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Published ahead of print on September 15, 2005, doi:10.1165/rcmb.2005-0236OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 565-573, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0236OC

Involvement of Discoidin Domain Receptor 1 in the Deterioration of Pulmonary Sarcoidosis

Wataru Matsuyama, Hideo Mitsuyama, Masaki Watanabe, Yuko Shirahama, Ikkou Higashimoto, Mitsuhiro Osame and Kimiyoshi Arimura

Division of Respiratory Medicine, Respiratory and Stress Care Center, Kagoshima University Hospital, Kagoshima, Japan

Correspondence and requests for reprints should be addressed to Wataru Matsuyama, M.D., Ph.D., Division of Respiratory Medicine, Respiratory and Stress Care Center, Kagoshima University Hospital, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan. E-mail: vega{at}xa2.so-net.ne.jp


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The prognosis of sarcoidosis with pulmonary infiltrates differs in each case, and several cytokines are reported to contribute to its deterioration. However, the detailed mechanism has not been fully elucidated. Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase activated by collagen and associated with cytokine production from inflammatory cells. We previously reported the functional expression of DDR1 on CD14-positive bronchoalveolar lavage fluid (BALF) cells in vivo. In this study, we hypothesized that DDR1 might be associated with the deterioration of pulmonary sarcoidosis (PS), and investigated 33 patients with sarcoidosis with pulmonary infiltrates, prospectively. We found that patients with deteriorated PS showed significantly higher DDR1 expression in CD14-positive BALF cells predominant with DDR1b isoforms. Activation of DDR1 induced monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) production in a p38 mitogen-activated protein kinase–dependent manner from CD14-positive BALF cells of patients with deteriorated sarcoidosis. DDR1 activation also induced NF-{kappa}B nuclear translocation in CD14-positive BALF cells of patients with deteriorated PS. The inhibitor of NF-{kappa}B inhibited the production of MCP-1 and MMP-9. We propose that DDR1 is associated with the deterioration of pulmonary sarcoidosis.

Key Words: chemokines • lung • NF-{kappa}B • monocyte chemoattractant protein-1 • matrix metalloproteinase-9


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Pulmonary sarcoidosis (PS) is a systemic disorder of unknown etiology characterized pathologically by the presence of noncaseating granulomas (1). Lungs and thoracic lymph nodes are involved in 90% of patients with PS with acute or insidious respiratory problems. Although resolution of the inflammatory process occurs spontaneously in 80% of cases, deterioration occurs in 10–20% of the cases (1–3). Especially, the morbidity and mortality are variable in patients with PS with lung parenchymal infiltrates (radiographic stages II and III) (2, 3). In the progression of PS, chemokines, such as monocyte chemoattractant protein-1 (MCP)-1 (4), and collagenase, such as matrix metalloproteinase (MMP)-9, are considered to contribute to irreversible pulmonary structural remodeling (4, 5). However, the detailed molecular mechanism, which leads to the deterioration of PS, has not been fully elucidated.

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase activated by binding with its ligand, collagen (6, 7). DDR1 has a unique extracellular domain that is homologous to discoidin 1 of Dictyostelium discoideum (8). DDR1 is constitutively expressed in normal tissues, such as lung, kidney, colon, and brain, and in tumor cells of epithelial origin, such as mammary, ovarian, and lung carcinomas (8). Five DDR1 isoforms (a, b, c, d, and e) can be generated by alternative splicing of the DDR1 gene (8, 9). We previously reported that the expression of two DDR1 isoforms, DDR1a and DDR1b, could be induced in vitro in human leukocytes. In vivo studies have shown that tissue-infiltrating mononuclear cells, particularly macrophages, were positive for DDR1 mRNA (10). The DDR1a and DDR1b isoforms differ from each other by an in-frame insertion of 111 bp that codes for an additional 37-aa peptide in the proline-rich juxtamembrane region. The 37-aa insertion in DDR1b contains the LXNPXY motif that corresponds to the consensus-binding motif of the Shc phosphotyrosine–binding domain (8). In addition, we recently found that the contribution of DDR1 activation, most likely DDR1b, for the production of MCP-1 and MMP-9 in CD14-positive bronchoalveolar lavage fluid (BALF) cells, occurs in a p38 mitogen-activated protein kinase (MAPK)–dependent manner in idiopathic pulmonary fibrosis (11).

