Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase whose ligand is collagen. Recently, we have reported the association of DDR1 in the cytokine production of human leukocytes in in vitro and in vivo expression in idiopathic pulmonary fibrosis. However, its role in in vivo inflammation has not been fully elucidated. Small interference RNA (siRNA) can induce specific suppression of in vitro and in vivo gene expression. In this study, using a bleomycin-induced pulmonary fibrosis mouse model, we administered siRNA against DDR1 transnasally and evaluated histological changes, cytokine expression, and signaling molecule activation in the lungs. Histologically, siRNA against DDR1 successfully reduced in vivo DDR1 expression and attenuated bleomycin-induced infiltration of inflammatory cells. Furthermore, it significantly reduced inflammatory cell counts and concentrations of cytokines such as MCP-1, MIP-1α, and MIP-2 in bronchoalveolar lavage fluid. Subsequently, bleomycin-induced up-regulation of TGF-β in bronchoalveolar lavage fluid was significantly inhibited, and collagen deposition in the lungs was reduced. Furthermore, siRNA against DDR1 significantly inhibited bleomycin-induced P38 MAPK activation in the lungs. Considered together, we propose that DDR1 contributes to the development of bleomycin-induced pulmonary inflammation and fibrosis.

Discoidin domain receptor 1 (DDR1)3 is a receptor tyrosine kinase that is activated by binding to its ligand-collagen, including collagen type IV (1, 2), a component of the extracellular matrix (ECM) of the lungs. DDR1 possesses a unique extracellular domain that is homologous to discoidin 1 of Dictyostelium discoideum (3). DDR1 is constitutively expressed in the normal tissues of organs, such as the lungs, kidneys, colon, and brain, as well as in the tumor cells of epithelial origin, such as those of mammary, ovarian, and lung carcinomas (3). We have previously reported that in vitro DDR1 expression could be induced in human leukocytes, including neutrophils, monocytes, lymphocytes, and macrophages (4). The tissue-infiltrating mononuclear cells in vivo, macrophages in particular, were positive for DDR1 (5). We have recently discovered that DDR1 activation up-regulates the production of chemokines, such as MCP-1, in macrophages. These chemokines play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF) in a P38 MAPK-dependent manner and are likely to contribute to the development of inflammatory responses in the tissue microenvironment (6). Furthermore, we found that increased DDR1 expression in alveolar macrophages of IPF patients and its activation induce the production of cytokines, including MCP-1 and matrix metalloproteinase-9, which are key molecules in the pathogenesis of IPF (7). These findings led us to hypothesize that DDR1 suppression might affect or attenuate inflammation in the lungs.

To suppress a specific gene, antisense or ribozyme-based techniques were used in the past decades. In addition, gene silencing using synthetic small interfering RNA (siRNA) has emerged as a powerful tool in the suppression of target gene expression (8). It has been reported recently that transnasal administration of siRNA is able to down-regulate protein expression (9) as well as the respiratory virus replication (10) specifically in the lungs. In this study, by using a bleomycin-induced pulmonary fibrosis mouse model, we investigated whether DDR1 suppression by siRNA is capable of attenuating bleomycin-induced lung inflammation.

This study was conducted using female C57BL/6 mice (weight, 17–20 g; age, 8 wk) in specific pathogen-free conditions and was approved by the Kagoshima University Ethics Committee for Animal Experiments.

The mice were anesthetized by i.p. administration of 1.5 mg of ketamine hydrochloride (Sankyo) and 0.3 mg of xylazine hydrochloride (Bayer), and the trachea was exposed via a cervical incision. Bleomycin (90 μg; Nippon Kayaku) was dissolved in 50 μl of saline and then instilled intratracheally with a 27-gauge needle. For additional experiments, the mice were ether anesthetized and then sacrificed by axillary artery exsanguination. Table I lists the oligonucleotides used in this study. Each siRNA was diluted in PBS and then administered intranasally after anesthetizing the animals, as described above.

Table I.

Oligonucleotides used in this study

NameTargetSequence
siRNA1 DDR1 5′-AAGTGCCGCTATGCCCTGGdTdT-3′ 
  5′-dTdTCCAGGGCATAGCGGCACTT-3′ 
siRNA2 DDR1 5′-AAGCTATCGGTTGCGTTACdTdT-3′ 
  5′-dTdTGTAACGCAACCGATAGCTT-3′ 
siRNA-Luc Luciferase 5′-CGUACGCGGAAUACUUCGAdTdT-3′ 
  3′-dTdTGCAUGCGCCUUAUGAAGCU-5′ 
Negative None 5′-UUCUCCGAACGUGUCACGUdTdT-3′ 
  3′-dTdTTAAGAGGCUUGCACAGUGCA-5′ 
NameTargetSequence
siRNA1 DDR1 5′-AAGTGCCGCTATGCCCTGGdTdT-3′ 
  5′-dTdTCCAGGGCATAGCGGCACTT-3′ 
siRNA2 DDR1 5′-AAGCTATCGGTTGCGTTACdTdT-3′ 
  5′-dTdTGTAACGCAACCGATAGCTT-3′ 
siRNA-Luc Luciferase 5′-CGUACGCGGAAUACUUCGAdTdT-3′ 
  3′-dTdTGCAUGCGCCUUAUGAAGCU-5′ 
Negative None 5′-UUCUCCGAACGUGUCACGUdTdT-3′ 
  3′-dTdTTAAGAGGCUUGCACAGUGCA-5′ 

BALF was obtained by cannulating the trachea with a 20-gauge needle and infusing the lungs four times with 1 ml of saline. The recovery of BALF ranged between 2.0 and 3.5 ml, with no significant difference in the volume recovered from each mouse. The BALF cells were collected after centrifugation (1000 × g, 10 min, 4°C). The supernatants were stored immediately at −80°C until use for further analysis.

