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Activation of Discoidin Domain Receptor 1 Facilitates the Maturation of Human Monocyte-Derived Dendritic Cells Through the TNF Receptor Associated Factor 6/TGF--Activated Protein Kinase 1 Binding Protein 1/p38 Mitogen-Activated Protein Kinase Signaling Cascade

Wataru Matsuyama^*, Michel Faure and Teizo Yoshimura^1,*

^* Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick, MD 21702; and SUGEN, Inc., South San Francisco, CA 94080

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Maturation of dendritic cells (DCs) is critical for their ability to stimulate resting naive T cells in primary immune responses. Previous studies demonstrated that collagen, such as type I collagen, could facilitate DC maturation; however, the basis of collagen-mediated DC maturation remains unclear. Discoidin domain receptor 1 (DDR1) is a nonintegrin collagen receptor constitutively expressed in a variety of epithelial cells, including tumor cells, and is inducible in leukocytes. In this study, we evaluated the role of DDR1 in DC maturation using human monocyte-derived DCs. Two DDR1 isoforms, DDR1a and DDR1b, were expressed in both immature and mature DCs. Activation of DDR1 on immature DCs resulted in their partial maturation; however, DDR1 activation markedly amplified TNF-- and LPS-induced phenotypic and functional maturation of DCs through activation of p38 mitogen-activated protein kinase (MAPK), suggesting the involvement of DDR1b in this process. Activation of DDR1b on differentiated DDR1b-overexpressing THP-1 cells or DDR1 on mature DCs induced the formation of TNFR associated factor 6 (TRAF6)/TGF--activated kinase 1 binding protein 1/p38 MAPK complex and p38 autophosphorylation. Transfection of differentiated DDR1b-overexpressing THP-1 cells with dominant negative TRAF6 completely abrogated DDR1b-mediated p38 MAPK phosphorylation, indicating a critical role of TRAF6 in DDR1b-mediated p38 MAPK activation. Taken together, our data suggest that DDR1b-collagen interaction augments the maturation of DCs in a tissue microenvironment through a unique TRAF6/TGF--activated kinase 1 binding protein 1/p38 MAPK signaling cascade and contributes to the development of adaptive immune responses.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DCs)² are professional APCs that play a predominant role in stimulating resting naive T cells in primary immune responses (1, 2). Most DCs in peripheral tissues have an immature phenotype, the prototype being Langerhans cells in the epidermis (2). Immature DCs (iDCs) can take up Ags, but do not present them efficiently to T cells. In response to microbial products or proinflammatory cytokines, iDCs mature and migrate from peripheral tissue sites to lymphatic organs using the CCR7 (3) through the extracellular matrix (ECM) and afferent lymphatics (4). As iDCs mature into mature DCs (mDCs), their capacity for Ag uptake is reduced, but they express high levels of MHC class I and class II molecules and costimulatory molecules that enable them to activate T cells (2). Previous studies demonstrated that collagen, the major component of the ECM, could promote the differentiation of murine DC precursors or human monocyte-derived iDCs into mDCs (5, 6, 7). Cell surface receptors, such as ₁ integrins, are well-known collagen receptors, and ligation of ₁ integrin could induce the expression of proinflammatory cytokines in monocytes (8). However, collagen-mediated maturation of monocyte-derived DCs occurred independently of the classical collagen receptors, ₁₁ or ₂₁ integrins (7). Furthermore, the expression of these integrins is low or undetectable on monocyte-derived macrophages and DCs (9, 10), suggesting the presence of as yet unidentified collagen receptors that may promote maturation of iDCs.

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase activated by the binding to its ligand, collagen (11, 12). We previously demonstrated that the expression of the two DDR1 isoforms, DDR1a and DDR1b, could be induced in human leukocytes, including neutrophils, monocytes, and lymphocytes. Overexpression of DDR1a in the human monocytic leukemic cell line, THP-1, promoted their migration through three-dimensional collagen lattices (13). Recently, we also observed that collagen-activation of DDR1b promoted PMA-induced differentiation of THP-1 cells through activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Activation of DDR1 endogenously expressed on monocyte-derived macrophages resulted in an increased level of HLA-DR expression (14). These findings led us to hypothesize that activation of DDR1, especially DDR1b, may contribute to the collagen-mediated maturation of iDCs to mDCs through activation of the p38 MAPK pathway, a pathway reported to be involved in DC maturation (15).

In the present study, we tested this hypothesis using human monocyte-derived DCs, and have found that the expression of DDR1a and DDR1b occurs in human DCs, and that activation of DDR1 on iDCs, in combination with other DC-maturation signals such as TNF- or LPS, up-regulates the expression of cell surface molecules characteristic of mDCs, including CD80, CD83, CD86, HLA-DR, CCR7, and MHC class I molecules. mDCs produced by activating DDR1 stimulated allogeneic MLR, produced the Th1 cytokine IL-12 p70, migrated to the CCR7 ligand, and primed CD8⁺ T lymphocytes to become CTL at significantly higher levels than DDR1-nonactivated mDCs. The effects of DDR1 activation appear to be caused by the recently described TGF--activated protein kinase 1 (TAK1) binding protein 1 (TAB 1)-mediated autophosphorylation of p38 MAPK (16) through DDR1b. This is the first report demonstrating that DDR1 plays a role in the maturation of DCs by activating its unique and distinct signaling pathway. Our study also suggests a contribution of DDR1-collagen interaction to the development of adaptive immune responses.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