The deterioration of PS is associated with the production, deposition, and proteolysis of the pulmonary extracellular matrix (2, 12), which is abundant with the DDR1 ligand, collagen (13). Therefore, we hypothesized that DDR1 might be associated with the prognosis of PS, and investigated DDR1 expression in patients with PS. We found that the CD14-positive BALF cells of patients with deteriorated PS expressed DDR1, with the DDR1b isoform being the predominant form, whereas DDR1a was predominant in patients with stable or improved PS. DDR1 activation in patients with deteriorated PS induced MCP-1 and MMP-9 production via a p38 MAPK–dependent pathway.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Patients
This study was reviewed and approved by the Kagoshima University Faculty of Medicine Committee on Human Research. This study included 33 consecutive patients with confirmed PS in whom lung involvement was evaluated with CT scanning from 1995 to 2003 in our institution. The criteria for the diagnosis of sarcoidosis included the following: a compatible clinical picture, consistent with American Thoracic Society guidelines (2, 3); histologic evidence of noncaseating granuloma; no mycobacterial infection present; and no exposure to aerocontaminants or medication known to cause granulomatous disorders. Histologic proof was obtained by bronchial and transbronchial biopsy in 27 patients, mediastinal biopsy in 3 patients, and open lung biopsy in 3 patients. Patients with bilateral hilar lymphadenopathy alone (stage I) and with pulmonary fibrosis (stage IV) were excluded from this study because spontaneous remission is common in stage I and 0% in Stage IV (2). To assess changes that have occurred over time, follow-up laboratory studies, chest radiographs, pulmonary function tests, and arterial blood gas analyses were performed at baseline, 3, 6, 12, 15, 18, and 24 mo. At baseline, no patients were treated with corticosteroids. Chest radiographs and pulmonary function tests were compared with the original baseline films, and were classified as improved (Group 1), stable (Group 2), or deteriorated (Group 3), based on the consensus review of two radiologists and physicians who had no knowledge of any of the other clinical data. Fourteen healthy volunteers (mean age = 57.8 ± 17.8 yr; male:female ratio = 6:8) also participated in this study. All patients and volunteers provided written informed consent to participate in the study.

BALF
The time point of bronchoscopy was regarded as the time point of diagnosis. A bronchofiberscope (Olympus BF type p20; Olympus, Tokyo, Japan) was wedged into the right B4 segment of the lung to collect BALF cells. Four 40-ml aliquots of sterile physiological saline were instilled at 37°C, and recovered by gentle suction. The recovered fluid was immediately filtered through sterilized gauze, and the lavage fluid was centrifuged in a cytometer (KN-70; Kubota Ltd., Tokyo, Japan) at 44 x g for 5 min and stained with May-Giemsa stain to identify cell populations.

Five hundred cells, excluding epithelial cells, were identified per slide to establish differential cell counts, and the counts were expressed in percentages. Concurrently, 1 x 105 cells were suspended in 50 µl of cold PBS containing 0.1% sodium azide, 10 ng/ml BSA, and 20 µg/ml of human IgG, and incubated for 10 min on ice. The cells were then incubated for an additional 15 min on ice with FITC-conjugated CD14 monoclonal antibody (M5E2; Becton Dickinson, Mountain View, CA), and mouse monoclonal anti–human CD29 antibody (MAR4, {beta}1-integrin; Becton Dickinson) or anti–human DDR1 mouse monoclonal IgG antibody (48B3; Santa Cruz Biotechnology, Santa Cruz, CA). The cells were washed with PBS, and incubated with biotin-conjugated goat anti–mouse IgG antibody for 15 min on ice. Cells were again washed with PBS, and incubated with phycoerythrin-conjugated streptavidin for 15 min on ice. At the end of the incubation period, 7AAD (Pharmingen, San Diego, CA) was added to each tube. The cells were washed with PBS, and subsequently analyzed by flow cytometry using a FACScan (Becton Dickinson). Dead cells, identified by the 7AAD incorporation, were gated out. Results were processed using the CellQuest software (Becton Dickson) as described previously (14). BALF were stored at –20°C for further analysis.

