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Published ahead of print on May 11, 2006, doi:10.1164/rccm.200603-333OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 420-427, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200603-333OC

Original Article

Discoidin Domain Receptor 1–deficient Mice Are Resistant to Bleomycin-induced Lung Fibrosis

Carmel Avivi-Green, Mayank Singal and Wolfgang F. Vogel

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

Correspondence and requests for reprints should be addressed to Wolfgang F. Vogel, Ph.D., Medical Sciences Building, Room 6342, 1 King's College Circle, Toronto, ON, M5S 1A8 Canada. E-mail: w.vogel{at}utoronto.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Discoidin domain receptor 1 (DDR1) is a tyrosine kinase activated by native collagens. Based on previous findings showing increased DDR1 expression in bronchoalveolar lavage cells from patients with idiopathic pulmonary fibrosis, we hypothesized that DDR1 mediates disease progression after lung injury.

Objectives: To investigate the inflammatory and fibrotic responses of DDR1 knockout and wild-type mice to bleomycin-induced lung injury.

Methods: Age- and sex-matched DDR1 knockout and wild-type C57BL/6 mice received a single intratracheal instillation of 2 U/kg bleomycin or saline, respectively. After 2 wk, lung inflammation and fibrosis were assessed using immunohistochemistry, real-time polymerase chain reaction, TUNEL assay, ELISA, fluorescence-activated cell sorting, and Western blot analysis.

Measurements and Main Results: Compared with wild-type animals, DDR1-null mice were largely protected against bleomycin-induced injury. Bleomycin-induced increases in collagen protein levels and tenascin-C mRNA levels were abrogated in knockout animals. Furthermore, myofibroblast expansion and apoptosis were much lower in these animals compared with their wild-type counterparts. Absence of inflammation in knockout mice was confirmed by lavage cell count and a cytokine ELISA. Western blot analysis of injured lung tissue revealed that DDR1-null mice failed to respond to the bleomycin insult with p38 MAPK activation, which was readily observed in wild-type mice.

Conclusions: DDR1 expression is a prerequisite for the development of lung inflammation and fibrosis. Blockade of DDR1 may therefore be a novel therapeutic intervention in patients with pulmonary fibrosis.

Key Words: kinases/phosphatases • lung • signal transduction • transgenic/knockout mice

Lung injury, caused by a wide range of insults, including environmental toxins, oxidants, immunologic factors, or viral infections, can progress to persistent inflammation and end-stage fibrosis. Idiopathic pulmonary fibrosis (IPF) is recognized as a disease with high morbidity and mortality among patients 65 yr and older, due to the lack of effective therapy (1). Hallmarks of IPF include an expansion of fibroblastic cells, excessive deposition of extracellular matrix and collagen in the lung interstitium, and gradual replacement of the alveoli by scar tissue. Despite recent gains in the understanding of the mechanisms underlying IPF, effective drug treatment regimens are still largely lacking (2).

Discoidin domain receptor 1 (DDR1) is unique among other receptor tyrosine kinases because it is activated by native collagens (3, 4). Under normal conditions, DDR1 is present in several tissues, including lung, colon, pancreas, and mammary gland. DDR1 expression is up-regulated in breast, lung, ovarian, and esophageal carcinomas (5–8). In the adult human lung, DDR1 expression is highest at the basolateral surface of the bronchial epithelium, which is in contact with the type IV collagen-rich basement membrane (9). DDR1 is critical for leukocyte migration and for the differentiation of monocytes into macrophages (10, 11).