Rabbit polyclonal Abs against human DDR1 (C-20) or TAB 1 (N-19 and C-20), and mouse mAbs against human Shc, TNFR associated factor (TRAF) 6, and TAK1 were from Santa Cruz Biotechnology (Santa Cruz, CA). The production of rabbit polyclonal Abs specific for DDR1a or DDR1b was previously described (14). Mouse monoclonal anti-DDR1 IgM (513) was raised against the entire extracellular domain of DDR1 (17). The Ab was produced by growing the hybridoma cells (513GA12) in protein-free medium (Protein Free Hybridoma Medium; Life Technologies, Rockville, MD). The isotype of the Ab produced is IgM. This Ab has the capacity to induce autophosphorylation of DDR1 (14). A biotinylated Ag-purified rabbit polyclonal Ab against the N-terminal region of human DDR1 (DMKGHFDPAKC) was a kind gift from Trans Genic (Kumamoto, Japan). A mouse mAb against phosphotyrosine (4G10) was from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal Abs against phosphorylated or nonphosphorylated p38, p38, MAPK kinase (MKK) 3/MKK6, activating transcription factor 2 (ATF2), phosphorylated ATF2, extracellular signal-regulated kinase (ERK), and phosphorylated ERK were from Cell Signaling Technology (Beverly, MA). Mouse mAbs against CD80, CD83, CD86, HLA-DR, and MHC class I molecules, PE-conjugated anti-CD8 mouse mAb, FITC-conjugated anti-IFN- mouse mAb, avidin-FITC, and a mouse monoclonal IgM were from BD PharMingen (San Diego, CA). A PE-conjugated anti-human CCR7 mouse monoclonal IgG was from R&D Systems (Minneapolis, MN). Sheep anti-mouse or anti-rabbit IgG coupled with HRP, [³H]thymidine and [⁵¹Cr]sodium chromate were from Amersham Pharmacia Biotech (Piscataway, NJ). PBS, RPMI 1640, recombinant protein G-agarose (PGA), and TRIzol Reagent were from Invitrogen. FCS was from HyClone Laboratories (Logan, UT). LPS (Escherichia coli LPS, 055:B5) was from Difco (Detroit, MI). Paraformaldehyde, polymixin B, human serum, and propidium iodide (PI) were from Sigma-Aldrich (St. Louis, MO). Human recombinant GM-CSF, IL-4, TNF-, and macrophage inflammatory protein-3 (MIP-3)/CCL19 were from PeproTech (Rocky Hill, NJ). SB203580 was from Biochem-Novabiochem (San Diego, CA). Protease inhibitor mixture tablets, complete mini, were from Roche (Indianapolis, IN).

Preparation of human monocyte-derived DCs

Human PBMC were isolated from leukapheresis preparations obtained by the Blood Bank, Clinical Center, National Institutes of Health (Bethesda, MD). The leukocyte-rich preparation was overlaid on Accu-prep in 50-ml tubes, and the tubes were centrifuged at 800 x g for 20 min at room temperature. PBMC fractions were collected, washed once with PBS at room temperature, and twice with RPMI 1640 containing 10% FCS (complete medium) at 4°C, and resuspended in the same medium. Monocytes were further purified by using iso-osmotic Percoll gradient. At this stage the purity of monocytes was higher than 90% (18). The cells (5 x 10⁶/ml) were allowed to adhere to the surface of plastic plates. After a 5-h incubation at 37°C, nonadherent cells were removed, and remaining adherent cells (>95% of cells were positive for CD14 by flow cytometry analysis) were subjected to the DC maturation protocol described previously (19, 20). Briefly, adherent cells were cultured in complete medium containing 50 ng/ml GM-CSF and 50 ng/ml IL-4 for 5 days, with cytokine added every other day, to obtain a population of iDCs. Final maturation to mDCs was induced by an additional 2-day incubation with TNF- (50 ng/ml) or LPS (1 µg/ml). A total of 5 µg/ml 513 Ab was used to activate DDR1. An unrelated mouse monoclonal IgM was used as control.

Flow cytometry analysis

The expression of cell surface molecules was evaluated by flow cytometry analysis. A total of 100,000 cells were suspended in 50 µl of cold PBS containing 0.1% sodium azide, 10 ng/ml BSA, and 20 µg/ml human IgG, incubated for 10 min on ice, and incubated with primary mouse mAbs or biotinylated rabbit polyclonal Ab for an additional 15 min on ice. Cells were washed with PBS and incubated with FITC-conjugated goat anti-mouse IgG or avidin-FITC for 15 min on ice. At the end of the incubation, PI was added to each tube to give the final concentration of 100 µM. The cells were washed with PBS and subsequently analyzed by flow cytometry using a FACScan (BD Biosciences, San Jose, CA). Dead cells, determined by the incorporation of PI, were gated out. Results were processed using CellQuest software (BD Biosciences). To determine the effect of SB203580, iDCs were pretreated with SB203580 or DMSO for 30 min.

Allogeneic MLR

Human T cells were purified from PBMCs of healthy donors by magnetic separation using CD3⁺ microbeads (Miltenyi Biotec, Auburn, CA). T cells (1 x 10⁵) were placed in 96-well plates with increasing numbers of DCs (500–10,000 cells) in 200 µl of complete medium. On day 4, 1 µCi/well [³H]thymidine was added, and incorporation of radioactivity was measured after 15 h of incubation. All tests were performed in triplicate. To evaluate the effect of p38 MAPK inhibitor, iDCs were pretreated with SB203580 or DMSO for 30 min.

Measurement of endotoxin and cytokine concentrations

The concentrations of endotoxin and cytokines were measured in the Lymphokine Testing Laboratory, Clinical Services Program, Science Applications International Corporation Frederick (Frederick, MD), with the QCL-1000 Chromogenic LAL Test kit (Cambrex, Walkersville, MD) and ELISA kits (R&D Systems), respectively. The sensitivities of the assays were <0.1–1.0 EU/ml for endotoxin, 5 pg/ml for IL-10, 0.5 pg/ml for IL-12 p70, and 15.6 pg/ml for IFN-.

Phagocytosis assay

DCs were plated into 96-well plates (10⁵ cells/well) in triplicate with 0.75 µm Fluoresbrite Yellow Green Carboxylate Microspheres (Polysciences, Warrington, PA) at the final concentration of 0.0027% (v/v) in complete medium, and then cultured for 8 h at 37°C in a humidified CO₂ incubator. Control plates were incubated at 4°C. The percentage of phagocytic cells was measured by flow cytometry using a FACScan.

Chemotaxis assay

Migration of DCs was assessed using a 48-well chemotaxis chamber (NeuroProbe, Cabin John, MD) with 5-µm pore size polycarbonate filters as previously described (21). Briefly, different concentrations of MIP-3/CCL19 were placed in the wells of the lower compartment of the chamber, and 50 µl of cell suspensions (1 x 10⁶ cells/ml) were added to the wells of the upper compartment. After a 90-min incubation at 37°C in a humidified CO₂ incubator, the filters were removed and stained, and the cells migrating across the filter were counted using the Baioquant semiautomatic counting system. The results were presented as the number of cells per high power field.