CD14-positive cells in BALF from each group were selected using magnetic beads (Miltenyi Biotec, Auburn, CA) as reported previously (11) and used for further analysis.

Immunohistochemistry
Biopsied lung tissues obtained from three patients with sarcoidosis of Group 3 were examined by immunohistochemical staining for DDR1 using a rabbit anti-DDR1 antibody (Santa Cruz Biotechnology) and visualized employing the DAB method, as described previously (15). Briefly, 4-µm-thick sections were mounted on poly-L-lysine–coated slides, dewaxed, and washed in Tris-buffered saline (pH 7.4) for 10 min. For optimal antigen retrieval, 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. After two washes in PBS with 1% saponin, the blocking reaction was performed as reported previously (16). Sections were incubated with primary antibody solution for 2 h at room temperature using a 1:50 dilution of the antibody. Negative-control slides were incubated with rabbit IgG (R&D Systems, Minneapolis, MN). Secondary biotinylated anti-Ig antibody (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 horseradish peroxidase (Amersham, Arlington Heights, IL) and then rinsed with deionized water. DAB substrate solution was added, and the mixture was incubated for 10 min. A brown-color reaction represented a positive result.

ELISA Assay
CD14-positive BALF cells (1 x 106 cells/ml) were incubated in 10% FCS RPMI 1640 (Invitrogen, Tokyo, Japan) with DDR1 agonistic antibody (513DDR1 ab) (17, 18) or 50 µg/ml of type I collagen (Sigma, St. Louis, MO) and cultured for 24 h. After culture, the supernatants were collected, and the concentrations of MCP-1, MMP-2 and MMP-9 were measured using ELISA kits (R&D Systems) according to the manufacturer's protocols.

To evaluate whether chemokine and cytokine production induced by DDR1 activation is dependent on p38 MAPK or NF-{kappa}B, we pretreated BALF cells with 10 µM of SB203580 (Biochem-Novabiochem, San Diego, CA) or 10 µg/ml of CAPE (Biochem-Novabiochem) for 30 min, followed by stimulation with collagen or DDR1 agonistic antibody.

We also measured MMP-2, MMP-9, and MCP-1 concentrations in the BALF from each group.

Cytokine concentrations were determined by linear regression from a standard curve using GraphPad software (Flow Laboratories, Helsinki, Finland), as described previously (19).

Gelatin Zymography
MMP-2 and MMP-9 activities were analyzed using gelatin zymography. Precast 7.5% polyacrylamide minigels containing 0.3% SDS and gelatin (1 mg/ml) were obtained from the YAGAI Research Center (YAGAI Research Center, Yamagata, Japan). A total of 15 µl of undiluted culture supernatant was mixed with an equal volume of 50 mM Tris–HCl buffer (pH 6.8) containing 2% SDS, 10% glycerol, and 0.01% bromophenol blue. Samples were loaded and electrophoresed at 10 mA for 20 min, followed by electrophoresis at 20 mA for 80 min, until the dye front reached the bottom of the gel. As a positive control, partially activated MMP-9 and MMP-2 samples were also loaded on the gels. After electrophoresis, the gels were agitated in 2.5% (vol/vol) Triton X-100 for 1 h to remove the SDS, followed by washing in 50 mM Tris–HCl buffer (pH 7.5) containing 200 mM NaCl for 1 h to restore enzymatic activity. The gels were incubated for 24 h at 37°C in 50 mM Tris–HCl buffer (pH 7.5) containing 200 mM NaCl, 5 mM CaCl2, 0.02% (wt/vol) Brij-35, and 0.01% NaN3 to allow proteolysis of the gelatin substrate. Finally, the gels were stained for 1 h with 0.1% Coomassie Brilliant Blue G-25 in 30% methanol and 10% acetic acid, followed by destaining for 3 h in 30% methanol and 5% acetic acid. Gelatinolytic activity was identified as clear bands against the blue background.

