. Time course for the gene expression of the three profibrotic markers in the rat bleomycin model. Rats were treated with bleomycin (2.2 mg/kg) on Day 0. At the time points indicated on the x axis, the central lobes of the left lung were collected (Day 0 = untreated control) and gene expression profiles for (A) transforming growth factor (TGF)-beta1, (B) fibronectin, and (C) pro-collagen I were determined by quantitative reverse transcriptase polymerase chain reaction (Taqman). The gene expression for each gene is indicated relative to endogenous 18S RNA control. The bars represent the standard error of the mean. *p <= 0.05, **p <= 0.01, ***p <= 0.001.

 

Figure 2
View larger version (86K):
[in this window]
[in a new window]
  		Figure 2. Collagen staining of representative lung sections taken from rats treated with 2.2 mg/kg bleomycin for up to 21 d. Rats were treated with bleomycin (2.2 mg/kg) on Day 0. At the time points indicated, 10 rats were killed and the lungs fixed in 4% paraformaldehyde before paraffin embedding. Sections (4 µM) were cut and baked onto 3-amino propyl tri ethoxysilene–coated slides. The sections were then stained with Masson's Trichrome stain, with which muscle and cells are stained red, nuclei are stained black, and collagens are stained blue. Each photomicrograph is representative for each group. BLM = bleomycin. Magnification x 250.

 
As previously described, bleomycin induces a gross inflammation of the airways; this sustained damage leads to the initiation of fibrotic processes (29, 30). To determine the duration of the bleomycin-induced inflammatory phase, protein levels of inflammatory cytokines, namely IL-1{alpha}, IL-1beta, IL-6, IL-10, and IFN-{gamma}, were measured in lysates of lung tissue obtained at each of the time points. As shown in Figure 3, IL-1{alpha}, IL-1beta, IL-6, and IFN-{gamma} exhibited similar kinetic of expression over the 21-d period. Cytokine levels increase rapidly, within the first 3 d and remain elevated up to Day 9. After Day 9, a significant decrease in expression is observed from Day 14 until the end of the experiment, ultimately returning to the baseline levels seen at Day 0. An exception to this trend was the time course profiles for IL-10. IL-10 showed no change in expression between Days 0 and 3 and a slight increase between Days 6 and 9. A significant drop in expression (p = 0.01) was observed at Day 14, after which the levels were raised again at Day 21 (data not shown).


Figure 3
View larger version (23K):
[in this window]
[in a new window]
  		Figure 3. Expression of inflammatory cytokines in the whole lung lysates of rats treated with 2.2 mg/kg bleomycin up to 21 d. The inflammatory cytokines investigated were (A) interleukin 1{alpha} (IL-1{alpha}), (B) IL-1beta, (C) IL-6, and (D) IFN-{gamma}. Rats were treated with bleomycin (10 U/kg) on Day 0. At the time points indicated, 10 rats were killed and part of the left lobe was snap frozen in liquid nitrogen and stored at –80°C until analysis. The lobe was disrupted in modified radioimmunoprecipitation buffer for protein extraction. The total protein concentration was measured and all normalized to a concentration of 1 mg/ml. The levels of inflammatory cytokines were then measured using a Bioplex multiplex cytokine assay. Bars: median values. *p <= 0.05, ***p <= 0.001.

 
Because the significant decrease in the expression of inflammatory mediator proteins after Day 9 is concomitant with an increase in pro-collagen I gene expression, a second peak of TGF-beta1 expression and collagen deposition, we hypothesized that there is a "switch" between an inflammatory and fibrotic phase of the model around Day 9. Therefore, we speculated that comparison of continuous pharmacologic treatment commencing at Day 0 with treatment beginning Day 10 would allow us to distinguish antiinflammatory and antifibrotic actions of test drugs. If selected compounds work by merely inhibiting the initial inflammation, they should prove active when administered over the entire Day 1 day period (= preventive treatment), whereas they should be ineffective if administered after the bulk of the inflammatory response has resolved (i.e. treatment starting at Day 10 = therapeutic treatment). In contrast, antifibrotic agents should prove effective irrespective of the treatment mode.