It was previously shown that exposure of a variety of cell lines to fibrillar (types I–V) or basement membrane (type IV) collagens results in a sustained activation of the DDR1 kinase function (for review, see Reference 12). The binding of the ShcA and Nck2 adaptor molecules to phosphorylated DDR1 triggers cell type–specific events, such as activation of the Src or p38 mitogen-activated protein kinase (MAPK) pathway, that, in turn, regulate cell migration and differentiation as well as matrix metalloproteinase (MMP) expression and collagen deposition (3, 13–16). DDR1 knockout mice exhibit normal embryonic and postnatal development, but female reproduction is severely impaired (17). Also, pregnant mice fail to lactate due to improper differentiation of the alveoli in the mammary gland (18). Injury of the carotid arteries in these animals, used as a model for atherosclerosis, revealed that DDR1 is essential for smooth muscle cell migration and neointima formation (19). Compared with normal cells, DDR1-null smooth muscle cells exhibit reduced migration and MMP-2 and MMP-9 secretion (20). DDR1 has also been shown to be up-regulated in cutaneous wound healing (21). Taken together, these data place DDR1 at the crossroad of signaling pathways essential in maintenance and repair of connective tissues.

In a recent study, CD14-positive cells in the bronchoalveolar lavage (BAL) fluid from patients with IPF were compared with samples from healthy volunteers or from patients with other lung diseases, and significantly higher levels of DDR1 were found in cells from patients with IPF (22). These observations prompted us to examine the effect of DDR1 gene knockout on lung fibrosis using the bleomycin-mediated injury mouse model. Our data provide compelling evidence that mice lacking DDR1 are protected against lung fibrosis.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
The generation of DDR1 knockout mice is described elsewhere (17). The original line generated in a 129Sv/ICR background has been backcrossed into the C57BL/6 strain for greater than 20 generations. For this study, sex- and age-matched knockout and wild-type mice were obtained by breeding heterozygote animals. The bleomycin injury experiment was performed three independent times using 2- to 3-mo-old male mice (four animals/group). A total of 66 mice were used for the entire study. Representative data from each experiment are presented. After anaesthetizing mice with isofluorane, the trachea was surgically exposed, and 2 U/kg bleomycin sulfate (1 U/ml in phosphate-buffered saline [PBS]) or PBS only was intratracheally instilled (23, 24). An average-weight mouse (25 g) therefore received 0.05 U bleomycin dissolved in 50 µl PBS. The incisions were closed with surgical staples, and all animals were allowed to recover for 14 d. Previous work has established this time interval to be the most suitable for measurement of lung fibrosis parameters (25). None of animals died after surgery. All animals received humane care in compliance with the "Guide to the Care and Use of Experimental Animals" issued by the Canadian Council of Animal Care. All animal procedures were performed in accordance with a protocol approved by the animal care committee of the University of Toronto.

BAL
Fourteen days after injury, animals were anesthetized with ketamine/xylazine. The trachea was cannulated with a 20-gauge, 1-in angiocatheter (Beckton-Dickinson, Mississauga, ON, Canada). BAL was performed, and three aliquots of 0.5-ml PBS obtained. Cells were pelleted by centrifugation, resuspended in PBS, and total cell count was determined using a hemocytometer (VWR, Mississauga, ON, Canada). A differential cell count was obtained after cytospin and staining with Diff-Quik (Dade Behring, Newark, DE).

Fluorescence-actived Cell Sorting Analysis
One million BAL cells were labeled with fluorescein isothiocyanate–conjugated F4/80 monoclonal antibody (Cedarlane Laboratories, Hornby, ON, Canada) and fixed with paraformaldehyde before flow cytometry. Alternatively, cells were incubated with DDR1 monoclonal antibody (YH1–10D4) followed by secondary phycoerythrin (PE)-conjugated donkey anti-mouse antibody before fixation. Flow cytometry was performed on a FACSCalibur (Becton Dickinson).

