The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hu, Y.
Right arrow Articles by Ivashkiv, L. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hu, Y.
Right arrow Articles by Ivashkiv, L. B.
The Journal of Immunology, 2006, 176: 6022-6033.
Copyright © 2006 by The American Association of Immunologists

Costimulation of Chemokine Receptor Signaling by Matrix Metalloproteinase-9 Mediates Enhanced Migration of IFN-{alpha} Dendritic Cells1

Yang Hu* and Lionel B. Ivashkiv2,{dagger},{ddagger}

* Graduate Program in Neuroscience, Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10021; {dagger} Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, New York, NY 10021; and {ddagger} Graduate Program in Immunology and Microbial Pathogenesis, Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Type I IFNs induce differentiation of dendritic cells (DCs) with potent Ag-presenting capacity, termed IFN-{alpha} DCs, that have been implicated in the pathogenesis of systemic lupus erythematosus. In this study, we found that IFN-{alpha} DCs exhibit enhanced migration across the extracellular matrix (ECM) in response to chemokines CCL3 and CCL5 that recruit DCs to inflammatory sites, but not the lymphoid-homing chemokine CCL21. IFN-{alpha} DCs expressed elevated matrix metalloproteinase-9 (MMP-9), which mediated increased migration across ECM. Unexpectedly, MMP-9 and its cell surface receptors CD11b and CD44 were required for enhanced CCL5-induced chemotaxis even in the absence of a matrix barrier. MMP-9, CD11b, and CD44 selectively modulated CCL5-dependent activation of JNK that was required for enhanced chemotactic responses. These results establish the migratory phenotype of IFN-{alpha} DCs and identify an important role for costimulation of chemotactic responses by synergistic activation of JNK. Thus, cell motility is regulated by integrating signaling inputs from chemokine receptors and molecules such as MMP-9, CD11b, and CD44 that also mediate cell interactions with inflammatory factors and ECM.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Type I IFNs (IFN-{alpha}/IFN-beta/IFN-{omega}) are pleiotropic cytokines with antiviral and immunoregulatory properties (1, 2). Type I IFNs augment innate immune responses by inducing an antiviral state and activating NK cells, and promote the transition from innate to acquired immunity by enhancing the differentiation and maturation of DCs. Type I IFNs drive acquired immune responses by promoting Th1 responses and enhancing B cell class switching, differentiation into plasma cells, Ab production, and immunological memory (3). Type I IFNs have been implicated in the development of autoimmunity and in the pathogenesis of systemic lupus erythematosus (SLE)3 (4), a systemic autoimmune disease characterized by elevated IFN production and loss of B and T cell tolerance that leads to autoantibody production. Mechanisms by which type I IFNs promote autoimmunity are not fully understood, but one attractive possibility is that type I IFNs promote maturation of dendritic cells (DCs) into cells termed IFN-{alpha} DCs. IFN-{alpha} DCs contribute to normal immune responses and to mouse lupus (5, 6, 7, 8, 9), have been observed in human SLE (10), and exhibit enhanced Ag uptake, cytokine production, and an increased capacity to activate T cells (4, 10, 11, 12, 13, 14, 15). The effects of IFNs on additional DC functions have not been thoroughly investigated.