The Antiinflammatory and Antifibrotic Actions of Prednisolone and Imatinib Mesylate in the Rat Bleomycin Model
We selected two drugs previously reported to be active in the bleomycin model, namely prednisolone, an antiinflammatory agent, and the potential antifibrotic agent imatinib mesylate, a PDGFR/c-abl/c-kit inhibitor, a potential antifibrotic agent (31) and tested their activity in the rat bleomycin model using the two treatment options mentioned. To rats treated with bleomycin, prednisolone or imatinib mesylate were administered once daily at their respective fully effective dose via oral gavage (see METHODS) either from Day 0 to Day 21 (preventive model) or from Day 10 to Day 21 (therapeutic model; Figure 4A). In both settings, administration of saline was used as a control. After 21 d, animals were killed and the level of fibrosis was determined by gene expression profiling of TGF-beta1 and pro-collagen I (Figure 4B).


Figure 4
View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. Activity of prednisolone and imatinib mesylate on profibrotic marker gene expression in the rat bleomycin model. (A) Experimental designs to determine the activity of prednisolone and imatinib mesylate as preventive or therapeutic agents in the bleomycin model. In both cases, rats were administered bleomycin (2.2 mg/kg) at Day 0. In the preventive model, treatment commenced at Day 1 and was continued daily until Day 21. In the therapeutic model, treatment commenced at Day 10 and was continued daily until Day 21. (B) On Day 22, the rats were killed and a part of the left lung lobe was processed for RNA extraction. The gene expression levels of TGF-beta1 and pro-collagen I was determined by quantitative reverse transcriptase polymerase chain reaction (Taqman). The gene expression for each gene is indicated relative to endogenous 18S RNA control. Bleo = bleomycin; Pred = prednisolone. Bars: median values.

 
As shown in Figure 4B, continuous treatment of rats with prednisolone and imatinib mesylate for 21 d after bleomycin treatment results in a strong reduction of pro-collagen I and TGF-beta1 gene expression. Conversely, when treatment started at Day 10 (therapeutic model), prednisolone did not effect the expression of pro-collagen I and only marginally reduced the expression of TGF-beta1 mRNA. In contrast, imatinib mesylate still blocked the expression of the two fibrotic markers, also in the therapeutic setting. The gene expression data are further supported by immunohistochemistry. As shown in Figure 5, compared with the untreated control group, the degree of collagen staining is significantly decreased in rats treated continuously for 21 d with prednisolone and imatinib mesylate. However, when steroid treatment started at Day 10, there was no reduction in fibrosis as suggested by the strong collagen staining, whereas treatment of rats with imatinib mesylate from Day 10 to Day 21 still resulted in a strong reduction of collagen deposition as evidenced by the consistent reversal to a near-normal lung morphology.


Figure 5
View larger version (84K):
[in this window]
[in a new window]
  		Figure 5. Collagen staining of representative lung sections taken from rats treated with bleomycin followed by 10 mg/kg prednisolone or 50 mg/kg imatinib mesylate in the preventive and therapeutic models. In both cases, rats (10 animals/group) were administered bleomycin (2.2 mg/kg) at Day 0. In the preventive model, treatment commenced at Day 1, and was continued daily until Day 21. In the therapeutic model, treatment commenced at Day 10 and was continued daily until Day 21. On Day 22, the rats were killed and the lungs fixed in 4% paraformaldehyde before paraffin embedding. Sections (4 µM) were cut and baked onto APES-coated slides. The sections were then stained with Masson's Trichrome stain, with which muscle and cells are stained red, nuclei are stained black, and collagens are stained blue. Each photomicrograph is representative for each group. Magnification x 250.