Immunohistology and Immunofluorescence
Lung tissue was inflated and fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-µm-thin sections. Sections were stained with hematoxylin and eosin or Masson trichrome stain (Sigma, Oakville, ON, Canada) according to the manufacturer's instructions. Sections were also processed for immunohistochemistry or immunofluorescence. For staining with anti-CD3, antigen retrieval was performed in 10 mM Na-citrate buffer at 100°C for 10 min. Endogenous peroxidase activity was blocked using 3% H2O2 in 70% methanol. Sections were incubated with anti-CD3 antibody (1:100; Dako, Glostrup, Denmark) followed by secondary biotinylated antibody, and labeling was visualized with the ABC kit detection (Vector Laboratories, Burlingame, CA). Sections were counterstained with hematoxylin and mounted with Permount (Fisher, Fair Lawn, NJ). Staining with fluorescein isothiocyanate–conjugated F4/80 antibody (1:1,000) was performed according to the manufacturer's protocol (Cedarlane Laboratories). Apoptotic cells were detected using the FragEL kit (Oncogene Research Products, Boston, MA). For immunostaining with anti-bromo-deoxyuridine (BrdU) antibody (1:200), the slides were pretreated with 20 µg/ml proteinase K for 1 min, washed with PBS, and denatured with 2 N HCl at 37°C for 15 min. For {alpha}-smooth muscle actin ({alpha}-SMA) staining, proteinase-treated sections were incubated with Cy3-conjugated anti–{alpha}-SMA antibody (1:500; Sigma) and counterstained with Hoechst.

Morphometric Analysis of Lung Sections
Mean alveolar surface was calculated from 10 randomly selected fields at 200x magnification as described previously (26, 27).

Statistical Analysis
All data are expressed as mean ± SEM. Student's t-test was used to compare group means. Values of p < 0.05 were accepted as significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Reduced Fibrosis in DDR1-null Lung Tissue after Bleomycin Injury
Sex- and age-matched groups of wild-type and DDR1-null mice received a single intratracheal dosage of bleomycin (2 U/kg). Two weeks postinjury, the lung tissue was isolated and subjected to histologic and ultrastructural analysis. Although interalveolar thickening of the septa and reduction of alveolar space was readily apparent in injured wild-type mice, injured DDR1-null mice presented with grossly normal lung morphology (Figure 1A). Mean alveolar surface, defined as the average area per alveolar space in a given section, was 9.0 x 103 µm2 in wild-type animals compared with 5.9 x 103 µm2 in knockout mice (p < 0.01). As depicted in Figure 1B, electron microscopy of bleomycin-injured lung tissue confirmed thickened septa and rough brush border cells in wild-type animals (26). In contrast, the ultrastructure of epithelial and endothelial cells was largely unaltered in bleomycin-injured DDR1-null mice and was almost indistinguishable from that seen in PBS-treated animals (data from PBS-treated animals not shown). To evaluate the extent of fibrosis after bleomycin instillation, we performed Masson trichrome staining. As shown in Figure 2A, collagen-rich nodular depositions were present in bleomycin-injured wild-type mice (Figure 2A). In contrast, injured DDR1-null mice displayed very little change in matrix deposition and were similar to the PBS-treatment groups. To further evaluate the effect of DDR1 genotype on fibrotic reactions, we quantified the amount of pepsin-extractable collagen within the lung tissue. Although the collagen content in wild type mice increased 4.6-fold after injury (p < 0.001), it remained near basal levels in DDR1-null mice (Figure 2B). Increased expression of the matrix protein tenascin-C is a reliable indicator of tissue fibrosis (28, 29). Real-time polymerase chain reaction analysis of RNA extracted from tissue of injured wild-type and DDR1-null mice confirmed that wild-type mice had elevated levels of tenascin-C, whereas knockout animals did not (Figure 2C). Quantification of fibrillin-1 RNA levels was included as control.