A key DC function is migration to sites where they carry out effector functions such as Ag presentation and inflammatory cytokine production (16). Emigration of DCs from tissues to lymph nodes, where they can either activate or tolerize T cells, has been extensively studied. Activation of immature DCs in peripheral tissues by infectious or inflammatory factors results in functional maturation that includes induction of costimulatory molecules and the lymphoid homing chemokine receptor CCR7. CCR7 mediates migration of mature DCs via lymphatics to lymph nodes in response to the chemokines CCL19 and CCL21 (16). In addition to migration to lymph nodes, immature DCs and DC precursors circulate in the blood and can be recruited to the spleen and peripheral tissues (17, 18, 19, 20, 21, 22, 23). This recruitment into peripheral tissues maintains DC homeostasis, and influx of DCs/DC precursors maintains DC numbers at inflammatory sites, where DCs can produce cytokines, capture Ags, and activate T cells locally or after migration to draining lymph nodes. Immature DCs and DC precursors do not express CCR7 and instead are recruited to sites of inflammation by inflammatory chemokines such as CCL3 (MIP-1{alpha}), CCL4 (MIP-1beta), and CCL5 (RANTES) that signal via CCR1 and CCR5 (24, 25). Although DC numbers in the blood are typically low, they can be markedly increased in autoimmune diseases. For example, numbers of CCL5-responsive DCs are elevated in the NZB/W model of SLE (26), and in the BXSB SLE model, DCs comprise 15% of peripheral blood cells (27). Emigration of these blood DCs into kidneys, where they secrete inflammatory cytokines and capture (auto)antigens, contributes to lupus nephritis (27). Inflammatory DCs present in the joints of patients with rheumatoid arthritis are also thought to arise at least in part from precursors that migrate into the joint synovium (28).

Migratory cells like DCs move along concentration gradients of chemokines that stimulate cells via seven-transmembrane, G protein-coupled chemokine receptors. The pertussis toxin (PTX)-sensitive G{alpha}i protein that regulates calcium influx is a key mediator of chemokine receptor function, and several signaling molecules activated downstream of chemokine receptors, including Pyk2, PI3K{gamma}, and MAPKs (ERKs, JNK, and p38), are important for cell motility (29, 30). In addition, interaction of cells with extracellular matrix (ECM) is important for migration across basement membranes and through tissues (31). Interaction of DCs with ECM is mediated by two classes of molecules, adhesion receptors and proteases. Adhesion receptors, such as integrins and the hyaluronan receptor CD44, mediate direct contact of cells with ECM components that provide a scaffold for cell movement through tissues, and also can induce additional signals that regulate cell motility. Proteases are widely believed to facilitate movement of cells through basement membranes and tissues by degrading ECM components. In addition, proteases can promote migration by activation of enzymatic cascades that effectively degrade ECM, and can regulate chemotaxis by cleaving chemokines (32). Matrix metalloproteinase-9 (MMP-9) has been shown to play a key role in DC migration in vivo and in migration through Matrigel, a model basement membrane, in vitro (33, 34, 35, 36, 37).

We were interested in defining mechanisms by which IFN-{alpha} could potentiate DC function and thereby contribute to enhanced immune responses and potentially to autoimmunity. In this study, we found that IFN-{alpha} DCs exhibit enhanced chemotaxis, and that enhanced migration of IFN-{alpha} DCs was mediated by MMP-9. Surprisingly, MMP-9 enhanced DC chemotaxis even in the absence of ECM by a mechanism that involved regulation of chemokine receptor signaling and function. These results identify a new mechanism for regulating DC migration that may be operative in immune responses characterized by high type I IFN or MMP-9 expression, including the human autoimmune diseases SLE and rheumatoid arthritis (38, 39, 40, 41).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and Abs

Chemokines were purchased from PeproTech. CD14-FITC, CD25-PE, CD40-FITC, CD80-PE, CD86-PE, CCR5-FITC, and their corresponding isotype control Abs, CCR5 neutralizing mAb (2D7) and CD44 Ab (IM7) were obtained from BD Pharmingen, anti-human RANTES, CCR1-PE, CCR2-PE and CCR3-PE were obtained from R&D Systems, and HLA-DR-PE was from Immunotech (Beckman Coulter). Anti-MMP-9 mAb (Ab-1, clone 6-6B), recombinant human tissue inhibitor of metalloproteinase-1 (TIMP-1), PTX, AKT inhibitor IV, SP600125, PD98059, SB203580 were obtained from Calbiochem. TNF{alpha} protease inhibitor 1 (TAPI-1) was obtained from Peptides International. CD11b mAb LM2/1 that recognizes the I domain of CD11b, the binding site of MMP-9, was obtained from BioSource International and M1/70 was from eBioscience. Phospho-AKT (Ser473), phospho-p38 (Thr180/Tyr182), phospho-ERK (Thr202/Tyr204), and phospho-JNK (Thr183/Tyr185) Abs were purchased from Cell Signaling Technology. {alpha}-Tubulin mAb was obtained from Sigma-Aldrich.