Western Blot Analysis
To detect DDR1 isoforms, 1 x 107 CD14-positive BALF cells, which were selected using magnetic beads (Miltenyi Biotec), were lysed on ice for 20 min in 1 ml of lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, and a cocktail of protease inhibitors (Roche, Indianapolis, IN). The lysates were centrifuged, and 20 µl of the supernatant was collected. Subsequently, 20 µl double-strength sample buffer (20% glycerol, 6% SDS, and 10% 2-mercaptoethanol) was added to the supernatants. The samples were boiled for 10 min. Proteins were analyzed on 8% polyacrylamide gels by SDS-PAGE and transferred electrophoretically to nitrocellulose membranes at 150 mA for 1 h using a semidry system. The membranes were incubated with rabbit IgGs that specifically recognize DDR1a (17), DDR1b (18), or both forms of DDR1 (Santa Cruz Biotechnology), or with anti–human actin monoclonal mouse IgG antibody (Santa Cruz Biotechnology), followed by sheep anti-rabbit or mouse IgG coupled with horseradish peroxidase (Amersham). Peroxidase activity was visualized by the Enhanced Chemiluminescence Detection System (Amersham). The intensities of DDR1 isoforms and actin were analyzed using the NIH Image (National Institutes of Health, Bethesda, MD), and then the relative amount of each DDR1 isoform (DDR1 amount ratio) in each patient was calculated.

To detect phosphorylation of p38 MAPK, CD14-positive cells were serum-starved in RPMI 1640 containing 1% FCS for 10 h, as previously described (11), and then activated with 513DDR1 ab or 50 µg/ml of type I collagen (Sigma). A total of 20 µl of cell lysate was directly mixed with 20 µl of sample buffer and analyzed. Phosphorylation of p38 MAPK was analyzed by Western blotting using rabbit polyclonal anti–phosphorylated p38{alpha} antibody or rabbit polyclonal anti-p38{alpha} antibody (Cell Signaling Technology, Beverly, MA) and sheep anti-rabbit IgG horseradish peroxidase (Amersham). Peroxidase activity was visualized by the Enhanced Chemiluminescence Detection System (Amersham).

Electrophoretic Mobility Shift Assay
1 x 108 CD14-positive BALF cells were incubated with 513DDR1 ab (17, 18) for 6 h, nuclear extracts prepared as previously described (17), and aliquots frozen at –80°C. For electrophoretic mobility shift assay, end-labeled 32P-oligonucleotide probes corresponding to the NF-{kappa}B binding site of the Ig {kappa}-chain gene (5'-AGTTGAGGGGACTTTCC CAGGC-3') was incubated with 5 µg of nuclear extracts in a 20-µl binding mixture (50 mM Tris-HCl, pH 7.4, 25 mM MgCl2, 0.5 mM DTT, 50% glycerol) at 4°C for 15 min The DNA–protein complexes were resolved on a 5% polyacrylamide gel. Gels were dried and then exposed to X-ray films.

Statistical Analysis
The Bonferroni-Dunn test with one-way factorial ANOVA and Pearson's correlation coefficient test were used for data analysis. A P value < 0.05 was considered significant. Values are presented as the mean ± SD unless otherwise stated.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Clinical Features
Twelve patients were improved (Group 1), 8 patients were stable (Group 2), and 13 patients were in a deteriorated condition (Group 3). Table 1 shows a comparison of clinical features. The percentages of lymphocyte and neutrophils were significantly higher and that of macrophages was significantly lower in patients with sarcoidosis than in healthy volunteers. There was no significant difference of BALF cellular populations among Group 1, Group 2, and Group 3. In Group 3, all patients were placed on corticosteroid treatment, whereas half the patients in Group 2, because of the worsening of non-PS symptoms, and none in Group 1, were placed on corticosteroid treatment. As we reported previously (11), CD14-positive BALF cells expressed DDR1. The DDR1-positive percentage was significantly higher in Group 3 than in the other groups. There was no significant difference of DDR1-positive percentage between radiographic stage II patients and stage III patients (stage II: 18.2 ± 8.9%, stage III: 16.1 ± 9.9%). There was no significant difference in {beta}1-integrin, another collagen receptor (20), positive percentage among all groups. The BALF levels of MCP-1 and MMP-9 were significantly higher in Group 3 than in other groups and healthy volunteers. The BALF levels of MCP-1 and MMP-9 in Group 3 patients showed significant positive correlation with the percentage of DDR1-positive cells in BALF (MCP-1: r = 0.675; P < 0.001. MMP-9: r = 0.619; P < 0.001, Pearson's correlation coefficient test). Also, BALF MCP-1 level showed significant correlation with that of MMP-9 in Group 3 patients (r = 0.632; P < 0.001, Pearson's correlation coefficient test). There were no significant differences in other clinical features investigated in this study.