Figure 1
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  		Figure 1. Histologic analysis of lung tissue after bleomycin administration revealed that there was substantial tissue damage, including interalveolar septum thickening, in wild-type mice, but DDR1-null tissue exhibited a nearly normal appearance. (A) Hematoxylin and eosin stain of paraffin sections obtained from DDR1 knockout and wild-type lungs 14 d after bleomycin injury (original magnification, 100x; scale bar, 150 µm). (B) Lung tissue from bleomycin-injured mice was processed for transmission electron microscopy as described in METHODS in the online supplement. Images of alveolar septa show ruffled brush borders in injured wild-type tissue, but smooth cell membranes in DDR1-null animals (original magnification, 3,500x; scale bar, 5 µm). Representative images from 12 animals per group are shown. KO = knockout; WT = wild-type.

 

Figure 2
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  		Figure 2. Unlike tissue from wild-type animals, DDR1-null tissue does not exhibit augmented extracellular matrix deposition. (A) Masson trichrome stain of paraffin sections from bleomycin and phosphate-buffered saline (PBS)–treated mice (original magnification, 200x; scale bar, 100 µm). (B) The collagen content of lung tissue from DDR1-null and wild-type mice 14 d after bleomycin treatment was determined by the Sircol assay as described in METHODS in the online supplement. Data are expressed as a percentage of the PBS-treated control (p < 0.001, n = 4/group). (C) RNA expression levels of tenascin-C and fibrillin-1 were determined by real-time polymerase chain reaction analysis. Results are expressed as percentages of the PBS-treated control (p = 0.009, n = 3/group).

 
Because it is well accepted that differentiation and expansion of myofibroblasts are associated with the development of bleomycin-induced pulmonary fibrosis (30), we labeled tissue sections for {alpha}-SMA, a marker for myofibroblasts and smooth muscle cells lining the airspaces and larger blood vessels. Consistent with the striking differences in matrix deposition seen between wild-type and DDR1-null mice, focal areas of intense {alpha}-SMA staining were found in larger numbers in the lung parenchyma of wild-type compared with bleomycin-injured DDR1-null mice (Figure 3).


Figure 3
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  		Figure 3. Reduced myofibroblast expansion in knockout mice treated with bleomycin for 14 d. Immunofluorescent labeling of {alpha}-smooth muscle actin (red) was performed, followed by nuclear counterstaining (blue). Note that smooth muscle actin–positive cell populations are more abundant in bleomycin-treated wild-type compared with DDR1-null samples (original magnification, 400x; insets, 200x; scale bar, 100 µm). A smooth muscle actin–positive blood vessel is shown in the PBS-treated group. Representative images from 12 animals per group are shown.

 
Bleomycin-induced Apoptosis Is Abolished in DDR1-null Mice
The progression to lung fibrosis entails complex cellular mechanisms, including activation, proliferation, and apoptosis. To address these functions in the bleomycin-induced lung injury, we quantified the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells in lung sections from wild-type and DDR1-null mice. As shown in Figure 4, the number of apoptotic cells was significantly decreased in the absence of DDR1 (Figure 4A). However, because there was an overall decrease in airspace in wild-type mice, as shown in Figure 1A, we hypothesized that the increased apoptosis observed in these animals is accompanied by increased proliferation. To test this hypothesis, BrdU was injected in bleomycin-injured mice 2 h before killing and lung sections were stained with a BrdU-specific antibody. Quantification of BrdU labeling revealed dramatically increased proliferation in injured wild-type animals, but not in injured DDR1-null mice, which exhibited numbers of proliferating cells comparable to PBS-treated animals (Figure 4B).


Figure 4
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  		Figure 4. Apoptosis and proliferation are higher in injured wild-type than DDR1 knockout mice. (A) Paraffin tissue sections from wild-type and knockout mice treated with bleomycin for 14 d were labeled for apoptotic cells as detailed in METHODS. Apoptotic cells were quantified by counting 10 fields at 400x magnification (n = 3/group). No TUNEL-positive cells were found in PBS-treated mice. The inset shows TUNEL-positive cells (green, arrows) and nuclear counterstain (blue) in a lung section from a bleomycin-injured wild-type mouse. (B) Bleomycin-injured animals were injected with BrdU 2 h before killing. Lung tissue sections were stained with an anti-BrdU antibody, and the number of positive cells per 10 fields determined (n = 3/group).