Human DC culture

Human monocyte-derived DCs were generated as described previously (42). Briefly, PBMC were isolated by density gradient centrifugation with Ficoll (Invitrogen Life Technologies) of buffy coats purchased from the New York Blood Center. CD14+ monocytes (>97% pure, as verified by flow cytometry) were obtained from PBMC immediately after isolation by positive selection with anti-CD14 magnetic beads, as recommended by the manufacturer (Miltenyi Biotec). A total of 106/ml of CD14+ cells was plated in 6-well plates in 3 ml of RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS (HyClone), human recombinant GM-CSF (1000 U/ml, Leukine; Immunex), and either human IL-4 (25 ng/ml; R&D Systems) or recombinant human IFN-{alpha}A (500 U/ml; BioSource International) to generate control DCs and IFN-{alpha} DCs, respectively. Cytokines were replenished on days 2 and 4 of culture and on day 5 nonadherent cells were analyzed by flow cytometry and used in migration assays. The experiments using human cells were approved by the Hospital for Special Surgery Institutional Review Board.

Murine bone marrow-derived DC (BM-DC) culture

Mice were purchased from The Jackson Laboratory, including MMP-9-deficient and matched control mice (FVB/NJ), and CD11b-deficient and matched control mice (C57BL/6J). Animal experiments were approved by the Hospital for Special Surgery Institutional Animal Care and Use Committee. BM-derived DCs were cultured as described (43) with minor modifications. Femurs and tibiae of 6- to 8-wk-old male mice were isolated from the surrounding muscle tissue. The intact bones were left in 70% ethanol for 5 min and then flushed with medium to obtain bone marrow cells. Cells were passed through a cell strainer (70-µm pore size; BD Biosciences) after vigorous pipetting and cultured in RPMI 1640 medium supplemented with 100 U/ml penicillin and streptomycin, 2 mM L-glutamine, 50 µM 2-ME, 10% heat-inactivated FCS (HyClone), 10 ng/ml recombinant murine (rm) GM-CSF, and 10 ng/ml rmIL-4 (PeproTech). Cytokines were replenished on days 3 and 6 of culture, and after 7 days of culture nonadherent cells were used for analysis.

In vitro migration assay

Cell migration was quantified in duplicate using 24-well Transwell inserts (6.5 mm) with polycarbonate filters (5-µm pore size; Corning Costar). The upper side of filters was coated with growth factor-reduced Matrigel (BD Biosciences) diluted in HBSS (50 µg/filter). DCs (0.5 x 106 in 100 µl of serum-free medium) were added to the upper chamber of the insert. The lower chamber contained 500 µl of serum-free medium with or without chemokine. The plates were incubated at 37°C in 5% CO2 for 4 h and cells that had migrated into the lower chamber were counted using flow cytometry with a fluorescent counting bead internal standard in each tube (Bangs Laboratories); 10,000 singlet beads were acquired in each sample.

Immunoblotting

Total cell extracts were obtained as described (44). Cell extracts corresponding to 3.3 x 105 cells were fractionated by 10% SDS-PAGE gel, transferred to polyvinylidene fluoride membranes (Millipore), and incubated with specific Abs; ECL was used for detection.

Gelatin zymography and MMP-9 ELISA

DCs were cultured in serum-free medium, supernatants were collected, and membrane proteins were extracted using a Native Membrane Protein Extraction kit (Calbiochem). Supernatant and membrane extracts were concentrated using Microcon centrifugal filters (Millipore) and proteins derived from 2 x 106 cells were loaded onto 10% SDS-PAGE gels containing 2 mg/ml gelatin (Sigma-Aldrich). Following electrophoresis, gels were washed twice with 2.5% Triton X-100 for 30 min and incubated in developing buffer overnight at 37°C. The gels were then stained with 0.5% Coomassie blue and destained before imaging. MMP-9 levels were measured using ELISA kits Biotrak RPN 2614 and RPN 2634 (Amersham Biosciences) that detect pro-MMP-9 and active MMP-9, respectively.