Preparation of tumor cell lysate

Confluent cultures of human melanoma cells (ATCC A375, HLA-A2⁺; American Type Culture Collection (ATCC), Manassas, VA) were incubated with 0.01% EDTA solution for 10 min, carefully detached with a cell scraper, washed twice in PBS, and resuspended at a density of 5 x 10⁶ cells/ml in a serum-free medium. The cell suspensions were lysed by five cycles of freezing (methanol and dry ice for 5 min) and thawing (room temperature for 5 min) (22). For removal of cell debris, the lysate were centrifuged at 300 x g for 10 min at 4°C. The supernatants were collected and passed through 0.2-µm filters. Lysates were tested for the contamination of endotoxin in the Lymphokine Testing Laboratory and were found to be free of any detectable level of endotoxin.

Generation of tumor lysate-pulsed mDCs and priming of CD8⁺ T cells

iDCs were generated from HLA-A2⁺ donors as described above. The cells were incubated with 100 µg/ml melanoma cell lysate at 5 x 10⁵ cells/ml for 4 h at 37°C, followed by TNF- alone, TNF- plus 513 Ab, or TNF- plus control IgM for an additional 48 h.

CD8⁺ T cells were isolated from PBMCs of HLA-A2⁺ donors by magnetic separation using CD8⁺ microbeads (Miltenyi Biotec). CD8⁺ T cells (1.5 x 10⁶) were added to 5 x 10⁴ DCs that were matured in various conditions, and they were cocultured for 7 days. IL-2 (40 IU/ml) was added on days 1 and 4. Nonadherent cells were collected (>95% were positive for CD8 by flow cytometry) and restimulated by freshly generated TNF--matured DCs at a ratio of 30:1 and incubated for 48 h.

Intracellular staining of IFN- in CD8⁺ T cells

The percentage of IFN--producing CD8⁺ cells was quantified by an intracellular staining technique (23). Briefly, nonadherent cells obtained after 48 h of restimulation were cultured in complete medium with brefeldin A (10 µg/ml) (Sigma-Aldrich) for 10 h. After incubation with brefeldin A, cells were incubated with PE-conjugated anti-CD8 mAb, washed with PBS, resuspended in PBS containing 4% paraformaldehyde, and then incubated for 20 min at 4°C. After washing with PBS, cells were resuspended in PBS containing 1% saponin and incubated with FITC-conjugated anti-IFN- mAb for 1 h at 4°C. The cells were washed in PBS containing 1% saponin, resuspended in PBS, and then analyzed by flow cytometry using a FACScan.

Cytotoxicity assay

Forty-eight hours after re-stimulation, the cytotoxic activity of CD8⁺ T cells was assessed in an 18-h ⁵¹Cr-release assay. Ten million melanoma cells (target cells) in single-cell suspension were incubated with 100 µCi [⁵¹Cr]sodium chromate/10⁶ cells for 1 h and washed five times with PBS. The labeled melanoma cells (5 x 10³ cells/well) were incubated with CD8⁺ T cells (effector cells) generated by coculturing with mDCs at E:T ratios ranging from 4:1 to 80:1 in 96-well plates. After an 18-h incubation, supernatants of each well were collected, and radioactivity was measured with a gamma counter (1480 WIZARTM 3"; PerkinElmer, Downer Grove, IL). Specific lysis was calculated by the formula: specific ⁵¹Cr-release = [(mean experimental cpm - mean spontaneous cpm)/(mean maximum cpm - mean spontaneous cpm)] x 100%, in which spontaneous release represents cpm in supernatants from wells containing target cells with medium only, and maximum release represents cpm in supernatants from wells containing target cells in medium with 2% Triton X-100. At the same time, the supernatants from the 18-h coculture of unlabeled melanoma cells with effector cells (1:40) were collected and subjected to IFN- ELISA in the Lymphokine Testing Laboratory. Data were presented as the mean ± SD from the data of three independent experiments.

Western blot analysis

To detect DDR1 isoforms expressed by DCs, cells were harvested on each day of the maturation process. Cells were washed three times with PBS, and 1 x 10⁷ cells 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 mixture of protease inhibitors. The lysates were spun, and the supernatants were collected and stored at -80°C until use. The samples were incubated with 20 µl of packed PGA for 1 h at 4°C. After centrifugation, supernatants were collected, mixed with 1 µg/ml polyclonal anti-human DDR1 Ab (C-20), and incubated for 1 h at 4°C. Twenty microliters of PGA were then added, and the samples were incubated for an additional 12 h. IgG-coupled PGA was washed with washing buffer containing 50 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol buffer three times, and 20 µl of double-strength sample buffer (20% glycerol, 6% SDS, 10% 2-ME) was added. Bound proteins were eluted by boiling for 10 min, analyzed on 8% polyacrylamide gels by SDS-PAGE, and transferred electrophoretically to nitrocellulose membranes at 150 mA for 1 h by a semidry system. The membranes were incubated with rabbit IgGs that recognize only DDR1a, only DDR1b, or both forms of DDR1 (C-20), followed by sheep anti-rabbit IgG coupled with HRP. Peroxidase activity was visualized by the ECL Detection System (Amersham Pharmacia Biotech).

To analyze the kinetics of DDR1 autophosphorylation, 1 x 10⁷ iDCs or TNF--induced mDCs were plated on dishes, serum-starved in RPMI 1640 containing 1% FCS for 10 h, and subsequently activated with 513 agonistic anti-DDR1 IgM (5 µg/ml) or control IgM (5 µg/ml) for various times. Cell lysates were prepared, subjected to immunoprecipitation with anti-DDR1 Ab (C-20), and tyrosine-phosphorylation of DDR1 was analyzed by Western blotting using mouse monoclonal anti-phosphotyrosine IgG and sheep anti-mouse IgG coupled with HRP. Peroxidase activity was visualized by the ECL detection system.

To detect phosphorylation of MAPKs, ATF2, or MKK3/6, iDCs and mDCs were activated with 513 Ab or control IgM for various times. Because TNF- is known to activate p38 MAPK (24), cells were washed six times with PBS before activation to minimize the effect of TNF-. Twenty microliters of cell lysates were directly mixed with 20 µl of sample buffer and then analyzed.

To determine the association of TRAF6, TAB 1, or TAK1 with p38 MAPK, cell lysates of DCs or PMA-treated DDR1a- or DDR1b-overexpressing THP-1 cells (13) were subjected to immunoprecipitation with anti-p38 MAPK, p38 MAPK, or TRAF6 Ab, and coimmunoprecipitation of TRAF6, TAB 1, TAK1, or p38 MAPK was evaluated using Abs against each protein.