 
Because imatinib mesylate is a combined PDGFR/c-abl/c-kit inhibitor (31), we wondered whether it might exert also an antiinflammatory function possibly due to inhibition of c-abl or c-kit kinases. To test this hypothesis, we treated rats intratracheally instilled with bleomycin with imatinib mesylate from Day 1 to Day 6 and analyzed the expression of inflammatory markers (namely, IL-1{alpha}, IL-1beta, IL-2, and IL-6) at Day 6. As a control, animals were treated with prednisolone over the same period. The results in Figure 6 show that compared with untreated rats, prednisolone resulted in a significant reduction in the levels of IL-1beta and IL-6 as expected for a corticosteroid. Interestingly, imatinib mesylate also strongly inhibited the expression of the inflammatory cytokines IL-1{alpha}, IL-1beta, and IL-6 to levels attained by prednisolone treatment. These data suggest an additional antiinflammatory function of imatinib mesylate, possibly through the action of c-kit or c-abl kinase inhibition.


Figure 6
View larger version (24K):
[in this window]
[in a new window]
  		Figure 6. Activity of prednisolone and imatinib mesylate on proinflammatory cytokine expression in the rat bleomycin model. (A) The experimental design to determine the activity of prednisolone and imatinib mesylate in the inflammatory phase of the model. Rats were treated with bleomycin (2.2 mg/kg) on Day 0, and treatment with prednisolone (2.2 mg/kg) or imatinib mesylate (50 mg/kg) commenced on Day 1 and continued until Day 6. (B) On Day 7, the rats were killed and a part of the left lobe was snap frozen in liquid nitrogen and stored at –80°C until analysis. The lobe was disrupted in RIPA buffer for protein extraction. The total protein concentrations were measured and all normalized to a concentration of 1 mg/ml. The levels of inflammatory cytokines were then measured using a Bioplex multiplex cytokine assay. Bars: median values.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Bleomycin-induced pulmonary fibrosis in rats is widely used as a model exhibiting a pathology similar to that found in human IPF. However, despite the fact that numerous agents targeting diverse signaling and molecular pathways inhibited fibrosis very effectively in this model, none of these molecules has demonstrated, as of today, clear efficacy in the treatment of IPF. Because in the bleomycin model, a pulmonary injury characterized by an inflammatory response leading to fibrosis is seen, we asked whether some drugs might show activity in this model solely by interfering with the initial inflammatory phase. We therefore performed time course experiments in the rat bleomycin model and determined the expression profiles of proinflammatory and profibrotic markers to define the onset and duration of inflammatory and fibrotic phases of the model. To our knowledge, this is the first published study to determine the time course of the development of inflammation and fibrosis in bleomycin-induced lung fibrosis. Using multiplex protein analysis, we showed that levels of inflammatory markers such as IL-1{alpha}, IL-1beta, IL-6, and IFN-{gamma} significantly increase during the first 9 d after bleomycin administration. These results confirm previously published data showing that bleomycin induces an initial inflammation (30, 32). After Day 9, a resolution of the inflammation seems to occur as exemplified by the reduction of cytokine expression down to control levels until the end of the experiment. In contrast, collagen deposition, as tested by Trichrome staining, is low up to Day 6 but increases strongly from about Day 9 up to a maximum at Day 21. Concomitantly, the expression pro-collagen I mRNA begins at Day 9. Taken together, the results suggest that in the rat bleomycin model, the "switch" between inflammation and fibrosis occurs on or just after Day 9. The two peaks observed for the expression of TGF-beta1 mRNA might reflect the proinflammatory and profibrotic functions of the growth factor.

Dividing the bleomycin model into two discrete phases permits one to discriminate between antiinflammatory and antifibrotic activity of test compounds. If selected compounds work by merely inhibiting the initial inflammation, they should prove active when administered over the entire period, whereas they should be ineffective if administered after the bulk of the inflammatory response has resolved. In contrast, drugs with antifibrotic activity should prove effective irrespective of the treatment mode.