 
Bleomycin-induced Inflammation Is Reduced in DDR1-null Mice
As an intrinsic feature of the bleomycin mouse model, fibrosis is triggered by substantial inflammation (31). To examine the extent of bleomycin-induced inflammation in DDR1-null animals, we stained lung sections with antibodies directed against markers for lymphocytes and macrophages. Bleomycin-injured wild-type animals displayed a 2.3-fold greater number of CD3-positive lymphocytes than their PBS-treated counterparts, whereas the difference between injured and noninjured knockout mice was only 1.2-fold (p <0.001; Figure 5C). Similarly, bleomycin injury induced a 3.5- and 2.6-fold increase in F4/80-positive macrophages per visual field in wild-type and DDR1-null mice, respectively (p = 0.014; Figure 5F). Consistent with these immunohistologic findings, we observed significantly higher levels of macrophages in BAL from control versus DDR1-null mice (Figure 6A). BAL lymphocyte and neutrophil counts showed a similar trend, but the difference was not statistically significant (Figure 6B, and data not shown). Importantly, flow cytometry confirmed that the percentage of cells from BAL expressing DDR1 as surface antigen, most likely macrophages, is higher in wild-type versus DDR1-null animals (Figure 6C).


Figure 5
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  		Figure 5. Knockout mice show less inflammation than wild-type mice after lung injury (bleomycin, 14 d). (A) Bleomycin-induced tissue infiltration by lymphocytes and macrophages was assessed by immunohistochemistry and immunofluorescence, respectively. (A, B) Representative images of lung tissue from an injured wild-type mouse that was stained with (A) anti-CD3 antibody or (B) preimmune serum as detailed in METHODS (scale bar, 50 µm). (C) Immunohistochemical labeling from bleomycin and PBS-treated groups was quantified and results from injured mice expressed as a percentage of the PBS-treated control (n = 4/group, p < 0.001). (D, E) Immunofluorescent labeling for F4/80, a macrophage marker, was performed on sections from injured lungs obtained from injured (D) wild-type and (E) DDR1-null mice (scale bar, 50 µm). (F) The number of F4/80-positive cells in 10 fields at 400x magnification was determined, and results expressed as percentage of the number present in PBS-treated controls (p = 0.014).

 

Figure 6
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  		Figure 6. Quantification of infiltrating immune cells in bronchoalveolar lavage (BAL) fluid found higher number of macrophages in wild-type than knockout mice treated with bleomycin for 14 d. Differential cell count was performed on cytospins of BAL samples, and the mean number of (A) macrophages (p = 0.05) and (B) lymphocytes (p = 0.06) are given. (C) Fluorescence-activated cell sorting analysis of pooled BAL samples from bleomycin and PBS-treated mice was performed for macrophages (F4/80) and DDR1. Note that the majority of wild-type BAL cells express DDR1 as surface antigen, and less than 2% of DDR1-null cells were sorted as false-positive cells.

 
A large number of cytokines, such as interleukins, are critically involved in triggering inflammatory responses during lung injury (30). As shown in Figure 7A, BAL supernatants contained significantly lower concentrations of interleukin (IL)-6 in DDR1-null mice compared with their wild-type counterparts as measured by ELISA (p = 0.04). In addition, there was a trend toward lower monocyte chemoattractant protein-1 levels in knockout versus wild-type mice; however, the difference was not statistically significant (Figure 7B).


Figure 7
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  		Figure 7. Evaluation of cytokines in BAL fluid after bleomycin injury for 14 d. The concentration of interleukin (IL)-6 (A) or monocyte chemoattractant protein (MCP)-1 (B) in BAL fluid was determined by ELISA (n = 4/group). BAL from wild-type animals contained markedly higher concentrations of IL-6 than their DDR1-null counterparts (p < 0.05). There was a similar trend for MCP-1 concentrations, but the difference was not statistically significant.