Expression of dominant negative (DN)-TRAF6

A mammalian expression vector for the Flag-DN-TRAF6 (289–522) fusion protein similar to the previously reported construct (25) was prepared as follows: a cDNA coding for the C-terminal portion of TRAF6 (289–522) was obtained by RT-PCR using a full-length human TRAF6 cDNA (I.M.A.G.E. 5272008; ATCC) and a pair of primers: forward primer 5'-GCGAATTCTCAGAGGTCCGGAATTTCCAG-3' and reverse primer 5'-CGAAGCTTCTATACCCCTGCATCAGTACTTCG-3'. PCR was performed by 15 cycles of denaturation (1 min at 94°C), annealing (1 min at 62°C), and extension (30 s at 68°C). The PCR product was then ligated into the EcoRI-HindIII site of the pCMVTag2 (Stratagene, La Jolla, CA). DDR1b-overexpressing THP-1 cells were treated with 10 nM PMA for 12 h and then transfected with 2 µg of the vector with or without insert using Effectene Transfection Reagent (Qiagen, Valencia, CA) for 24 h, rinsed with PBS, incubated for an additional 12 h in RPMI 1640 containing 1% FCS, and subsequently activated with 50 µg/ml collagen for 60 min. A total of 100,000,000 cells were lysed, and the cell lysates were subjected to Western blotting.

Statistical analysis

Statistical analyses were performed by Bonferroni/Dunn with One Way Factorical ANOVA. Data are shown as the mean ± SD.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of DDR1a and DDR1b is induced during DC maturation

To study a potential role of DDR1 in collagen-mediated DC maturation, we first examined the expression of DDR1 in DCs. We incubated monocytes in the presence of IL-4 and GM-CSF for 5 days to produce iDCs, and for an additional 2 days in the presence of IL-4, GM-CSF, and TNF- to produce mDCs. As shown in Fig. 1A, a high level of DDR1a was already expressed on day 1. The level of DDR1a reached a peak on day 2 and gradually decreased thereafter. In contrast, the level of DDR1b gradually increased and reached the highest level on day 7. Because total DDR1 levels were similar between days 3 and 7, it appears that DDR1a is the dominant isoform during the early stage of DC maturation, whereas DDR1b becomes the dominant isoform in mDCs. By flow cytometry analysis, we confirmed cell surface expression of DDR1 on both iDCs and mDCs, but not on monocytes (Fig. 1B). There was no significant difference in the percentage of cells expressing cell surface DDR1 between iDCs and mDCs (Fig. 1C).

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Expression of DDR1 in DCs. A, Detection of DDR1a and DDR1b by Western blotting. Ten million monocytes were plated in each dish and incubated for 5 days in the presence of 50 ng/ml GM-CSF and 50 ng/ml IL-4 to obtain a population of immature monocyte-derived DCs. For terminal maturation, iDCs were treated with TNF- (50 ng/ml) from days 5 to 7. Cells were collected each day, and cell lysates were prepared. The lysates were subjected to immunoprecipitation with anti-DDR1 Ab (C-20), and bound proteins were analyzed. Membranes were probed with either anti-DDR1 (C-20), anti-DDR1a, or anti-DDR1b Ab. Representative data of three experiments with cells from three different donors are shown. B, Expression profiles of cell surface DDR1 on DCs by flow cytometry. Representative data of three experiments with cells from three different donors are shown. C, Percentages of DDR1-positive iDCs and mDCs. Data are shown as means ± SD of three experiments with cells from three different donors.

To determine the role of DDR1 in DC maturation, we treated iDCs with either 513 agonistic anti-DDR1 Ab (IgM) or control IgM for 2 days in the presence of GM-CSF and IL-4. We then evaluated the expression of cell surface molecules, including CD80, CD83, CD86, HLA-DR, CCR7, and MHC class I molecules by flow cytometry and compared them with that of TNF--induced mDCs. As shown in Fig. 2, A and B, DDR1 activation with 513 Ab significantly up-regulated cell surface expression of CD83 (p < 0.01) and CD86 (p < 0.01), but not of CD80, HLA-DR, CCR7, or MHC class I molecules on iDCs. The level of CD83 expressed on 513 Ab-treated iDCs was similar to that expressed on TNF--induced mDCs, whereas the levels of CD86 on 513 Ab-treated iDCs were lower. When 513 Ab was used in combination with 50 ng/ml TNF- or 1 µg/ml LPS, 513 Ab further up-regulated TNF--induced expression of CD80 (p < 0.01), CD83 (p < 0.05), CD86 (p < 0.01), HLA-DR (p < 0.01), CCR7 (p < 0.01), and MHC class I molecules (p < 0.01, p < 0.05). The 513 Ab preparation we used contained no detectable level of endotoxin, and the addition of 50 µg/ml polymixin B had no effect (Fig. 2C). As shown in Fig. 2, D and E, the effects of 513 Ab were more evident with lower concentrations of TNF- or LPS. These results indicate that activation of DDR1 by itself induces partial phenotypic maturation of DCs and is not sufficient to induce full maturation of DCs; however, activation of DDR1 markedly amplifies TNF-- and LPS-induced phenotypic maturation of DCs.

View larger version (71K): [in this window] [in a new window]   FIGURE 2. Effects of DDR1 activation on the expression of CD80, CD83, CD86, HLA-DR, CCR7, and MHC class I molecules on DCs. Monocyte-derived iDCs were treated for 2 days with 513 agonistic anti-DDR1Ab (5 µg/ml) and control IgM (5 µg/ml) in the presence or absence of 50 ng/ml TNF- or 1 µg/ml LPS. The expression of CD80, CD83, CD86, HLA-DR, CCR7, and MHC class I molecules was analyzed by flow cytometry. A, Representative profiles of three individual experiments with almost identical results are shown. B, The percentages of CD80-, CD83-, CD86-, HLA-DR-, CCR7-, or MHC class I-positive cells were quantified. C, Effect of polymixin B on 513 Ab-induced amplification of CD80, CD83, CD86, HLA-DR, CCR7, and MHC class I molecule expression. A total of 50 µg/ml polymixin B was used. D and E, Different concentrations of TNF- or LPS were used in combination with 5 µg/ml 513 Ab to induce DC maturation, and the percentages of CD80-, CD83-, CD86-, HLA-DR-, CCR7-, or MHC class I Ag-positive cells were quantified. Statistical analysis was performed by Bonferroni/Dunn with One Way Factorial ANOVA. *, p < 0.01, n = 3. **, p < 0.05, n = 3.