We have chosen prednisolone and imatinib mesylate as prototype antiinflammatory or antifibrotic compounds, respectively. Systemic corticosteroids are the recommended and most prescribed therapy for IPF and associated lung fibroses (32–34). However, they are of little or no benefit in the majority of patients. There are some studies that have shown that for corticosteroids to be effective, treatment needs to be started early in the progression of the disease. However, in practical terms, this is unlikely to be a scenario clinicians encounter, because most IPF patients normally present with symptoms after significant fibrosis has occurred. Because imatinib mesylate is a selective PDGFR/cAbl/cKit receptor kinase inhibitor (31), we hypothesized that by blocking PDGFR and c-Abl it would interfere directly with fibroblast biology. PDGF has been shown to be secreted by activated fibroblasts, macrophages, platelets, smooth muscle, and damaged epithelial cells in asthma. Furthermore, it is able to induce stress fiber formation and increased motility in fibroblasts.

Our results clearly show that treatment with imatinib mesylate does result in significant attenuation of fibrosis, independent on the treatment regime, whereas prednisolone is only active when treatment occurred during the inflammatory phase of the model. Obviously, only imatinib mesylate is able to interfere with the fibrotic phase. Recent studies by Daniels and colleagues (21) have shown imatinib mesylate to be effective in attenuating bleomycin-induced fibrosis. The authors suggest that fibroblasts respond to TGF-beta by stimulating c-Abl kinase activity independently of Smad2/3 phosphorylation or PDGFR activation, and that c-Abl inhibition is therefore crucial for the activity of imatinib in the bleomycin model. Because of the lack of selective pharmacologic tools, we cannot define which of the three kinases blocked by imatinib mesylate is the target for the antifibrotic activity seen or whether the inhibition of all three enzymes is required.

A key difference between Daniels and colleagues (21) and the present study is the point at which pharmacologic treatment occurred. We have shown that, in contrast to prednisolone, treatment with imatinib mesylate is also efficacious in the fibrotic phase of the model. To test whether imatinib mesylate would also exert antiinflammatory activity, we administered it in the initial inflammatory phase of the model and showed that it possesses significant antiinflammatory activity comparable to high-dose prednisolone. Antiinflammatory activities of imatinib mesylate have been described before (35) and might be explained by the known functions of c-Abl and c-kit in various inflammatory processes (36, 37).

In summary, this study revisits the most commonly used animal model for pulmonary fibrosis. Our results indicate that previous studies using the bleomycin-induced fibrosis model in rats did not discriminate between, antiinflammatory or antifibrotic mechanisms (or a combination of the two). The adapted model in the present study resembles more closely the clinical setting physicians are faced with, in which patients with fibrosis present long after the resolution of inflammation. Furthermore, we reconfirmed that PDGFR/c-abl/c-kit inhibition may represent a novel antifibrotic therapy with the option to slow or reverse the rapid decline in lung health that most often leads to death. In the case of asthma and chronic obstructive pulmonary disease, decreasing or reversing fibrosis may help in making current antiinflammatory therapies more effective.


   FOOTNOTES
 
Originally Published in Press as DOI: 10.1164/rccm.200505-717OC on January 13, 2006

Conflict of Interest Statement: N.I.C. is an employee of Boehringer Ingelheim Pharma GmbH and Co. KG. A.S. is an employee of Boehringer Ingelheim Pharma GmbH and Co. KG. J.E.P. is an employee of Boehringer Ingelheim Pharma GmbH and Co. KG.