BALF cells were obtained from the bleomycin-treated mice (day 14). The cells (2 × 106) were maintained under aerobic conditions in 5% CO2 at 37°C in 6-well culture dishes containing 6 ml of DMEM supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM glutamine (JRH Biosciences). To evaluate the suppressive effects of siRNA, we transfected each siRNA construct into the mice BALF cells by using Transfectene Reagents (Qiagen), according to the manufacturer’s protocol.

The excised lungs were immediately fixed with 10% formaldehyde neutral buffer solution for 48 h and then embedded in paraffin. Sagittal sections were cut at 2-μm thickness and stained with H&E and Masson-trichrome stains. The total lung area of the sections was used for fibrotic scale microscope evaluation (Olympus; BX50F4). The criteria for grading lung fibrosis were in accordance with the method reported by Ashcroft et al. (11): grade 0, normal lung; grade 1, minimal fibrous thickening of alveolar or bronchiolar walls; grade 3, moderate thickening of walls without obvious damage to the lung architecture; grade 5, increased fibrosis with definite damage to the lung architecture and formation of fibrous bands or small fibrous masses; grade 7, severe distortion of architecture and large fibrous area; and grade 8, total fibrous obliteration of the field. The severity of fibrotic changes in each lung section was assessed as the mean score of severity from the observed microscopic fields. The grade of lung fibrosis was scored on a scale from 0 to 8 by examining 10 randomly chosen regions per sample at a magnification of ×200 by four pathologists who were blinded to the treatment. After examining the entire section, the mean of the scores from all the fields was considered as the fibrotic score.

Lung tissues were examined by immunohistochemical staining for DDR1 using a rabbit anti-DDR1 Ab (Santa Cruz Biotechnology) by using the diaminobenzidine method, as described previously (12). In brief, 4-μm-thick sections were mounted on poly-l-lysine-coated slides, dewaxed, and washed in TBS (pH 7.4) for 10 min. For optimal Ag retrieval, the sections were pressure cooked in 0.01 M citrate buffer (pH 6.0) for 90 s. Endogenous peroxidase activity was blocked using a 3% hydrogen peroxide solution in methanol for 10 min. The blocking reaction was performed after two washes in PBS with 1% saponin. The sections were incubated with the primary Ab solution for 2 h at room temperature by using a 1/50 dilution of the Ab. Negative control slides were incubated with rabbit IgG (R&D Systems). Secondary biotinylated anti-Ig Ab (R&D Systems) was added, and the mixture was incubated for 30 min at room temperature. After washing, the sections were incubated with streptavidin conjugated to HRP (Amersham) and then rinsed with deionized water. Diaminobenzidine substrate solution was added, and the mixture was incubated for 10 min. A brown-colored reaction represented a positive result.

To detect DDR1 and actin, we lysed 1 mg of lung tissue from each mouse or 2 × 106 mice BALF cells in 1 ml of lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, and a mixture of protease inhibitors (Roche) by using a hypersonic homogenizer (UD-200; Tomy). We used rabbit IgGs that recognize the DDR1 of humans and mice (Santa Cruz Biotechnology) or anti-actin mouse IgG mAb (Santa Cruz Biotechnology). To detect P38 MAPK phosphorylation, we lysed 1 mg of lung tissue from each mouse and used rabbit polyclonal anti-phosphorylated mouse P38α Ab or rabbit polyclonal anti-mouse P38α Ab (Cell Signaling Technology). To detect β1 integrin, we lysed 1 mg of lung tissue from each mouse and used anti-mouse β1 integrin rat mAb (R&D Systems). We performed the Western blot analysis, as described previously (4, 6, 13). In brief, the lysates were centrifuged, and 20 μl of the supernatant was collected. Subsequently, 20 μl of double-strength sample buffer (20% glycerol, 6% SDS, and 10% 2-ME) was added to each of supernatants. The samples were boiled for 10 min. Proteins were analyzed on 8 or 12% polyacrylamide gels by SDS-PAGE and transferred electrophoretically to nitrocellulose membranes at 150 mA for 1 h by using a semidry system. The membranes were incubated with each primary Ab, followed by sheep anti-rabbit or mouse IgG coupled with HRP (Amersham Biosciences). Peroxidase activity was visualized using the Enhanced Chemiluminescence Detection System (Amersham). The density of the bands was measured using a freeware image analysis software (NIH Image, version 1.62; National Institutes of Health 〈http://rsb.info.nih.gov/nih-image/〉).