View larger version (39K): [in this window] [in a new window]   FIGURE 2A. Continued.

The most important function of DCs is to present Ags to naive T cells. We treated iDCs with 513 Ab or control IgM for 2 days in the presence or absence of TNF- and/or LPS, and evaluated their capacity to stimulate allogeneic MLR. Our data confirmed that iDCs only weakly stimulate allogeneic MLR, and activation of DDR1 with 513 Ab had no effect (Fig. 3A). mDCs induced with TNF- or LPS markedly stimulated allogeneic MLR in a cell concentration-dependent manner. TNF- and LPS had an additive effect on the functional maturation of DCs. It was of great interest that activation of DDR1 with 513 Ab markedly increased the capacity of mDCs induced with TNF- (p < 0.001), LPS (p < 0.001), or TNF- plus LPS (p < 0.001) to stimulate allogeneic MLR. As shown in Fig. 3B, the release of IL-12 p70 from LPS-induced mDCs was also up-regulated 2- to 3-fold by activation of DDR1 (p < 0.001) without affecting the release of IL-10. Activation of iDC with 513 Ab alone did not induce the release of either IL-12 or IL-10 (data not shown).

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Effects of DDR1 activation on the functional maturation of DCs. A, iDCs were incubated in the presence of GM-CSF (50 ng/ml) and IL-4 (50 ng/ml) for 2 days with various combinations of TNF- (50 ng/ml), LPS (1 µg/ml), 513 agonistic anti-DDR1 Ab, or control IgM. The resultant cell populations were used to evaluate their capacity to stimulate allogeneic MLR. Data were analyzed by Bonferroni/Dunn with One Way Factorial ANOVA. *, p < 0.001, n = 3. B, iDCs were incubated in the presence of GM-CSF (50 ng/ml) and IL-4 (50 ng/ml) for 2 days with various combinations of LPS (1 µg/ml), 513 anti-DDR1 Ab (5 µg/ml), and control IgM (5 µg/ml). Culture supernatants were collected, and cytokine levels were measured by ELISA. Data were analyzed by Bonferroni/Dunn with One Way Factorial ANOVA. *, p < 0.0001, n = 8. C, iDCs were incubated in the presence of GM-CSF (50 ng/ml) and IL-4 (50 ng/ml) for 2 days with various combinations of TNF-, 513 agonistic anti-DDR1 Ab, or control IgM. The numbers of migrated cells in response to MIP-3/CCL19 were counted. Data were analyzed by Bonferroni/Dunn with One Way Factorial ANOVA. *, p < 0.001, n = 3.

To further evaluate the effect of DDR1 activation on the functional maturation of DCs, we examined DC-dependent priming of MHC class I-restricted CD8⁺ T cells. We prepared iDCs from monocytes of HLA-A2⁺ donors pulsed with cell lysates of HLA-A2⁺ human melanoma cells, and then induced their final maturation with TNF- in the presence of 513 Ab or control mouse IgM. These tumor Ag-loaded mDCs were used to prime HLA-A2⁺ CD8⁺ T cells. We first evaluated an Ag-specific response of the primed CD8⁺ T cells by detecting intracellular IFN- after re-priming by tumor Ag-loaded mDCs induced with TNF- alone. As shown in Fig. 4A, the percentage of IFN-- positive CD8⁺ T cells primed by mDCs that were induced with 513 Ab plus TNF- was approximated 3-fold higher than that of the cells primed by mDCs induced with TNF- alone.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Effects of DDR1-activation on the generation of tumor-specific CD8+ CTLs. A, CD8+ T lymphocytes (HLA-A2+) were cocultured with tumor lysate-pulsed mDCs (HLA-A2+) for 7 days. After restimulation with tumor lysate-pulsed mDCs, the presence of IFN- was analyzed by flow cytometry using an intracellular staining technique. Representative profiles of three individual experiments with almost identical results are shown. B, The percentages of IFN--positive CD8+ T cells were quantified. C, 51Cr release assay was performed with 51Cr-labeled malignant melanoma cells (HLA-A2+), and CTLs were generated using various mDC preparations. Data were analyzed by Bonferroni/Dunn with One Way Factorial ANOVA. *, p < 0.01, n = 3. D, Release of IFN- from CTLs generated using different mDC preparations was quantified by ELISA. Data were analyzed by Bonferroni/Dunn with One Way Factorial ANOVA. *, p < 0.01, n = 3.

Activation of DDR1 induces phosphorylation of p38 MAP kinase, but not MKK3/6, in DCs

We recently reported that the activation of the DDR1b isoform, but not the DDR1a isoform, facilitated the differentiation of THP-1 cells and primary macrophages. Collagen activation of DDR1b induced its autophosphorylation, followed by the recruitment of the adaptor protein Shc to the juxtamembrane domain of the receptor and subsequent activation of p38 MAPK in differentiated THP-1 cells. Interestingly, activation of p38 MAPK was independent of its upstream kinases, MKK3 and 6 (14). These previous observations led us to the hypothesis that DDR1-mediated amplification of DC maturation is also regulated by the p38 MAPK pathway activated through DDR1b, independent of MKK3 and 6. To test this hypothesis, we first evaluated the kinetics of DDR1 autophosphorylation and the recruitment of Shc in response to 513 Ab or control IgM. In both iDCs (Fig. 5A) and mDCs (Fig. 5B), autophosphorylation of DDR1 was detected at 30 min (lane 3), and DDR1 remained phosphorylated at 120 min (lane 6). Treatment with control IgM did not induce autophosphorylation of DDR1 in either iDCs or mDCs (lanes 1–6). In addition to tyrosine phosphorylated DDR1, we detected another tyrosine-phosphorylated protein, Shc. DDR1 remained phosphorylated in mDCs 8 h after 513 Ab activation (data not shown); however, coimmunoprecipitation of Shc was seen only between 30 and 90 min, identical with that observed in differentiated DDR1b-overexpressing THP-1 cells (14). The recruitment and phosphorylation of Shc were significantly higher in mDCs than in iDCs (Fig. 5C).