 
Bleomycin-induced p38 Activation Is Absent in DDR1-null Mice
Previous studies of monocytes and BAL cells suggest that DDR1 regulates inflammatory and fibrotic responses via the p38 MAPK pathway (22, 32). In wild-type animals, DDR1 protein levels increased 1 d after bleomycin instillation (Figure 8A). Moreover, p38 phosphorylation was induced in a time-dependent manner after bleomycin instillation (Figure 8B). Levels of phosphorylated p38 were lower in knockout animals, relative to wild-type animals at 2 and 14 d after injury (Figure 8C). These findings may help account for the protective effect of a DDR1-null background.


Figure 8
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  		Figure 8. Time course of bleomycin-induced changes in DDR1 protein levels and p38 phosphorylation. Wild-type mice were killed 1, 3, 7, or 14 d after bleomycin injury. (A) One milligram of lung protein lysate was subjected to lectin-affinity chromatography followed by anti-DDR1 Western blotting. Quantification relative to the untreated animal is presented. (B) Total protein lysates were probed with antibodies specific for activated p38 MAPK (phospho-p38, top panel). The blot was stripped and subsequently reprobed for total p38 (middle panel) or GAPDH (lower panel). (C) Phosphorylation of p38 was measured in wild-type and DDR1-null mice 2 or 14 d after bleomycin treatment. All Western blot experiments were repeated three times and a set of representative data is shown here.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mice lacking the collagen receptor DDR1 are resistant to bleomycin-induced lung inflammation and fibrosis. Specifically, reduced inflammation in DDR1-null mice was shown by quantifying the numbers of lymphocytes and macrophages within the tissue and BAL and also by measuring IL-6 concentration in the lavage supernatant. Absence of fibrosis in injured DDR1-null mice was assessed histochemically using Masson trichrome stain and by measuring collagen protein and tenascin-C RNA concentrations. These data provide convincing evidence that the DDR1 collagen receptor may have an important role in lung fibrosis.

Signal transduction pathways downstream of DDR1 have been studied in a variety of cell types and tissues. Collectively, the protracted but sustained activation of DDR1 by collagen recruits a selected group of intermediate adaptor proteins, such as ShcA, Shp-2, Nck2, and FRS-1, which trigger activation of the p38 and p42/p44 MAPK cascades (3, 14, 15, 33). Here we show that p38 MAPK is activated by bleomycin in wild-type mice, but not in DDR1-null mice. The importance of p38 activation in pulmonary inflammation is supported by the fact that stimulation of DDR1-expressing alveolar macrophages from the BAL of patients with IPF with a DDR1-agonistic antibody results in p38 phosphorylation (22). Moreover, small-molecule inhibitors specific for active p38 reduce inflammation and fibrosis in the bleomycin mouse model (34, 35). In addition, nuclear factor-{kappa}B has not only been implicated as a target of activated DDR1 but was also found to be centrally involved in mediating bleomycin-induced lung injury (31, 32).

The time course of cellular and molecular events after bleomycin-induced lung injury are well established (25, 36, 37). The initial exposure of the lung epithelium to bleomycin generates reactive oxygen species, which trigger cellular apoptosis (38). Because DDR1 is primarily expressed in the pulmonary epithelium at the cell–matrix contact, it is tempting to speculate that the collagen-mediated DDR1 signaling is involved in mediating caspase cleavage or up-regulation of Fas ligand (9, 39). After the initial insult, tissue damage and secretion of cytokines elicit an influx of inflammatory cells, which trigger disease progression. As shown here, a high percentage of lavage cells express DDR1 at their surface, which may be important for the efficient tissue infiltration of macrophages through a collagen-rich basement membrane. However, because the number of CD3- and F4/80-reactive cells was markedly reduced not only in BAL but also within the lung parenchyma of injured DDR1-null mice relative to their wild-type counterparts, the lack of inflammatory response in these animals may be caused by lower levels of recruited immune cells, rather than by defects in transepithelial migration.