Received in original form May 6, 2005; accepted in final form January 9, 2006


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dacic S, Yousem SA. Histologic classification of idiopathic chronic interstitial pneumonias. Am J Respir Cell Mol Biol 2003;29:S5–S9.[Medline]
  2. Eddy AA. Molecular insights into renal interstitial fibrosis. J Am Soc Nephrol 1996;7:2495–2508.[Abstract]
  3. Wynn TA. Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat Rev Immunol 2004;4:583–594.[CrossRef][Medline]
  4. Nadrous HF, Myers JL, Decker PA, Ryu JH. Idiopathic pulmonary fibrosis in patients younger than 50 years. Mayo Clin Proc 2005;80:37–40.[Medline]
  5. Brewster CE, Howarth PH, Djukanovic R, Wilson J, Holgate ST, Roche WR. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 1990;3:507–511.[Medline]
  6. Roche WR, Beasley R, Williams JH, Holgate ST. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1989;1:520–524.[CrossRef][Medline]
  7. Holgate ST, Davies DE, Lackie PM, Wilson SJ, Puddicombe SM, Lordan JL. Epithelial-mesenchymal interactions in the pathogenesis of asthma. J Allergy Clin Immunol 2000;105:193–204.[CrossRef][Medline]
  8. Jeffery PK. Remodeling in asthma and chronic obstructive lung disease. Am J Respir Crit Care Med 2001;164:S28–S38.[Abstract/Free Full Text]
  9. Ramos C, Montano M, Garcia-Alvarez J, Ruiz V, Uhal BD, Selman M, Pardo A. Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ in growth rate, apoptosis, and tissue inhibitor of metalloproteinases expression. Am J Respir Cell Mol Biol 2001;24:591–598.[Abstract/Free Full Text]
  10. Uhal BD, Joshi I, True AL, Mundle S, Raza A, Pardo A, Selman M. Fibroblasts isolated after fibrotic lung injury induce apoptosis of alveolar epithelial cells in vitro. Am J Physiol 1995;269:L819–L828.[Medline]
  11. Davies DE, Holgate ST. Asthma: the importance of epithelial mesenchymal communication in pathogenesis: inflammation and the airway epithelium in asthma. Int J Biochem Cell Biol 2002;34:1520–1526.[CrossRef][Medline]
  12. Zhang S, Smartt H, Holgate ST, Roche WR. Growth factors secreted by bronchial epithelial cells control myofibroblast proliferation: an in vitro co-culture model of airway remodeling in asthma. Lab Invest 1999;79:395–405.[Medline]
  13. Coker RK, Laurent GJ, Shahzeidi S, Lympany PA, du Bois RM, Jeffery PK, McAnulty RJ. Transforming growth factors-beta 1, -beta 2, and -beta 3 stimulate fibroblast procollagen production in vitro but are differentially expressed during bleomycin-induced lung fibrosis. Am J Pathol 1997;150:981–991.[Abstract]
  14. Vasquez R, Marien BJ, Gram C, Goodwin DG, Menzoian JO, Raffetto JD. Proliferative capacity of venous ulcer wound fibroblasts in the presence of platelet-derived growth factor. Vasc Endovascular Surg 2004;38:355–360.[Abstract/Free Full Text]
  15. Keerthisingam CB, Jenkins RG, Harrison NK, Hernandez-Rodriguez NA, Booth H, Laurent GJ, Hart SL, Foster ML, McAnulty RJ. Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-beta in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice. Am J Pathol 2001;158:1411–1422.[Abstract/Free Full Text]
  16. Mattey DL, Dawes PT, Nixon NB, Slater H. Transforming growth factor beta 1 and interleukin 4 induced alpha smooth muscle actin expression and myofibroblast-like differentiation in human synovial fibroblasts in vitro: modulation by basic fibroblast growth factor. Ann Rheum Dis 1997;56:426–431.[Abstract/Free Full Text]