Flow cytometry and calcium flux

Cells were analyzed using flow cytometry as described previously (45). Analysis was done using a FACSCalibur flow cytometer with CellQuest software (BD Biosciences). Ca2+ flux was measured using Fluo-3 AM (Molecular Probes). DCs were loaded with 5 µM Fluo-3 AM for 30 min at 37°C. Cells were then washed twice with HBSS and diluted in prewarmed complete medium before FACSCalibur analysis. The fluorescence of resting cells was measured for 30 s to obtain a baseline, and then CCL5 was added and data was acquired for another 3 min. The results were analyzed using FCSPress 1.4 software (<www.FCSPress.com>).

Gene expression analysis

Microarray analysis using U95Av2 oligonucleotide microarrays was performed according to the instructions of the manufacturer (Affymetrix) as previously described (46). Data were analyzed using Affymetrix Suite 5.0 and Genespring (Silicon Genetics). For real-time quantitative PCR, total RNA was extracted using a RNeasy Mini kit (Qiagen), and 1 µg of total RNA was treated with RNase-free DNase before reverse transcription into cDNA using a First Strand cDNA synthesis kit (Fermentas). Real-time quantitative PCR was performed as previously described (47) using iQ SYBR-Green Supermix and the iCycler iQ thermal cycler (Bio-Rad). Relative expression was normalized relative to levels of GAPDH or beta-actin. The generation of only the correct size amplification products was confirmed using agarose gel electrophoresis. Oligonucleotide primers were as follows: GAPDH, 5'-GTGAAGGTCGGAGTCAAC-3' and 5'-TGGAATTTGCCATGGGTG-3'; beta-actin, 5'-GGATGCAGAAGGAGATCACTG-3' and 5'-CGATCCACACGGAGTACTTG-3'; CCR1, 5'-GTGCCAGAAGGTGAACGAGAGG-3' and 5'-TCCAACCAGGCCAATGACAAATAC-3'; CCR5, 5'-TTCTCTTCTGGGCTCCCTACAACA-3' and 5'-CAGCAGTGCGTCATCCCAAGAG-3'. MMP-9, 5'-TGCCCGGACCAAGGATACAGT-3' and 5'-AGGCCGTGGCTCAGGTTCAGG-3'; CCR7 5'-TCCCACAGACTCAAATGCTC-3' and 5'-TTCCTCACCAAGCCAAGAAG-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Increased migration of IFN-{alpha} DCs mediated by CCR5 and CCR1

We wished to investigate the effects of IFN-{alpha} on the phenotype of human myeloid DCs. Similar to the approaches taken in previous reports (11, 12, 13, 14, 15), IFN-{alpha} DCs were generated from human CD14+ monocytes by culturing cells for 5 days with IFN-{alpha} and GM-CSF. Control monocyte-derived DCs were obtained using the well-established approach of culture with IL-4 and GM-CSF (16). The cell surface phenotype of DCs was verified using flow cytometry. As expected, control DCs were CD14 negative and expressed low levels of the activation markers and costimulatory molecules CD25, CD40, CD80, CD86, and HLA-DR that increased with maturation after addition of LPS (Fig. 1, top panels, and Table I). Consistent with previous reports (11, 12, 13, 14, 15), IFN-{alpha} DCs expressed higher levels of CD40, CD80, CD86, and HLA-DR relative to control DCs before LPS stimulation (Fig. 1). This partially mature phenotype of IFN-{alpha} DCs in the absence of any additional maturation stimuli likely explains the enhanced Ag-presenting capacity of these cells, as previously reported (10, 11, 12, 13, 14, 15) and confirmed in our laboratory (data not shown).