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  		TABLE 1. COMPARISON OF CLINICAL FEATURES IN EACH GROUP

 
CD14-Positive BALF Cells from Deteriorated Patients with Sarcoidosis Expressed DDR1b
The total amount of DDR1 protein was higher per CD14-positive 1 x 107 BALF cells in Group 3 patients (Figure 1) and DDR1b was the predominant isoform in this group, whereas DDR1a was predominant in the other two groups. In Group 3 patients, total DDR1 and DDR1b amount ratios were significantly higher than in the other two groups (P < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA). In Group 3 patients, DDR1b amount ratio was significantly higher than the DDR1a amount ratio (P < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA).



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  		Figure 1. Comparison of discoidin domain receptor (DDR1) amount in CD14-positive bronchoalveolar lavage fluid (BALF) cells in each group. The total DDR1 amount in patients with deteriorated pulmonary sarcoidosis (PS) (Group 3) was significantly higher than in other groups. The amount of DDR1b in patients with deteriorated PS (Group 3) was also significantly higher than in other groups ([A] representative data. [B] comparison among three groups. *P < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA). In patients with deteriorated PS (Group 3), DDR1b amount ratio was significantly higher than the DDR1a amount ratio ([B] **P < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA).

 
Immunohistochemical Analysis of DDR1 Expression
As shown in Figure 2, infiltrating inflammatory cells in the sarcoidosis lesion of Group 3 patients stained positive for DDR1. Multinuclear giant cells also stained positive for DDR1. Samples from three different patients of Group 3 showed the same results.



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  		Figure 2. Immunohistochemistry of the biopsied lung of a patient with PS in Group 3. Inflammatory cells in the sarcoidosis lesion showed positive staining for DDR1. Multinuclear giant cells also stained positive for DDR1 (A and C: x300; B and D: x500, original magnification; C and D: negative control for DDR1).

 
Collagen and DDR1 Agonistic Antibody Stimulation Induced p38 MAPK Phosphorylation
DDR1 stimulation with DDR1 agonistic antibody induced p38 MAPK phosphorylation in CD14-positive BALF cells from Group 3 patients, but not in the other groups (Figure 3A). The results of collagen stimulation were the same as those obtained with DDR1 agonistic antibody (data not shown). The phosphorylation level was significantly higher in Group 3 than in the other groups (Figure 3B).



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  		Figure 3. p38 Mitogen-activated protein kinase (MAPK) in CD14- positive BALF cells of patients with deteriorated PS (Group 3) was phosphorylated, whereas no phosphorylation was seen in other groups (A representative data; B: comparison among three groups. *P < 0.001, Bonferroni-Dunn test with one-way factorial ANOVA).

 
DDR1 Activation Upregulated MCP-1 Production from CD14-Positive BALF Cells of Deteriorated Patients with Sarcoidosis
As shown in Figure 4, stimulation with collagen or DDR1 agonistic antibody significantly upregulated MCP-1 production from CD14-positive BALF cells in Group 3 patients. In preliminary experiments, we cultured CD14-positive cells for various time points (4, 8, 12, 24, 36, and 48 h), and concluded that culture for 24 h is the optimal because MCP-1 levels peaked at 24 h and remained elevated in Group 3 patients (data not shown). In the other groups, collagen or DDR1 agonistic antibody stimulation did not upregulate MCP-1 production. MCP-1 production was significantly inhibited by adding the p38 MAPK inhibitor, SB203580.



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  		Figure 4. Stimulation with DDR1 agonistic antibody or collagen induced monocyte chemoattractant protein (MCP)-1 production from CD14-positive BALF cells of patients with deteriorated PS (*P < 0.01, compared with no addition, control IgM, Group 1 or Group 2, analyzed by Bonferroni-Dunn test with one-way factorial ANOVA). This MCP-1 production was significantly inhibited by adding p38 MAPK inhibitor, SB203580 (**P < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA).