View larger version (37K): [in this window] [in a new window]   FIGURE 5. DDR1 phosphorylation and the recruitment of Shc to DDR1 in immature and mDCs in response to 513 Ab. Ten million iDCs or mDCs were serum-starved in RPMI 1640 containing 1% FCS for 10 h on culture dishes and then incubated for various times in the presence of 5 µg/ml 513 anti-DDR1 Ab or control IgM. Cell lysates were prepared and subjected to immunoprecipitation with anti-DDR1 Ab (C-20). Bound proteins were analyzed by Western blotting. A, Membranes prepared from iDCs were probed with either anti-phosphotyrosine Ab, anti-DDR1 Ab (C-20), or anti-Shc Ab. Arrows indicate phosphorylated DDR1 or Shc. B, Membrane prepared from mDCs was probed with either anti-phosphotyrosine Ab, anti-DDR1 Ab (C-20), or anti-Shc Ab. Arrows indicate phosphorylated DDR1 or Shc. C, Cell lysates of iDCs or mDCs prepared from the same donor were simultaneously subjected to immunoprecipitation, and the recruitment of Shc to DDR1 was compared. Representative data of three individual experiments using cells from three different donors are shown.

View larger version (45K): [in this window] [in a new window]   FIGURE 6. Phosphorylation of p38 MAPK, ATF2, but not ERK or MKK3/6, in iDCs or mDCs in response to 513 Ab. Ten million iDCs or mDCs were plated on each dish and serum-starved in RPMI 1640 containing 1% FCS for 10 h. A total of 5 µg/ml 513 anti-DDR1 Ab or control IgM was subsequently added, and cell lysates were prepared after incubation for additional times. Twenty microliters of cell lysates were directly mixed with 20 µl of sample buffer and subjected to Western blotting. A, Membranes from iDCs were blotted with Abs specific for phospho-p38 MAPK, p38 MAPK, phospho-ATF2, or ATF2. B, Membranes from mDCs were blotted with Abs specific for phospho-p38 MAPK, p38 MAPK, phospho-ATF2, or ATF2. C, Membranes from mDCs were blotted with Abs specific for phospho-ERK or ERK. D, Membranes from mDCs were blotted with Abs specific for phospho-MKK3/6 or MKK3/6. Representative data of three individual experiments using cells from three different donors are shown.

Activation of DDR1b induces the formation of TRAF6/TAB 1/p38 protein complex and subsequent p38 autophosphorylation

It was recently reported that the interaction of p38 MAPK, but not other forms of p38 MAPK, with TAB 1 leads to autophosphorylation and activation of p38 MAPK independently of MKKs, and these proteins could form a protein complex with TRAF6 (16). To examine a potential involvement of this alternative pathway inDDR1b-mediated p38 MAPK activation, we used differentiated DDR1b-overexpressing THP-1 cells as a model (14). We first evaluated whether DDR1b-mediated p38 MAPK phosphorylation was caused by autophosphorylation. Because SB203580 blocks autophosphorylation of p38 MAPK, but not transphosphorylation by MKKs (16), this inhibitor enabled us to discriminate between the two phosphorylation events. As shown in Fig. 7A, p38 MAPK was phosphorylated in response to collagen (lane 2), and it was almost completely inhibited by SB203580 (lane 4). DMSO had no effect (lane 6). Thus, DDR1b-mediated activation of p38 MAPK in differentiated THP-1 cells was indeed caused by the autophosphorylation of p38 MAPK.

View larger version (44K): [in this window] [in a new window]   FIGURE 7. DDR1b-mediated autophosphorylation of p38 MAPK and its association with TRAF6 and TAB 1 in differentiated THP-1 cells. A, DDR1b-overexpressing cells were incubated with PMA for 12 h, treated with SB203580 (10 µM) or DMSO for 30 min, and then activated with collagen. Cell lysates were subjected to immunoprecipitation with anti-p38 MAPK Ab, and the phosphorylation of p38 MAPK was analyzed by Western blotting. B, Differentiated DDR1a- or DDR1b-overexpressing THP-1 cells were activated with collagen for various times. Cell lysates were subjected to immunoprecipitation with anti-p38 MAPK Ab, and coimmunoprecipitation of TRAF6, TAB 1, and TAK1 was analyzed by Western blotting with Ab specific for each protein. C, Differentiated DDR1b-overexpressing THP-1 cells were activated with collagen for 60 min. Cell lysates were subjected to immunoprecipitation with anti-TRAF6 Ab, and coimmunoprecipitation of p38 was analyzed by Western blotting. D, Differentiated DDR1b-overexpressing THP-1 cells were activated with collagen for various times. Cell lysates were subjected to immunoprecipitation with anti-TRAF6 Ab, and coimmunoprecipitation of TAB 1 isoforms was analyzed by Western blotting. E, Differentiated DDR1a- or DDR1b-overexpressing THP-1 cells were activated with 513 Ab for various times. Cell lysates were subjected to immunoprecipitation with anti-p38 MAPK Ab, and coimmunoprecipitation of TRAF6, TAB 1, and TAK1 was analyzed by Western blotting with Ab specific for each protein. Representative results of two to three individual experiments with similar results are shown.

We evaluated the functional role of TRAF6 in DDR1b-mediated p38 MAPK activation by overexpressing a DN form of TRAF6 (25) in differentiated DDR1b-overexpressing THP-1 cells (Fig. 7F). Expression of DN-TRAF6 completely abrogated collagen-induced p38 MAPK phosphorylation (Fig. 7G, lane 4), indicating that TRAF6 plays a critical role in DDR1b-mediated activation of p38 MAPK. Taken together, our results indicate that DDR1b-mediated activation of p38 MAPK in differentiated THP-1 cells was caused by p38 autophosphorylation, and it was regulated through the TRAF6/TAB 1/p38 signaling cascade. Ab that specifically recognizes TAB 1 is currently not available.

Finally, we used mDCs to determine whether 513Ab-induced p38 MAPK activation detected in mDCs was also caused by the TAB 1-mediated p38 autophosphorylation. As shown in Fig. 8A, p38 MAPK was phosphorylated in response to 513 Ab (lane 2), and it was almost completely inhibited by SB203580 (lane 4). DMSO had no effect (lane 6). Activation of DDR1 with 513 Ab induced association of p38 MAPK with TRAF6 (Fig. 8, B and C, lanes 3–6) and TAB 1 (Fig. 8, D, lanes 3–6, and E, lane 3) in mDCs. TAB 1 included in this protein complex reacted with Ab against the N terminus, but not against the C terminus, of TAB 1, despite the fact that TAB 1 recognized by these two Abs was present in mDCs (data not shown). TAK1 was not detected in this complex (Fig. 8F). Thus, the signaling events detected in 513 Ab-activated mDCs are identical with that in collagen-activated, differentiated DDR1b-overexpressing THP-1 cells.