  17. Zeisberg M, Strutz F, Muller GA. Role of fibroblast activation in inducing interstitial fibrosis. J Nephrol 2000;13:S111–S120.[CrossRef][Medline]
  18. Desmouliere A, Chaponnier C, Gabbiani G. Tissue repair, contraction, and the myofibroblast. Wound Repair Regen 2005;13:7–12.[CrossRef][Medline]
  19. Buoro S, Ferrarese P, Chiavegato A, Roelofs M, Scatena M, Pauletto P, Passerini-Glazel G, Pagano F, Sartore S. Myofibroblast-derived smooth muscle cells during remodelling of rabbit urinary bladder wall induced by partial outflow obstruction. Lab Invest 1993;69:589–602.[Medline]
  20. Moore BB, Kolodsick JE, Thannickal VJ, Cooke K, Moore TA, Hogaboam C, Wilke CA, Toews GB. CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury. Am J Pathol 2005;166:675–684.[Abstract/Free Full Text]
  21. Daniels CE, Wilkes MC, Edens M, Kottom TJ, Murphy SJ, Limper AH, Leof EB. Imatinib mesylate inhibits the profibrogenic activity of TGF-beta and prevents bleomycin-mediated lung fibrosis. J Clin Invest 2004;114:1308–1316.[CrossRef][Medline]
  22. Chua F, Gauldie J, Laurent GJ. Pulmonary fibrosis: searching for model answers. Am J Respir Cell Mol Biol 2005;33:9–13.[Abstract/Free Full Text]
  23. Dik WA, McAnulty RJ, Versnel MA, Naber BA, Zimmermann LJ, Laurent GJ, Mutsaers SE. Short course dexamethasone treatment following injury inhibits bleomycin induced fibrosis in rats. Thorax 2003;58:765–771.[Abstract/Free Full Text]
  24. Iyer SN, Gurujeyalakshmi G, Giri SN. Effects of pirfenidone on procollagen gene expression at the transcriptional level in bleomycin hamster model of lung fibrosis. J Pharmacol Exp Ther 1999;289:211–218.[Abstract/Free Full Text]
  25. Marshall RP, Gohlke P, Chambers RC, Howell DC, Bottoms SE, Unger T, McAnulty RJ, Laurent GJ. Angiotensin II and the fibroproliferative response to acute lung injury. Am J Physiol Lung Cell Mol Physiol 2004;286:L156–L164.[Abstract/Free Full Text]
  26. Aono Y, Nishioka Y, Inayama M, Ugai M, Kishi J, Uehara H, Izumi K, Sone S. Imatinib as a novel antifibrotic agent in bleomycin-induced pulmonary fibrosis in mice. Am J Respir Crit Care Med 2005;171:1279–1285.[Abstract/Free Full Text]
  27. Greenberg DB, Reiser KM, Last JA. Correlation of biochemical and morphologic manifestations of acute pulmonary fibrosis in rats administered paraquat. Chest 1978;74:421–425.[CrossRef][Medline]
  28. Yi ES, Lee H, Yin S, Piguet P, Sarosi I, Kaufmann S, Tarpley J, Wang NS, Ulich TR. Platelet-derived growth factor causes pulmonary cell proliferation and collagen deposition in vivo. Am J Pathol 1996;149:539–548.[Abstract]
  29. Genovese T, Mazzon E, Di Paola R, Muia C, Threadgill MD, Caputi AP, Thiemermann C, Cuzzocrea S. Inhibitors of poly(ADP-ribose) polymerase modulate signal transduction pathways and the development of bleomycin-induced lung injury. J Pharmacol Exp Ther 2005;313:529–538.[Abstract/Free Full Text]
  30. Kradin RL, Sakamoto H, Jain F, Zhao LH, Hymowitz G, Preffer F. IL-10 inhibits inflammation but does not affect fibrosis in the pulmonary response to bleomycin. Exp Mol Pathol 2004;76:205–211.[CrossRef][Medline]
  31. Fabian MA, Biggs WH III, Treiber DK, Atteridge CE, Azimioara MD, Benedetti MG, Carter TA, Ciceri P, Edeen PT, Floyd M, et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotechnol 2005;23:329–336.[CrossRef][Medline]