 
DDR1 Stimulation Induced Active MMP-9 Production in CD14-Positive BALF Cells from Deteriorated Patients with Sarcoidosis
As shown in Figure 5A, collagen or DDR1 agonistic antibody stimulation also induced MMP-9 production from CD14-positive BALF cells from Group 3 patients. In preliminary experiments, we cultured CD14-positive cells for various time points (4, 8, 12, 24, 36, and 48 h) and decided that culture for 24 h is the optimal time point because the production levels of MMP-9 peaked at 24 h and decreased to basal level at 48 h, whereas the levels of MMP-2 and tissue inhibitors of metalloproteinases (TIMPs) did not change (data not shown). Active MMP-9 was produced from CD14-positive BALF cells (Figure 5B). This active MMP-9 production was significantly inhibited by adding the p38 MAPK inhibitor, SB203580. Neither collagen nor DDR1 agonistic antibody upregulated MMP-2, TIMP-1, or TIMP-2 production from CD14-positive BALF cells obtained from other groups or from healthy volunteers (data not shown).



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  		Figure 5. Matrix metalloproteinase (MMP) production by the activation of DDR1. Stimulation with DDR1 agonistic antibody or collagen induced MMP-9 production from CD14-positive BALF cells of patients with deteriorated PS (A: *P < 0.01, compared with no addition, control IgM, Group 1 or Group 2, analyzed by Bonferroni-Dunn test with one-way factorial ANOVA). This MMP-9 production was significantly inhibited by adding p38 MAPK inhibitor, SB203580 (**P < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA). Stimulation with DDR1 agonistic antibody or collagen did not induce MMP-2. (B) Stimulation with DDR1 agonistic antibody or collagen induced active MMP-9 production from CD14-positive BALF cells of patients with deteriorated PS.

 
DDR1 Activation of the BALF Cells from Patients with Deteriorated PS Induced Nuclear Translocation of NF-{kappa}B
DDR1 activation of CD14-positive BALF cells from patients with deteriorated PS induced nuclear translocation of NF-{kappa}B (Figure 6). This translocation did not occur in other groups (data not shown). Preliminarily, we examined 3, 6, 9, and 12 h, and decided that 6 h was optimal, because the translocation peaked at 6 h and then decreased (data not shown). The translocation was inhibited by adding p38 MAPK inhibitor. These experiments were repeated using samples from five different patients, and similar results were obtained.



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  		Figure 6. DDR1 activation induced NF-{kappa}B translocation in CD14- positive BALF cells from patients with deteriorated PS (Group 3). This NF-{kappa}B translocation was inhibited by adding p38 MAPK inhibitor, SB203580 (A: representative data; B: data from five patients in each group; *P < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA).

 
MCP-1 and MMP-9 Production from BALF Cells of Patients with Deteriorated PS Was NF-{kappa}B–Dependent
DDR1-induced MCP-1 and MMP-9 production from BALF cells of patients with deteriorated PS was significantly inhibited by adding the NF-{kappa}B inhibitor, CAPE (Figure 7). The buffer alone (DMSO) did not inhibit MCP-1 or MMP-9 production.



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  		Figure 7. MCP-1 and MMP-9 production from CD14-positive BALF cells of patients with deteriorated PS (Group 3) were significantly inhibited by adding the NF-{kappa}B inhibitor, CAPE (*P < 0.01, Bonferroni-Dunn test with one-way factorial ANOVA).