Figure 1
View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 1. Cell surface phenotype of human monocyte-derived control DCs and IFN-{alpha} DCs. DCs were generated as described in Materials and Methods and the expression of CD14, CD25, CD40, CD80, CD86, and HLA-DR was analyzed by flow cytometry before (shaded histograms) or after (dotted line) 2 days of stimulation with 10 ng/ml LPS. One representative experiment of three is shown.

 

View this table:
[in this window]
[in a new window]
  		Table I. DC surface markers

 
We then investigated whether IFN-{alpha} enhanced additional DC functions that could contribute to immune responses. IFN-{alpha} DCs did not produce increased amounts of inflammatory cytokines after exposure to a variety of activation and maturation stimuli (data not shown). The effect of IFN-{alpha} on DC migratory ability was then assessed using a Transwell system in which the membrane separating the upper and lower chambers was coated with Matrigel. This is a standard system for assessing DC migration in which the Matrigel serves as a model basement membrane. This experimental system assesses the capacity of DCs to respond to chemotactic stimuli added to the lower chamber and to traverse ECM. We measured migration of DCs in response to CCL5/RANTES, a chemokine that mediates migration of DCs into inflammatory sites and activates the chemokine receptors CCR1, CCR3, and CCR5 (24, 25, 48). Substantially greater numbers of IFN-{alpha} DCs than control DCs migrated from the upper chamber (to which cells were added) across the Matrigel barrier and into the lower chamber (to which CCL5 had been added) (Fig. 2A). This enhanced chemotaxis of IFN-{alpha} DCs to CCL5 was reproduced with DCs from 12 different blood donors and was statistically significant (p < 0.001, paired Student t test). The additional CCR1/CCR5 ligand CCL3/MIP-1{alpha} also induced IFN-{alpha} DC chemotaxis (Fig. 2B). In contrast, CCL2, CCL11, CCL20, CCL21 (ligand for CCR7), and CXCL10 did not induce chemotaxis (Fig. 2B), which is consistent with previous reports (24, 49). The lack of chemotactic response of IFN-{alpha} DCs to CCL21 could be explained by low CCR7 expression, comparable to that expressed by immature control DCs (Fig. 2C). CCR7 expression increased comparably on control and IFN-{alpha} DCs after maturation with LPS (Fig. 2C), and LPS-matured control and IFN-{alpha} DCs had a robust chemotactic response to CCL21 (data not shown). Low CCR7 expression by IFN-{alpha} DCs before further maturation with LPS suggests that IFN-{alpha} DCs have different migratory properties compared with DCs matured with activating stimuli such as TLR ligands. Overall, the results demonstrate enhanced migration of IFN-{alpha} DCs in response to inflammatory chemokines previously shown to recruit DCs to inflammatory sites.


Figure 2
View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 2. Increased migration of IFN-{alpha} DCs across Matrigel. DCs were added to the upper chambers of Transwell inserts that were coated with Matrigel and cells that migrated into the lower chamber were enumerated after 4 h of incubation in serum-free medium in the absence or presence of chemokines in the lower chamber. A, Increased migration of IFN-{alpha} DCs in response to CCL5. Results are presented as the mean and SEM obtained from 12 independent experiments; p < 0.001 as determined by paired Student’s t test. B, Responses of IFN-{alpha} DCs to various chemokines. A representative experiment of three is shown. C, CCR7 expression was measured using real-time PCR and normalized relative to GAPDH.