Total RNA was extracted from 1 mg of lung tissue or 2 × 106 BALF cells by using TRIzol reagent, and Northern blotting was performed, as described previously (14). The clones of mouse DDR1 cDNA of TGF-β, MCP-1, MIP-2, and MIP-1α were donated by T. Yoshimura (National Cancer Institute, Frederick, MD). Each cDNA was labeled with [α-32P]dCTP using the Rediprime II Random Prime Labeling System (Amersham).

The concentrations of TGF-β, MCP-1, MIP-2, and MIP-1α in 1 mg of homogenized lung tissue were measured by using ELISA kits (R&D Systems) according to the manufacturer’s protocols.

The lungs were harvested on day 21 after bleomycin administration. Each lung homogenate (0.5 ml) was digested in 1 ml of 6 N HCl for 8 h at 120°C. Five microliters of citrate/acetate buffer (5% citric acid, 7.24% sodium acetate, 3.4% sodium hydroxide, and 1.2% glacial acetic acid (pH 6.0)) along with 100 μl of chloramines T solution (282 mg of chloramines T, 2 ml of n-propanol, 2 ml of H2O, and 16 ml of citrate/acetate buffer (pH 6.0)) were added to 5 μl of the sample and incubated for 20 min. Next, 100 μl of Ehrlich’s solution (2.5 g of 4-(dimethylamino)benzaldehyde, 9.3 ml of n-propanol, and 3.9 ml of 70% perchloric acid) was added to each sample and incubated for 15 min at 65°C. The OD was determined at 550 nm on a DU 640 spectrophotometer (Beckman Instruments). Commercially available hydroxyproline (Sigma-Aldrich) was used to construct a standard curve.

The Mann-Whitney U test and Bonferroni-Dunn test with one-way factorial ANOVA were used. Kaplan-Meier analysis was used for survival analysis. A p value below 0.05 was considered to be significant. Values have been presented as the mean ± SD, unless otherwise stated. SD was calculated using Microsoft Excel software.

We first transfected various concentrations of siRNA (0.1, 1, 2, 5, and 10 nM) into mice BALF cells and found that transfection with 2 nM siRNA1 and siRNA2 significantly inhibited in vitro DDR1 mRNA and protein expression in mouse BALF cells (Fig. 1, a and b). The DDR1 expression was not inhibited by siRNA-Luc. Based on these results, we used siRNA1 for additional experiments.

FIGURE 1.

The effect of siRNA on in vitro DDR1 expression. The BALF cells obtained from bleomycin-treated mice on day 7 expressed endogenous DDR1 (a, lane 1). a, The two upper panels show Northern blot analysis, and the two lower panels show Western blot analysis. Three days after the transfection of 5 nM of each construct, the DDR1 mRNA and protein expression levels were significantly lower than in those with siRNA1 (a, lane 3) and siRNA2 (a, lane 4) when compared with the negative control (a, lane 2) and siRNA-Luc (a, lane 5). b, Shows the statistical result of DDR1/actin protein density ratio. The inhibitory effect on protein expression was statistically significant (∗, p < 0.01; Bonferroni-Dunn test with one-way factorial ANOVA, n = 5 in each group; b).

FIGURE 1.

The effect of siRNA on in vitro DDR1 expression. The BALF cells obtained from bleomycin-treated mice on day 7 expressed endogenous DDR1 (a, lane 1). a, The two upper panels show Northern blot analysis, and the two lower panels show Western blot analysis. Three days after the transfection of 5 nM of each construct, the DDR1 mRNA and protein expression levels were significantly lower than in those with siRNA1 (a, lane 3) and siRNA2 (a, lane 4) when compared with the negative control (a, lane 2) and siRNA-Luc (a, lane 5). b, Shows the statistical result of DDR1/actin protein density ratio. The inhibitory effect on protein expression was statistically significant (∗, p < 0.01; Bonferroni-Dunn test with one-way factorial ANOVA, n = 5 in each group; b).

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To evaluate whether transnasal administration is an adequate method for the suppression of in vivo DDR1 expression, we examined pulmonary DDR1 expression by Western blot analysis. We administered each type of siRNA on day 3 and collected the lungs on day 5. As shown in Fig. 2, siRNA1 significantly inhibited DDR1 expression in the lung tissue, while siRNA-Luc did not exert any inhibition. The minimum dose required to obtain adequate suppression of DDR1 expression was 5 nM. Therefore, we used 5 nM siRNA1 for additional experiments. The suppressive effect of DDR1 expression in the lung continued until day 9, and the maximum effect was observed on day 5 (Fig. 2, c and d). Based on these findings, we administered siRNA every 3 days to obtain continuous DDR1 suppression (Fig. 3, a and b). The DDR1-suppressive effect was specific to the lungs (Fig. 3 c).

FIGURE 2.