View larger version (34K): [in this window] [in a new window]   FIGURE 8. DDR1-mediated autophosphorylation of p38 MAPK and its association with TRAF6 and TAB 1 in mDCs. A, mDCs induced with TNF- (50 ng/ml) were pretreated with 10 µM SB203580 or DMSO for 30 min, rinsed with PBS, and activated with 513 anti-DDR1 Ab. Cell lysates were prepared and subjected to immunoprecipitation with anti-p38 MAPK, and phosphorylation of p38 MAPK was analyzed by Western blotting. B, mDCs were activated with 513 anti-DDR1 Ab for various times, and cell lysates were prepared. The lysates were subjected to immunoprecipitation with anti-p38 MAPK Ab, and coimmunoprecipitation of TRAF6 was analyzed by Western blotting with Ab specific for TRAF6. C, mDCs were activated with 513 anti-DDR1 Ab for various times, and cell lysates were prepared. The lysates were subjected to immunoprecipitation with anti-TRAF6, and coimmunoprecipitation of p38 MAPK was analyzed by Western blotting with Ab specific for p38 MAPK. D, mDCs were activated with 513 anti-DDR1 Ab for various times, and cell lysates were prepared. The lysates were subjected to immunoprecipitation with anti-p38 MAPK Ab, and coimmunoprecipitation of TAB 1 was analyzed by Western blotting with Abs specific for the N terminus or C terminus of TAB 1. E, Cell lysates were prepared from TNF--induced mDCs before and after a 60-min activation with 513 anti-DDR1 Ab. The lysates were subjected to immunoprecipitation with anti-p38 MAPK Ab, and coimmunoprecipitation of TAB 1 was analyzed by Western blotting with Abs specific for the N terminus or C terminus of TAB 1. Cell lysates were also prepared from 1 x 107 unstimulated human PBMC and subjected to immunoprecipitation with anti-TAB 1 Ab (C terminus). The immunoprecipitated proteins were used as a control for TAB 1 detection (lane 1). F, Cell lysates were prepared from TNF--induced mDCs before and after a 60-min activation with 513 anti-DDR1 Ab. The lysates were subjected to immunoprecipitation with anti-p38 MAPK Ab, and coimmunoprecipitation of TAK1 was analyzed by Western blotting with Ab specific for TAK1. Cell lysates were also prepared from 1 x 107 unstimulated human PBMC and subjected to immunoprecipitation with anti-TAK1 Ab. The immunoprecipitated proteins were used as a control for TAK1 detection (lane 1). Representative data of at least two experiments are shown.

To evaluate the involvement of the p38 MAPK pathway in DDR1-mediated up-regulation of CD80, CD83, CD86, HLA-DR, CCR7, and MHC class I expression, we pretreated iDCs with SB203580 before inducing their maturation with TNF- alone or TNF- plus 513 Ab. As shown in Fig. 9A, SB203580 almost completely inhibited TNF--induced expression of CD80, CD83, CD86, HLA-DR, CCR7, and MHC class I expression and its amplification with 513 Ab. Stimulation of allogeneic MLR by mDCs produced with either TNF- alone or TNF- plus 513 Ab was also almost completely abrogated by SB203580 (Fig. 9B). Thus, our data indicate that TNF--induced DC maturation and its amplification by DDR1 activation was dependent on the p38 MAPK pathway.

View larger version (42K): [in this window] [in a new window]   FIGURE 9. Effects of SB203580 on phenotypic and functional maturation of DCs. Monocyte-derived iDCs were pretreated with SB203580 or DMSO for 30 min and then incubated for 2 days in the presence of GM-CSF (50 ng/ml), IL-4 (50 ng/ml), and TNF- (50 ng/ml), with 5 µg/ml 513 agonistic anti-DDR1Ab or control IgM. A, The expression of CD80, CD83, CD86, HLA-DR, CCR7, and MHC class I molecules was analyzed by flow cytometry. B, Ag-presenting capacity of each cell population was evaluated by allogeneic MLR. Data were analyzed by Bonferroni/Dunn with One Way Factorial ANOVA. *, p < 0.001, n = 3; **, p < 0.01, n = 3.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Activation of DDR1 alone is not sufficient to induce full maturation of iDCs into mDCs. However, when iDCs were activated with 513 Ab along with TNF- or LPS, resulting mDCs expressed high levels of MHC class I and class II molecules and costimulatory molecules, including CD80, CD83, and CD86. As aconsequence, these highly mDCs stimulated allogeneic MLR at a significantly higher level than mDCs produced without DDR1-activation. mDCs use MHC class I molecules to present foreign Ags, such as tumor Ags, to CD8⁺ T cells (40). In our study, CD8⁺ T cells primed by Ag-loaded DDR1-activated mDCs exhibited 3-fold higher CTL activity, as determined by the ⁵¹Cr release assay, than those generated by mDCs without DDR1-activation. One of the major issues in the field of anti-tumor immunity is how to generate an efficient tumor Ag-specific immune response. It is clear that CD8⁺ CTLs are the most potent anti-tumor effector cells and the subject of many studies (41). Our results strongly suggest that highly mDCs produced by activating DDR1 with DDR1 agonists, such as agonistic anti-DDR1 Ab, in combination with other agents that are capable of inducing DC maturation provide a useful means of inducing efficient anti-tumor CTLs in vitro. In addition, mDCs that are loaded with soluble proteins in vitro can also prime CD8⁺ T cells in vivo (42, 43). Several clinical trials have been performed using in vitro generated DCs; however, some problems still exist. Especially, improvements in optimizing the maturation and activation of DCs for the enhancement of anti-tumor efficiency of DC-based anti-tumor vaccine need to be established (43, 44). Activation of DDR1 provides a way of enhancing the maturation of DCs and subsequent anti-tumor activity. Up-regulated CCR7 expression on mDCs would also enhance their migration to the secondary lymphoid organs for better priming of CD8⁺ T cells. Our next goal is to investigate the effectiveness of DDR1-activated mDC-based vaccine for tumor treatment using mouse tumor transplantation models.