 
Corticosteroid Significantly Inhibited DDR1-induced Cytokine Production from CD14-Positive BALF Cells but Did Not Change the DDR1 Expression Amount or Isoform Pattern
The use of corticosteroid is common in the treatment of patients with deteriorated PS (1). Therefore, we examined the effect of collagen and corticosteroid against DDR1-induced cytokine production from CD14-positive BALF cells of Group 3 patients. As shown in the online supplement, incubation of CD14-positive BALF cells with dexamethasone (Sigma; 0.1 µM, Figure E1A in the online supplement) significantly inhibited the DDR1 agonistic antibody–induced production of MCP-1 and MMP-9 from CD14-positive BALF cells of Group 3 patients. Preliminarily, we incubated CD14-positive BALF cells in various concentration of dexamethasone (0.01, 0.1, 0.5, and 1 µM), and concluded that the concentration of 0.1 µM was optimal because the inhibitory effect peaked at this concentration. The incubation with dexamethasone (Figure E1B) and collagen (Figure E1C), which increase in the PS lesion (1), showed no effect on the DDR1 expression amount or expression pattern of DDR1 isoforms in any group.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
This is the first report describing the role of DDR1 in PS. The prognosis of PS differs in each chest radiographic stage. Especially, in patients in stages II and III, spontaneous remission occurs in 40–70% (stage II) and in 10–20% (stage III), whereas other patients were stable or deteriorated during the clinical course (2). However, the molecular mechanism of deterioration has not been fully elucidated. Our study showed that the patients with deteriorated PS had higher DDR1 expression amounts with predominantly DDR1b isoforms than the improved or stable patients with PS. Furthermore, incubation of CD14-positive BALF cells with collagen, which is a ligand of DDR1 (7) that increases in the deteriorated PS lesion (1), did not affect the DDR1 expression amount or expression pattern. Of course, we cannot deny the possible existence of other factors that can regulate DDR1 expression. However, we believe that DDR1b is involved in the deterioration of PS. Because DDR1b, not DDR1a, has a binding site for Shc, an adaptor protein that has a variety of roles in cell signaling (21), this isoform can conduct signals to the nucleus (22). In addition, DDR1b was reported to be associated with the production of cytokines (11), which are associated with the pathogenesis of PS (4, 12). Because the scope of our study is too small to draw definitive conclusions, further studies must be undertaken to evaluate the prognostic value of DDR1 in PS.

Our study showed that DDR1 activation induced MCP-1 and MMP-9 production from CD14-positive BALF cells of patients with deteriorated PS. In addition, DDR1-positive percentage in BALF cells showed significant positive correlations between BALF MCP-1 and MMP-9 levels. There have been several reports showing an association of MCP-1 and MMP-9 levels with the clinical features of PS. In PS, sputum MMP-9 levels were highly correlated with the CD4/CD8 ratio in BALF and pulmonary diffusion capacity (12). Actually, patients with PS with pulmonary fibrosis show high MMP-9 concentration in BALF (5). MMPs are a family of zinc- and calcium-dependent endopeptidases capable of proteolytically degrading many of the components of the extracellular matrix (23), and TIMPs are the endogenous inhibitors of MMPs (24). In pulmonary diseases, MMPs are believed to be associated with wound repair of bronchial epithelial cells (25). The imbalance of MMPs and TIMPs is thought to contribute to the development of interstitial lung diseases (26). In PS, this imbalance was thought to be associated with cytotoxic CD8 T-lymphocytes and tissue damage (12). In the lung, alveolar macrophages, which can express endogenous DDR1 (11), are the main cellular source of MMP-9 (27). On the other hand, MCP-1 belongs to a C-C chemokine superfamily of small proteins that are important in recruiting and activating leukocytes during inflammation, including monocytes, immature dendritic cells, basophils, natural killer cells, and a subset of T lymphocytes (28). MCP-1 binds and signals through a seven-transmembrane–spanning G protein–coupled receptor, CCR2 (29). MCP-1 is produced by various types of cells. In the lung, it has been reported that MCP-1 mRNA and protein are strongly expressed in epithelial cells, macrophages, and endothelial cells (30), which express endogenous DDR1 (11, 31). MCP-1 expression has been studied in BALF in interstitial lung disease, but, until recently, these studies have been predominantly in fibrosing alveolitis (30) rather than in sarcoidosis (32). Recently, an increase in MCP-1 was reported in serum samples from sarcoidosis patients (33). In addition, increased MCP-1 protein levels in BALF has been reported in patients with PS with chest radiographic stage II disease and patients with PS with persistent and recurrent disease, and it has been suggested that MCP-1 is associated with the formation of granuloma or alveolitis in PS (4, 34). DDR1 was reported to be necessary for MMP-9 production (35). Activation of DDR1 can induce MCP-1 production from macrophages via the p38 MAPK pathway (11, 17). Taken together, we propose that DDR1 is associated with the molecular pathogenesis of deterioration in PS.