The effect of transnasal administration of siRNA1 on day 3 against lung DDR1 expression. a, Shows the DDR1/actin protein density ratio at each concentration of siRNA1. DDR1 siRNA1 was administered on day 3, and the lungs were obtained on day 5. The minimum dose of siRNA1 that was required to obtain significant inhibition of DDR1 protein expression in the lung on day 5 was 5 nM (∗, p < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA, n = 5 in each group; a). b, Shows the representative data obtained from Western blot analysis for DDR1. DDR1 siRNA1 significantly reduced lung DDR1 expression, whereas siRNA-Luc did not affect lung DDR1 expression (b). c and d, Show the suppressive effect of the transnasal administration of DDR1 siRNA1 (5 nM) on day 3 only (c shows the representative data obtained from Western blot analysis for lung DDR1 expression, and d shows the statistical result of DDR1/actin protein density). The suppressive effect of lung DDR1 expression continued until day 9, and the maximum effect was observed on day 5 (∗, p < 0.01; ∗∗, p < 0.05, compared with bleomycin alone, Bonferroni-Dunn test with one-way factorial ANOVA, n = 5 in each group; d).

FIGURE 2.

The effect of transnasal administration of siRNA1 on day 3 against lung DDR1 expression. a, Shows the DDR1/actin protein density ratio at each concentration of siRNA1. DDR1 siRNA1 was administered on day 3, and the lungs were obtained on day 5. The minimum dose of siRNA1 that was required to obtain significant inhibition of DDR1 protein expression in the lung on day 5 was 5 nM (∗, p < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA, n = 5 in each group; a). b, Shows the representative data obtained from Western blot analysis for DDR1. DDR1 siRNA1 significantly reduced lung DDR1 expression, whereas siRNA-Luc did not affect lung DDR1 expression (b). c and d, Show the suppressive effect of the transnasal administration of DDR1 siRNA1 (5 nM) on day 3 only (c shows the representative data obtained from Western blot analysis for lung DDR1 expression, and d shows the statistical result of DDR1/actin protein density). The suppressive effect of lung DDR1 expression continued until day 9, and the maximum effect was observed on day 5 (∗, p < 0.01; ∗∗, p < 0.05, compared with bleomycin alone, Bonferroni-Dunn test with one-way factorial ANOVA, n = 5 in each group; d).

Close modal
FIGURE 3.

The effect of transnasal every 3 days administration of siRNA1 on lung DDR1 expression (a and b). The transnasal administration of 5 nM siRNA1 every 3 days continuously inhibited lung DDR1 expression, while the siRNA-Luc did not affect it (a shows representative data obtained from Western blot analysis; b shows the statistical result of DDR1/actin protein ratio in the lungs; ∗, p < 0.01, compared with bleomycin alone, Bonferroni-Dunn test with one-way factorial ANOVA, n = 5 in each group). Bleomycin treatment induced lung DDR1 expression that peaked on day 14 and then showed a decrease (a and b). c, Shows the amount of DDR1 expression in each organ on day 14. The inhibitory effect of siRNA1 was specific to the lungs.

FIGURE 3.

The effect of transnasal every 3 days administration of siRNA1 on lung DDR1 expression (a and b). The transnasal administration of 5 nM siRNA1 every 3 days continuously inhibited lung DDR1 expression, while the siRNA-Luc did not affect it (a shows representative data obtained from Western blot analysis; b shows the statistical result of DDR1/actin protein ratio in the lungs; ∗, p < 0.01, compared with bleomycin alone, Bonferroni-Dunn test with one-way factorial ANOVA, n = 5 in each group). Bleomycin treatment induced lung DDR1 expression that peaked on day 14 and then showed a decrease (a and b). c, Shows the amount of DDR1 expression in each organ on day 14. The inhibitory effect of siRNA1 was specific to the lungs.

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We started the administration of each type of siRNA on day 3; following this, administration was performed every 3 days. Histological examinations revealed that DDR1 siRNA apparently attenuated the inflammation and pulmonary fibrosis (Fig. 4,a). Although the histological changes on day 3 were almost similar across the three groups, bleomycin-treated mice (without siRNA administration), DDR1 siRNA1-administered bleomycin-treated mice, and siRNA-Luc-administered bleomycin-treated mice, the infiltration of the inflammatory cells decreased after the administration of siRNA1. On day 21, the lung tissue showed slight infiltration of inflammatory cells in the DDR1 siRNA1-administered bleomycin-treated mice. In contrast, in the bleomycin-treated mice and the siRNA-Luc-administered bleomycin-treated mice, the bleomycin-induced inflammation progressed and resulted in severe pulmonary fibrosis on day 21 (Fig. 4,a). After day 14, the pulmonary fibrosis score of DDR1 siRNA1-administered bleomycin-treated mice was significantly lower than that of siRNA-Luc-administered bleomycin-treated mice and that of bleomycin-treated mice (p < 0.01; Fig. 4,b). When half the dose of bleomycin (45 μg) was administered on day 0, the pulmonary fibrosis score of DDR1 siRNA1-administered half dose bleomycin-treated mice on day 7 was significantly lower than that of half dose bleomycin-treated mice. On day 21, the amount of hydroxyproline was significantly lower in the DDR1 siRNA1-administered bleomycin-treated mice than that in the siRNA-Luc-administered bleomycin-treated mice and in the bleomycin-treated mice (p < 0.01; Fig. 4,c). The administration of siRNA1 alone did not affect the amount of hydroxyproline present in the lungs. Starting the siRNA1 administration after day 7 did not attenuate the bleomycin-induced lung inflammation (data not shown). The body weight on day 3 was almost the same across the three groups. After day 14, the body weight of DDR1 siRNA1-administered bleomycin-treated mice was significantly higher than those of siRNA-Luc-administered bleomycin-treated mice and the bleomycin-treated mice (Fig. 4 d). No mice died in the DDR1 siRNA1-administered bleomycin-treated mice group, while seven mice died in the siRNA-Luc-administered bleomycin-treated group and six mice died in the bleomycin-treated group. One mouse died in the half dose bleomycin (45 μg)-treated group, whereas no deaths were recorded in the siRNA1-administered half dose bleomycin-treated group.