 
We then investigated whether increased migration of IFN-{alpha} DCs in response to CCR1/CCR3/CCR5 ligands could be explained, at least in part, by increased expression of these receptors. IFN-{alpha} induced expression of CCR1 and CCR5 mRNA (Fig. 3A) and cell surface expression of CCR1 and CCR5 proteins (Fig. 3B); although induction of cell surface CCR5 expression was modest it was reproducible. CCR2 cell surface expression was not detected on either control or IFN-{alpha} DCs (Fig. 3B), consistent with the lack of a chemotactic response to CCL2 (Fig. 2B). The CCL5 receptor CCR3 was not expressed on DCs (Fig. 3B). These results suggested that chemotaxis of IFN-{alpha} DCs in response to CCL5 was mediated by CCR1 and/or CCR5. Because chemokine receptors are not always functional (50), we wished to determine the relative roles of CCR1 and CCR5 in mediating CCL5 responses of IFN-{alpha} DCs. We blocked CCR5 function using a CCR5 Ab and desensitized CCR1 responses by using the CCR1-specific ligand CCL23/MIP-3 to down-regulate CCR1, but not CCR5, cell surface expression (Fig. 3C). The CCR5-blocking Ab suppressed CCL5-induced IFN-{alpha} DC migration by ~60%, whereas down-regulation of CCR1 had a more modest effect (Fig. 3D). The combination of anti-CCR5 and CCR1 desensitization was additive and nearly completely blocked CCL5-induced migration of IFN-{alpha} DCs (Fig. 3D). Overall, these results demonstrate a role for CCR5 and CCR1 in mediating CCL5-induced migration of IFN-{alpha} DCs. Modestly increased expression of these receptors may contribute to enhanced chemotaxis, but is unlikely to explain the dramatically increased chemotaxis of IFN-{alpha} DCs. In addition, the results reveal that the IFN-{alpha} DC phenotype combines partial maturation (and thus enhanced APC function) with a migratory pattern similar to that of immature DCs, thereby suggesting a mechanism for enhanced recruitment of highly functional DCs into inflammatory sites.


Figure 3
View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 3. CCL5-induced IFN-{alpha} DC migration is mediated by CCR1 and CCR5. A, mRNA levels of CCR1 and CCR5 in control DC and IFN-{alpha} DC were measured by real-time PCR. B, Cell surface expression of chemokine receptors was measured by flow cytometry. Open histograms, isotype control staining; shaded histograms, staining with CCR Abs. One representative experiment of three is shown. C, Down-modulation of IFN-{alpha} DC cell surface CCR1 by CCL23. D, CCL5-induced migration of IFN-{alpha} DCs is mediated by CCR5 and CCR1. CCR5 was blocked using neutralizing Abs and CCR1 was down-regulated using CCL23 as in C before performing migration assays. Results are presented as the mean and SEM obtained from three independent experiments.

 
MMP-9 expression is elevated in IFN-{alpha} DCs and promotes migration

To identify additional factors important for IFN-{alpha} DC migration through ECM, we used microarray analysis. These experiments confirmed increased expression of multiple canonical IFN-inducible genes in IFN-{alpha} DCs relative to control DCs, including genes whose expression was also elevated in blood cells of SLE patients (51, 52, 53) (data not shown). A small number of genes involved in cell migration were expressed at elevated levels in IFN-{alpha} DCs (Table II). Most striking was the increased expression of MMP-9, a metalloprotease with gelatinase function that degrades many ECM molecules, including collagen and laminin. MMP-9 has been implicated in cell migration, including DC migration, and in invasion of tissues by tumor cells (32, 33, 34, 35, 36, 37, 54). The major known mechanism of MMP-9 action in cell migration is digestion of ECM, although MMP-9 mobilizes stem cells by cleaving cell surface Kit ligand (55) and can thus potentially regulate migration by cleaving cell surface receptors or their ligands. We chose to further investigate the role of MMP-9 in migration of IFN-{alpha} DCs.