The p38 MAPK pathway has been reported to play a critical role in the maturation of human monocyte-derived iDCs to mDCs in response to CpG-DNA, LPS, TNF-, and contact sensitizers such as dinitrochlorobenzene and NiSO₄ (15, 45, 46). The effects of DDR1 activation on DC maturation was also dependent on the p38 MAPK pathway. We recently reported that activation of the DDR1b isoform, but not DDR1a isoform, induces the activation of p38 MAPK (14), strongly suggesting that the activation of DDR1b, the major isoform expressed on mDCs, is responsible for 513 Ab-induced activation of p38 MAPK and amplification of DC maturation. In this study, we further investigated the molecular mechanisms regulating the DDR1b-mediated p38 MAPK activation and have shown that the activation of DDR1b induces the formation of the TRAF6/TAB 1/p38 protein complex and p38 autophosphorylation in both differentiated THP-1 cells and mDCs. TRAF6 is a critical signaling molecule regulating DDR1b-mediated activation of p38 MAPK. During the signaling of IL-1R, Toll-like receptors, CD40, and TRANCE-R, TRAF6 is used as an important molecule to transfer signals to their downstream signaling pathways (47, 48). The major difference in the signaling between DDR1b and these receptors is that the TRAF6 protein complex induced by DDR1b activation contains TAB 1, instead of TAB 1. Interestingly, TAB 1 lacks the TAK1-binding site located in the C terminus; therefore, it is unable to bind to TAK1. Therefore, TAK1 is not a target of the DDR1b signaling. Taken together, our results indicate that the DDR1b signaling in mDCs uses the novel TRAF6/TAB 1/p38 MAPK cascade to amplify their maturation.

In contrast to the p38 MAPK signaling, the ERK signaling pathway appears to negatively regulate the phenotypic and functional maturation of monocyte-derived DCs, and the balance of activation levels between p38 and ERK may regulate the initial commitment of naive T cells toward Th1 or Th2 subsets (49). In our study, mDCs prepared in the presence of LPS and 513 Ab secreted 2- to 3-fold higher level of the Th1 cytokine IL-12 p70, the functional form of IL-12. Transcription of the IL-12 p40 gene in DCs has been reported to be regulated by p38 MAPK (50). DDR1 activation did not affect the secretion of the Th2 cytokine IL-10 or phosphorylation of ERKs. Therefore, DDR1 signaling contributes to the development of Th1 responses by activating p38 MAPK, but not ERKs.

In addition to DDR1b, DDR1a is also expressed during DC maturation. DDR1a expression was rapid, reached a peak on day 2, and gradually decreased from day 3 as the cells became more mature. Our previous findings using DDR1a-overexpressing THP-1 cells (13) suggest that cells in an early stage of DC maturation have the greater capacity to migrate through the ECM than terminally matured DCs. It was previously reported that the adaptor protein fibroblast growth factor receptor substrate 2 was capable of binding to the juxtamembrane domain of DDR1a using a chimeric receptor that consisted of the extracellular domain of platelet-derived growth factor receptor,the transmembrane and juxtamembrane domain of DDR1a, and thekinase domain of TrkA (51). However, we were not able to detect either the recruitment or phosphorylation of fibroblast growth factor receptor substrate 2 after activation of DDR1 in DCs (our unpublished data). Additional studies are necessary to determine the role of DDR1a expressed on DCs and to unveil as yet uncharacterized DDR1a signaling pathways.

Activation of DDR1 signaling in DCs is regulated at least at two levels. First, the expression of DDR1 needs to be induced. Secondly, DDR1 signaling, especially DDR1b signaling, requires additional signals, such as TNF- or LPS, to be able to maximally activate p38 MAPK. Thus, DDR1b signaling occurs only when the maturation of iDCs is induced through activation of other receptors during immune responses and DDR1b acts as a costimulating receptor for DC maturation. Another potentially important mechanism involved in the regulation of the DDR1 signaling is the physical state of collagen optimal for DDR1-activation. Normal tissues are composed of a fibrillar mesh of ECM, including polymerized type I collagen. In inflammatory conditions such as atherosclerosis, polymerized collagen fibrils are absent from intermediate stages of lesion development that instead contain thin and disordered collagen fibers and collagen fragments. Previous studies have indicated that the triple helical configuration of collagen is required to serve as a DDR1 ligand and also for DDR1 activation (11, 12). However, it is not clear whether normal intact collagen can activate DDR1, and it will be important to clarify the form of collagen optimal to activate DDR1. Considering the fact that collagen is abundant and present everywhere in the ECM, tight regulation of DDR1 activation is necessary to avoid unwanted signaling through DDR1.

In conclusion, we have identified DDR1, most likely DDR1b, as a novel coreceptor involved in DC maturation, and have demonstrated that activation of DDR1, in combination with TNF- or LPS, results in the production of highly potent mDCs. We have also revealed that the DDR1b signaling pathway is unique and regulated by the recently described TAB 1-mediated alternative pathway of p38 MAPK activation. Further studies addressing the role and signaling pathways of DDR1 will provide new information regarding the involvement of the ECM protein, collagen, in the development of immune responses and bring a new insight into the mechanisms regulating this complex system in a tissue microenvironment.

	Acknowledgments

 
We are grateful to Dr. Joost J. Oppenheim for his invaluable comments, and to Dr. Morihiro Watanabe for his technical assistance (National Cancer Institute-Frederick, Frederick, MD). We are also grateful to Trans Genic for a kind gift of anti-DDR1 Ab and to the National Cancer Institute, Center for Cancer Research Fellows Editorial Board for their excellent editing during the preparation of this manuscript.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Teizo Yoshimura, Building 559, Room 9, National Cancer Institute, Frederick, MD 21702. E-mail address: yoshimur{at}mail.ncifcrf.gov

² Abbreviations used in this paper: DC, dendritic cell; iDC, immature DC; mDC, mature DC; ECM, extracellular matrix; DDR1, discoidin domain receptor 1; TAK1, TGF--activated protein kinase 1; TAB 1, TAK1 binding protein 1; MAPK, mitogen-activated protein kinase; ATF2, activating transcription factor 2; ERK, extracellular signal-regulated kinase; TRAF, TNFR associated factor; MKK, MAPK kinase; PI, propidium iodide; MIP-3, macrophage inflammatory protein-3; DN, dominant negative; PGA, protein G-agarose.

Received for publication April 30, 2003. Accepted for publication July 29, 2003.

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