FIGURE 4.

Pathological changes after the administration of siRNA1 or siRNA-Luc (a). We started the administration of each siRNA on day 3, and it was administrated every 3 days. Bleomycin treatment induced intense infiltration of inflammatory cells on day 3 in all groups. The inflammation progressed and inflammatory cells infiltrated almost the entire lungs on day 14; this finally led to pulmonary fibrosis on day 21 (left panels). The administration of siRNA1 reduced the amount of inflammatory cell infiltration on day 7. The area of inflammatory cell infiltration was apparently smaller in the siRNA1-administered bleomycin-treated mice (center panels) than in the bleomycin-treated mice (left panels) and the siRNA-Luc-administrated bleomycin-treated mice (right panels). In the siRNA1-administrated bleomycin-treated mice, the area of inflammatory cell infiltration was smaller on day 21 than on day 7 (arrows). The administration of siRNA-Luc did not decrease the infiltration of inflammatory cells, and pulmonary fibrosis occurred on day 21 (right panels). The pulmonary fibrosis score of siRNA1-administered bleomycin-treated mice was significantly lower than those of the bleomycin-treated mice and the siRNA-Luc-administered bleomycin-treated mice (b, average ± SDs of 16 mice; ∗, p < 0.01; ∗∗, p < 0.05, Bonferroni-Dunn test with one-way factorial ANOVA). The amount of hydroxyproline on day 21 was significantly lower in the siRNA1-administered bleomycin-treated mice (c; n = 12 in each group; ∗, p < 0.01, Mann-Whitney U test). The body weight on day 3 was almost the same among all of the three groups. After day 14, the body weight of the DDR1 siRNA1-administered bleomycin-treated mice was significantly higher than that of the siRNA-Luc-administered bleomycin-treated mice and the bleomycin-treated mice (∗, p < 0.01; ∗∗, p < 0.05, Bonferroni-Dunn test with one-way factorial ANOVA; d, n = 21 on day 3 in each group).

FIGURE 4.

Pathological changes after the administration of siRNA1 or siRNA-Luc (a). We started the administration of each siRNA on day 3, and it was administrated every 3 days. Bleomycin treatment induced intense infiltration of inflammatory cells on day 3 in all groups. The inflammation progressed and inflammatory cells infiltrated almost the entire lungs on day 14; this finally led to pulmonary fibrosis on day 21 (left panels). The administration of siRNA1 reduced the amount of inflammatory cell infiltration on day 7. The area of inflammatory cell infiltration was apparently smaller in the siRNA1-administered bleomycin-treated mice (center panels) than in the bleomycin-treated mice (left panels) and the siRNA-Luc-administrated bleomycin-treated mice (right panels). In the siRNA1-administrated bleomycin-treated mice, the area of inflammatory cell infiltration was smaller on day 21 than on day 7 (arrows). The administration of siRNA-Luc did not decrease the infiltration of inflammatory cells, and pulmonary fibrosis occurred on day 21 (right panels). The pulmonary fibrosis score of siRNA1-administered bleomycin-treated mice was significantly lower than those of the bleomycin-treated mice and the siRNA-Luc-administered bleomycin-treated mice (b, average ± SDs of 16 mice; ∗, p < 0.01; ∗∗, p < 0.05, Bonferroni-Dunn test with one-way factorial ANOVA). The amount of hydroxyproline on day 21 was significantly lower in the siRNA1-administered bleomycin-treated mice (c; n = 12 in each group; ∗, p < 0.01, Mann-Whitney U test). The body weight on day 3 was almost the same among all of the three groups. After day 14, the body weight of the DDR1 siRNA1-administered bleomycin-treated mice was significantly higher than that of the siRNA-Luc-administered bleomycin-treated mice and the bleomycin-treated mice (∗, p < 0.01; ∗∗, p < 0.05, Bonferroni-Dunn test with one-way factorial ANOVA; d, n = 21 on day 3 in each group).

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Immunohistological examinations revealed that infiltrating inflammatory cells, alveolar macrophages, and bronchoepithelial cells of bleomycin-treated mice expressed endogenous DDR1. DDR1 siRNA1 apparently suppressed DDR1 expression of these cells on day 7 (Fig. 5,a). The administration of siRNA-Luc did not affect DDR1 expression, and the suppressive effect of DDR1 siRNA1 remained for 21 days (Fig. 5 b).

FIGURE 5.