  32. Richeldi L, Davies HR, Ferrara G, Franco F. Corticosteroids for idiopathic pulmonary fibrosis. Cochrane Database Syst Rev 2003;3:CD002880.
  33. Field SK. Steroids and/or cytotoxic agents should be used early in the management of patients with. Can Respir J 2004;11:212–213.[Medline]
  34. Luppi F, Cerri S, Beghe B, Fabbri LM, Richeldi L. Corticosteroid and immunomodulatory agents in idiopathic pulmonary fibrosis. Respir Med 2004;98:1035–1044.[CrossRef][Medline]
  35. Berlin AA, Lukacs NW. Treatment of cockroach allergen asthma model with imatinib attenuates airway responses. Am J Respir Crit Care Med 2005;171:35–39.[Abstract/Free Full Text]
  36. Tsang CM, Wong CK, Ip WK, Lam CW. Synergistic effect of SCF and TNF-{alpha} on the up-regulation of cell-surface expression of ICAM-1 on human leukemic mast cell line (HMC)-1 cells. J Leukoc Biol 2005;78:239–247.[Abstract/Free Full Text]
  37. Sawada J, Shimizu S, Tamatani T, Kanegasaki S, Saito H, Tanaka A, Kambe N, Nakahata T, Matsuda H. Stem cell factor has a suppressive activity to IgE-mediated chemotaxis of mast cells. J Immunol 2005;174:3626–3632.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Respir. Crit. Care Med.Home page
N. Pottier, C. Chupin, V. Defamie, B. Cardinaud, R. Sutherland, G. Rios, F. Gauthier, P. J. Wolters, Y. Berthiaume, P. Barbry, et al.
Relationships between Early Inflammatory Response to Bleomycin and Sensitivity to Lung Fibrosis: A Role for Dipeptidyl-Peptidase I and Tissue Inhibitor of Metalloproteinase-3?
Am. J. Respir. Crit. Care Med., December 1, 2007; 176(11): 1098 - 1107.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Tabata, R. Tabata, Y. Kadokawa, S. Hisamori, M. Takahashi, M. Mishima, T. Nakano, and H. Kubo
Thalidomide Prevents Bleomycin-Induced Pulmonary Fibrosis in Mice
J. Immunol., July 1, 2007; 179(1): 708 - 714.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
N. I. Chaudhary, G. J. Roth, F. Hilberg, J. Muller-Quernheim, A. Prasse, G. Zissel, A. Schnapp, and J. E. Park
Inhibition of PDGF, VEGF and FGF signalling attenuates fibrosis
Eur. Respir. J., May 1, 2007; 29(5): 976 - 985.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
A. U. Wells and C. M. Hogaboam
Update in Diffuse Parenchymal Lung Disease 2006
Am. J. Respir. Crit. Care Med., April 1, 2007; 175(7): 655 - 660.
[Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
R. Vittal, H. Zhang, M. K. Han, B. B. Moore, J. C. Horowitz, and V. J. Thannickal
Effects of the Protein Kinase Inhibitor, Imatinib Mesylate, on Epithelial/Mesenchymal Phenotypes: Implications for Treatment of Fibrotic Diseases
J. Pharmacol. Exp. Ther., April 1, 2007; 321(1): 35 - 44.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
C. Tabata, Y. Kadokawa, R. Tabata, M. Takahashi, K. Okoshi, Y. Sakai, M. Mishima, and H. Kubo
All-trans-Retinoic Acid Prevents Radiation- or Bleomycin-induced Pulmonary Fibrosis
Am. J. Respir. Crit. Care Med., December 15, 2006; 174(12): 1352 - 1360.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
200505-717OCv1
173/7/769    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chaudhary, N. I.
Right arrow Articles by Park, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chaudhary, N. I.
Right arrow Articles by Park, J. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2006 American Thoracic Society