Additional roles of DDR1 in later stages of fibrosis need to be considered as well. It has been proposed that pulmonary fibrosis is an unlimited and overshooting repair process, largely mediated by myofibroblasts (40). DDR1 activity may constitute a perpetual "on-signal," which has been proposed to prevent proper reepithelialization, to lead to persistent repair through mesenchymal cells, and to culminate in fibrosis. Alternatively, the initial event may not solely be an inflammatory injury but an immune response to a previously unrecognized collagen antigen, which sets off an entire cascade of events. In accordance with the data presented here, we hypothesize that, in the absence of DDR1, injured epithelial cells fail to secret factors such as transforming growth factor-beta, which are key molecules in the differentiation and survival of myofibroblasts (41). As a consequence, tissue repair in DDR1-null mice is associated with limited myofibroblast expansion and hence less fibrosis, allowing for healing of the epithelial compartment to restore normal lung function. This hypothesis is supported by recent studies showing that blocking of integrin-mediated adhesion responses with a protein kinase inhibitor prevents bleomycin-induced myofibroblast accumulation and fibrosis, but not inflammatory responses (42). Because both bronchoalveolar macrophages and pulmonary epithelial cells express DDR1, it is unclear which cell type is responsible for the protective outcome in knockout mice. Additional experiments, such as in tissue-specific DDR1 knockout mice, will be necessary to address this question.

The specific function for DDR1 in lung tissue repair was recently investigated in lymphangioleiomyomatosis (LAM), a rare and sporadic disease of unknown etiology, which affects women of reproductive age (43). Lung samples from patients with LAM contained elevated DDR1 levels in ciliated epithelial cells, alveolar macrophages, and smooth muscle cells (44). These findings were corroborated by the fact that patients with LAM also exhibited high MMP-2 and MMP-9 activities and high MMP-1 levels. However, forced overexpression of DDR1 in cultured smooth muscle cells leads to a decrease in type I collagen synthesis, an observation at odds with the bleomycin-induced DDR1 induction and excess collagen deposition shown here. Potentially, tissue-specific responses modulate DDR1 signaling outcomes, leading to variable effects on collagen degradation and neosynthesis.

While this article was being finalized for submission, Matsuyama and colleagues published similar work describing the protective effects of DDR1 knockdown in the bleomycin-injury model (45). Of note, transnasal application of siRNA was used to suppress DDR1 function during early stages of bleomycin-induced lung injury. In agreement with the data presented here, RNA interference-mediated DDR1 suppression resulted in reduced cytokine production and diminished infiltration of alveolar macrophages. These changes were accompanied by reduced fibrosis 2 wk after bleomycin injury, as described here. The use of a DDR1 gene knockout mouse, which avoids the commonly observed ambiguities in protein knockdown levels achieved by RNA interference, validates these findings. The current work extends these findings by identifying apoptosis and myofibroblast expansion as critical factors associated with lung fibrosis, by demonstrating DDR1 surface expression in BAL cells, and by showing that bleomycin injury leads to increased expression of the noncollagenous matrix molecule tenascin-C, which may be equally important in the development of fibrosis in the lung parenchyma.


   Acknowledgments
 
The authors thank Diana Koo, Vera Cherepanov, Theo Moraes, and Jack Haitsma for experimental help. They thank Jeremy Scott and Gregory Downey for expert advice on the interpretation of lung pathology. Expert technical assistance of Catherine Ramsay is also acknowledged.


   FOOTNOTES
 
Supported by the Canada Research Chair Program and the Premier's Research Excellence Award.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200603-333OC on May 11, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 7, 2006; accepted in final form May 11, 2006


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 METHODS
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 DISCUSSION
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