Immunohistochemical analysis of lung DDR1 expression. a, Shows the immunohistochemical analyses of DDR1 expression on day 7 (A, bleomycin alone; B, negative control; C, bleomycin + siRNA1); b, shows those on day 21 (A and B, bleomycin alone; C and D, negative control; E and F, bleomycin + siRNA1; G and H, bleomycin + siRNA-Luc). We started the administration of each siRNA on day 3, and it was repeated every 3 days. In the bleomycin-treated mice, alveolar macrophages (arrowheads), infiltrating inflammatory cells (arrowheads), and bronchoepithelial cells (arrows) expressed endogenous DDR1 on days 7 and 21 (arrows, a, A and b, A and B). The administration of the negative control alone did not affect endogenous DDR1 expression and did not induce pulmonary inflammation on day 7 (a, B) and on day 21 (b, C and D). The administration of siRNA1 inhibited the DDR1 expression in alveolar macrophages (arrowheads), infiltrating inflammatory cells (arrowheads), and bronchoepithelial cells (arrows) on day 7 (a, C). This inhibitory effect continued until day 21 (b, E and F). The administration of siRNA-Luc did not inhibit the endogenous DDR1 expression on day 21 (b, G and H). Representative data of 10 different mice of each group (a, ×400 original magnification; b, left panels, ×150 original magnification, and right panels, ×400 original magnification).

FIGURE 5.

Immunohistochemical analysis of lung DDR1 expression. a, Shows the immunohistochemical analyses of DDR1 expression on day 7 (A, bleomycin alone; B, negative control; C, bleomycin + siRNA1); b, shows those on day 21 (A and B, bleomycin alone; C and D, negative control; E and F, bleomycin + siRNA1; G and H, bleomycin + siRNA-Luc). We started the administration of each siRNA on day 3, and it was repeated every 3 days. In the bleomycin-treated mice, alveolar macrophages (arrowheads), infiltrating inflammatory cells (arrowheads), and bronchoepithelial cells (arrows) expressed endogenous DDR1 on days 7 and 21 (arrows, a, A and b, A and B). The administration of the negative control alone did not affect endogenous DDR1 expression and did not induce pulmonary inflammation on day 7 (a, B) and on day 21 (b, C and D). The administration of siRNA1 inhibited the DDR1 expression in alveolar macrophages (arrowheads), infiltrating inflammatory cells (arrowheads), and bronchoepithelial cells (arrows) on day 7 (a, C). This inhibitory effect continued until day 21 (b, E and F). The administration of siRNA-Luc did not inhibit the endogenous DDR1 expression on day 21 (b, G and H). Representative data of 10 different mice of each group (a, ×400 original magnification; b, left panels, ×150 original magnification, and right panels, ×400 original magnification).

Close modal

The total BALF cell count along with the macrophage, lymphocyte, and neutrophil counts were significantly lower in the DDR1 siRNA1-administered bleomycin-treated mice than those in the siRNA-Luc-administered bleomycin-treated mice and the bleomycin-treated mice (Fig. 6,a). MCP-1, MIP-2, and MIP-1α mRNA expression and protein production were significantly inhibited by the DDR1 siRNA1 administration (Fig. 6, b and c). In all groups, TGF-β mRNA expression and protein production were almost the same on day 7; however, these were significantly lower on days 14 and 21 in the DDR1 siRNA1-administered bleomycin-treated mice than those in the siRNA-Luc-administered bleomycin-treated mice and the bleomycin-treated mice (Fig. 6, b and c).

FIGURE 6.

BALF cell analysis (a), Northern blot analysis of cytokine mRNA expression in the lungs (b), and cytokine concentration in 1 mg of lung tissue (c) in each group. In BALF analysis, total cell counts, macrophage counts, neutrophil counts, and lymphocyte counts significantly decreased after the administration of siRNA1 (a, n = 12 in each group; ∗, p < 0.01; ∗∗, p < 0.05, Bonferroni-Dunn test with one-way factorial ANOVA). Bleomycin treatment induced intense expression of TGF-β, MIP-2, MCP-1, and MIP-1α mRNA in the lung (b). We started administration of each siRNA on day 3, and it was repeated every 3 days. Administration of siRNA1 inhibited the expression of these cytokine mRNAs. The concentrations of MIP-2, MCP-1, and MIP-1α in 1 mg of lung tissue were significantly inhibited by the administration of siRNA1 from days 7 to 21 (c, n = 12 in each group; ∗, p < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA). In all groups, the concentration of TGF-β was approximately the same on day 7; however, it was significantly inhibited on days 14 and 21 in the siRNA1-administered bleomycin-treated mice (c, n = 12 in each group; ∗, p < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA).

FIGURE 6.