Although there have been numerous reports about cytokine profiles in PS, little is known about the signaling pathway in PS. There have been several reports describing the involvement of NF-{kappa}B in PS. In PS, the activity of peroxisome proliferator–activated receptor-{gamma}, a ligand-activated transcription factor, is too low to suppress NF-{kappa}B activity and, therefore, NF-{kappa}B activity is upregulated (36); this upregulated NF-{kappa}B is considered to be associated with cytokine production from BALF cells (37). DDR1 activation can induce chemokine production from PMA-treated THP-1 cells, which have characteristics of macrophages (38), in an NF-{kappa}B–dependent manner (17). Our study shows that DDR1 activation of BALF cells from patients with deteriorated PS induced significant upregulation of nuclear translocation of NF-{kappa}B. In addition, MCP-1 and MMP-9 production by DDR1 activation was NF-{kappa}B dependent. Therefore, we believe that DDR1 might be one of the upstream signaling molecules of the NF-{kappa}B pathway in PS. Our study also shows that DDR1 activation induced p38 MAPK phosphorylation. Little is known about MAPKs, such as p38 MAPK, in PS. The p38 MAPK is associated with the production of several cytokines, such as MCP-1 (17), which is associated with deterioration of PS. Of course, we cannot determine whether p38 MAPK is important in the deterioration of PS; we cannot deny the possible association of MAPKs with PS deterioration, and further studies of MAPKs in PS are necessary.

Corticosteroids are the common drugs used to treat deteriorated PS (1). In our study, incubation of CD14-positive BALF cells with corticosteroids did not affect the amount or pattern of DDR1 expression; however, corticosteroids inhibited DDR1 agonistic antibody–induced MCP-1 and MMP-9 production from CD14 BALF cells of Group 3 patients. It is well known that corticosteroids can inhibit intracellular signaling that activates NF-{kappa}B (39, 40). Therefore, we believe that corticosteroids can inhibit intracellular DDR1 signaling that activates NF-{kappa}B. Examination of DDR1 signaling in the presence or absence of corticosteroids might be useful in clarifying the nature of intracellular DDR1 signaling in PS.

Cell surface receptors, such as {beta}1-integrins, are another well known collagen receptor, and ligation of {beta}1-integrin can induce the expression of proinflammatory cytokines in monocytes (20). However, the expression of these integrins is low or undetectable on monocyte-derived macrophages (41, 42), suggesting the presence of collagen receptors other than {beta}1-integrins that may promote cytokine production in macrophages. Indeed, the present study shows a significant difference in the DDR1 expression levels between patients with deteriorated PS and other groups, whereas there was no significant difference in the {beta}1-integrin–positive percentage of alveolar macrophages. Taken together, we believe that the contribution of DDR1 to the deterioration of PS is greater than that of {beta}1-integrin.

In conclusion, our study showed the contribution of DDR1 to the deterioration of PS. In Asian PS, human leukocyte antigen–DRB1–associated genetic factors influence the risk for the development of sarcoidosis and disease progression (43), and is associated with the risk of persistent sarcoidosis (44). The gene locus of human leukocyte antigen–DRB1 (6p21.3, Online Mendelian Inheritance in Man [OMIM] no. 142,857) is very close to that of DDR1 (6p21.3 OMIM no. 600,408) (45). Furthermore, butyrophilin-like protein 2 (BTNL-2, 6p21.3, OMIM no. 606,000), the gene locus of which is very close to that of DDR1, is also the risk of sarcoidosis in Europe (46). Therefore, we believe that DDR1 might be closely associated with the pathogenesis of PS. Our next goal is to clarify whether the DDR1 gene has linkage and association with the pathogenesis of PS.


   Acknowledgments
 
The authors thank Dr. Teizo Yoshimura (Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland) for his invaluable comments. They also wish special thanks to Mrs. Rumi Matsuyama (Third Department of Internal Medicine, Kagoshima University, Kagoshima, Japan) for her excellent help.


   Footnotes
 
This work was supported by the Grant-in-Aid for Scientific Research (16790447) from the Japan Society for the Promotion of Science (JSPS), and grants from the Sumitomo Foundation (040010), the Kanae Foundation for Life & Socio-Medical Science, the Nagao Memorial Fund, and the Uehara Memorial Foundation.

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: 10.1165/rcmb.2005-0236OC on September 15, 2005

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 June 29, 2005

Accepted in final form August 15, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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