BALF cell analysis (a), Northern blot analysis of cytokine mRNA expression in the lungs (b), and cytokine concentration in 1 mg of lung tissue (c) in each group. In BALF analysis, total cell counts, macrophage counts, neutrophil counts, and lymphocyte counts significantly decreased after the administration of siRNA1 (a, n = 12 in each group; ∗, p < 0.01; ∗∗, p < 0.05, Bonferroni-Dunn test with one-way factorial ANOVA). Bleomycin treatment induced intense expression of TGF-β, MIP-2, MCP-1, and MIP-1α mRNA in the lung (b). We started administration of each siRNA on day 3, and it was repeated every 3 days. Administration of siRNA1 inhibited the expression of these cytokine mRNAs. The concentrations of MIP-2, MCP-1, and MIP-1α in 1 mg of lung tissue were significantly inhibited by the administration of siRNA1 from days 7 to 21 (c, n = 12 in each group; ∗, p < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA). In all groups, the concentration of TGF-β was approximately the same on day 7; however, it was significantly inhibited on days 14 and 21 in the siRNA1-administered bleomycin-treated mice (c, n = 12 in each group; ∗, p < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA).

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DDR1 activation induces P38 MAPK phosphorylation via a unique pathway (4, 13), and the activation of P38 MAPK occurs in the bleomycin-treated mouse model (15). Therefore, to evaluate whether P38 MAPK activation was affected by DDR1 suppression, we examined P38 MAPK phosphorylation. On day 3, P38 MAPK was activated. On day 7, P38 MAPK phosphorylation in the lungs was significantly down-regulated in the DDR1 siRNA1-administered bleomycin-treated mice than in the siRNA-Luc-administered mice and bleomycin-treated mice. This phenomenon continued for 21 days (Fig. 7,a). DDR1 siRNA1 did not have any effect on the amount of P38 MAPK (Fig. 7,a) and β1 integrin, another collagen receptor (16) (Fig. 7 c). During the observation period, the amount of β1 integrin did not show any difference across the three groups (data not shown).

FIGURE 7.

The effect of transnasal administration of siRNA1 against phosphorylation of P38 MAPK in the lungs (a and b). Bleomycin treatment resulted in P38 MAPK phosphorylation, and administration of siRNA1 down-regulated the P38 MAPK phosphorylation (a, representative data of six different mice in each group). We started administration of each siRNA on day 3, and it was repeated every 3 days. b, Shows the statistical result of P38 MAPK phosphorylation in the lungs from each group. The total amount of P38α from 1 mg of lung tissue did not differ between the groups. The phosphorylation of P38α was significantly down-regulated in siRNA1-administered bleomycin-treated mice than in siRNA-Luc-administered bleomycin-treated mice and the bleomycin-treated mice (b, ∗, p < 0.01, compared with bleomycin alone, Bonferroni-Dunn test with one-way factorial ANOVA). c, Shows the effect of DDR1 siRNA1 on the expression of β1 integrin in the lung. DDR1 siRNA1 did not affect the amount of β1 integrin (c, 1 mg of lung tissue on day 7; representative data of six different mice in each group).

FIGURE 7.

The effect of transnasal administration of siRNA1 against phosphorylation of P38 MAPK in the lungs (a and b). Bleomycin treatment resulted in P38 MAPK phosphorylation, and administration of siRNA1 down-regulated the P38 MAPK phosphorylation (a, representative data of six different mice in each group). We started administration of each siRNA on day 3, and it was repeated every 3 days. b, Shows the statistical result of P38 MAPK phosphorylation in the lungs from each group. The total amount of P38α from 1 mg of lung tissue did not differ between the groups. The phosphorylation of P38α was significantly down-regulated in siRNA1-administered bleomycin-treated mice than in siRNA-Luc-administered bleomycin-treated mice and the bleomycin-treated mice (b, ∗, p < 0.01, compared with bleomycin alone, Bonferroni-Dunn test with one-way factorial ANOVA). c, Shows the effect of DDR1 siRNA1 on the expression of β1 integrin in the lung. DDR1 siRNA1 did not affect the amount of β1 integrin (c, 1 mg of lung tissue on day 7; representative data of six different mice in each group).

Close modal

This study showed that DDR1 suppression by appropriately designed transnasal administration of siRNA has an anti-inflammatory effect specifically in the lung. There are several reports that show the involvement of DDR1 in malignant tumor proliferation and migration (17, 18, 19). Regarding nonmalignant diseases, DDR1 is involved in ECM remodeling in atherosclerosis and lymphangiomyomatosis (20), as well as in the proliferative stage of renal disorders (21). In the lungs, bronchoepithelial cells (22) as well as alveolar macrophages (7) express DDR1. Our recent study showed that infiltrating inflammatory cells in IPF express endogenous DDR1, and its interaction with collagen contributes to the secretion of chemokines, including MCP-1 (7). In the bleomycin-induced pulmonary fibrosis model, the DDR1 ligand (collagen) shows an increase from an early time point after the initial alveolar wall injury and then its level plateaus (23). In this process, the inflammatory cells infiltrate the lesion through the ECM that is composed of collagen and reach the lesion (24). In addition, infiltrating fibroblasts produce procollagen, and this leads to collagen deposition (25). Thus, the bleomycin model simplifies the activation of DDR1. The siRNA against DDR1 apparently inhibited the endogenous DDR1 expression in bronchoepithelial cells, alveolar macrophages, and infiltrating inflammatory cells on day 7, the limiting point of DDR1 siRNA effectiveness. In addition, siRNA did not affect the expression of β1 integrin, which is another important collagen receptor in inflammatory responses (26). Therefore, we believe that DDR1 suppression in infiltrating cells and bronchoepithelial cells is responsible for the successful attenuation of bleomycin-induced lung inflammation. Once DDR1 is activated, it induces intense inflammatory cytokine production (6). In our study, starting siRNA administration after day 7, when the destruction of lung structure begins (23), did not attenuate the bleomycin-induced lung inflammation. These results indicate that DDR1 contribution might occur during the early stages of bleomycin-induced lung injury.

DDR1 suppression down-regulated the production of MCP-1, MIP-2, MIP-1α, and TGF-β in the lungs. Activation of DDR1 in human macrophages induced MCP-1 and MIP-2 production in a P38 MAPK-dependent manner (6). MCP-1 is a major chemoattractant for monocytes in inflammation and immune responses (27). MCP-1 can be detected in the BALF of IPF patients (28) and has been suggested to be associated with the pathogenesis of IPF (29). Alveolar macrophages and epithelial cells are the main cellular sources of MCP-1 production in IPF (30). Anti-MCP-1 gene therapy attenuates bleomycin-induced pulmonary fibrosis in mice (31). MIP-2 is a murine functional homologue of IL-8, and is therefore a potent chemoattractant for neutrophils and plays a pivotal role in acute inflammation by recruiting and activating neutrophils (32). In the bleomycin model, infiltrating inflammatory cells are a cellular source of MIP-2, and neutralization of MIP-2 attenuates bleomycin-induced pulmonary fibrosis (33). MIP-1α regulates the trafficking and activation of select subgroups of inflammatory cells, modulates the adhesion of leukocytes to the endothelium, and contributes to leukocyte recruitment into the lungs (34). Through these functions, MIP-1α contributes to the development of bleomycin-induced pulmonary fibrosis (35). TGF-β is a critical mediator in lung injury (36). Bleomycin can induce significant TGF-β production by bronchoepithelial cells, fibroblasts, and alveolar macrophages (37, 38). The mechanism by which DDR1 suppression led to the down-regulation of TGF-β production is unclear; however, we believe that this may be an indirect effect of DDR1 suppression because TGF-β inhibition occurred on day 14, and not on day 7. DDR1 is also associated with leukocyte migration (5). Therefore, we believe that decreased DDR1 activation and inhibition of the inflammatory cell infiltrate are responsible for the decreased cytokine production, an important factor in the development of bleomycin-induced lung inflammation.

In our study, siRNA against DDR1 suppressed the phosphorylation of the P38 MAPK pathway. P38 MAPK is activated in the lung tissue of a murine IPF model (15). In addition, the inhibition of P38 MAPK can ameliorate murine bleomycin-induced pulmonary fibrosis (39). Monocyte-derived macrophages secrete inflammatory chemokines such as MCP-1 in a P38 MAPK-dependent manner (6). Thus, the suppression of P38 MAPK phosphorylation that was observed in our study may contribute to the attenuation of bleomycin-induced lung inflammation. Although we cannot deny the possibility that the blunting of cytokine responses may secondarily affect this response, we believe that DDR1 might be one of the upstream signaling molecules of P38 MAPK in bleomycin-induced fibrosis because DDR1 activation can induce P38 MAPK phosphorylation via a unique signaling pathway (4, 6, 13).

In conclusion, we have shown that DDR1 suppression could decrease chemokine and cytokine production in bleomycin-induced pulmonary fibrosis. DDR1 has been reported to play an important role in regulating the attachment to collagen, chemotaxis, proliferation, and matrix metalloproteinase production by smooth muscle cells; DDR1 also contributes to collagen deposition in an arterial wound repair model (40). It is possible that DDR1 has other functions and contributes to collagen deposition in pulmonary fibrosis. In our study, lung epithelial cells expressed DDR1. The lung epithelial cells closely attached to ECM, which is abundant in collagen, and play a role in regulating the immunity in the lung through monocyte chemoattractant activity and by releasing several chemokines such as IL-8, MCP-1, and RANTES (41, 42, 43). Although we did not examine the biological function of DDR1 in lung epithelial cells in this study, we believe that there is a possibility that DDR1 in lung epithelial cells contributes to pulmonary fibrosis. Because the distribution of ECM is known to be associated with the activation of inflammatory cells, this interaction is considered to be a key event that results in intraluminal fibrosis (44). Further studies on the role of DDR1 might provide an insight into the clarification of the pathogenesis of pulmonary fibrosis.

We appreciate Dr. Teizo Yoshimura (Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD) for his invaluable contribution to this study. We also extend special thanks to Rumi Matsuyama for her excellent technical help.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This study was supported by a grant-in-aid for scientific research (16790447) from Japan Society for the Promotion of Science, a grant from The Sumitomo Foundation (040010), a grant from Nagao Memorial Fund, a grant from Uehara Memorial Foundation, and a grant from Kanae Foundation for Socio-Medical Science.

3

Abbreviations used in this paper: DDR1, discoidin domain receptor 1; BALF, bronchoalveolar lavage fluid; ECM, extracellular matrix; IPF, idiopathic pulmonary fibrosis; siRNA, small interference RNA.

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