View this table:
[in this window]
[in a new window]
  		Table II. Examples of genes important for cell migration that are up-regulated in IFN-{alpha} DC

 
First, we confirmed the dramatic increase in MMP-9 expression in IFN-{alpha} DCs in additional blood donors using real-time quantitative PCR to measure MMP-9 mRNA (Fig. 4A) and ELISA to measure soluble MMP-9 in culture supernatants (Fig. 4B, left panel). We then used zymography to measure MMP-9 enzymatic function. Zymography detected higher levels of pro-MMP-9 in IFN-{alpha} DC culture supernatants (Fig. 4B, upper right panel, that represents a short exposure). Longer exposure of the zymogram revealed enzymatic activity of the shorter active form of MMP-9 in culture supernatants of IFN-{alpha} DCs, but not control DCs (Fig. 4B, lower right panel). MMP-9 protein was also detected in culture supernatants obtained from the 4-h chemotaxis assays (data not shown). MMP-9 can bind to cell surface receptors, and a role for cell surface-bound MMP-9 in migration has been described (56, 57). Zymography performed with cell membrane preparations revealed pro-MMP-9 and active MMP-9 enzymatic activity in membranes of IFN-{alpha} DCs, but not control DCs (Fig. 4C, left panel). In addition, ELISA specific for active MMP-9 detected higher levels of cell-associated active MMP-9 in IFN-{alpha} DCs relative to very low levels (0.05 ng/ml) in control DCs (Fig. 4C, right panel). Taken together, these results demonstrate increased MMP-9 expression and activity in IFN-{alpha} DCs.


Figure 4
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 4. MMP-9 expression is elevated in IFN-{alpha} DC and is required for migration through Matrigel. A, MMP-9 mRNA levels in control DC and IFN-{alpha} DC were measured by real-time PCR. B, Pro-MMP-9 in culture supernatants was measured using an ELISA (left panel) and zymography (right panel, which shows shorter and longer exposures of the same zymogram). C, Enzymatic activity of MMP-9 in membrane extracts was measured using zymography (left panel) and cell-associated active MMP-9 was measured by ELISA (right panel). D, MMP-9 activity was inhibited by adding a neutralizing Ab (5 µg/ml), TIMP-1 (5 nM), and TAPI-1 (100 µM) 30 min before migration assays. A representative experiment of three (left panel) or four (right panel) is shown. E, Migration assays were conducted using murine BM-DCs from control and MMP-9-deficient mice; n = 3, p < 0.05 as determined by paired Student’s t test.

 
Next, we determined the effects of blocking MMP-9 function on IFN-{alpha} DC migration using three different approaches. A neutralizing Ab specific for MMP-9 nearly completely suppressed CCL5-induced IFN-{alpha} DC migration through Matrigel (Fig. 4D). In addition, migration also blocked by TIMP-1, a protein that binds to and inactivates the catalytic function of several MMPs including MMP-9, and by TAPI-1 and TAPI-2, peptide inhibitors of MMPs (Fig. 4D, left panel, and data not shown). Migration of control DCs was minimally suppressed by inhibiting MMP-9 (Fig. 4D, right panel; mean inhibition by anti-MMP-9 was <20% and was not statistically significant). These results suggest that increased expression of MMP-9 in IFN-{alpha} DCs contributes to their increased migration. We wished to use DCs from MMP-9-deficient mice to confirm the role of MMP-9 in migration. Murine bone marrow-derived and splenic DCs expressed a more mature phenotype than did human control monocyte-derived DCs, expressed MMP-9, and were responsive to the CCR7 ligand CCL19, but not to CCL5 (data not shown). This pattern of responsiveness is consistent with a previous report (37). Deficiency of MMP-9 resulted in decreased CCL19-induced DC migration (Fig. 4E), confirming a role for MMP-9 in DC migration as previously reported (32, 33, 34, 35, 36, 37, 58). The results, taken together, support a role for increased expression and proteolytic activity of MMP-9 in the enhanced migration of IFN-{alpha} DCs.

MMP-9 increases DC chemotaxis even in the absence of ECM

We wished to investigate the mechanism by which MMP-9 increases IFN-{alpha} DC migration, and to address the notion that MMP-9 facilitates migration by proteolytic digestion of ECM. We first tested the prediction that MMP-9-mediated enhancement of migration would no longer be apparent in the absence of an ECM barrier to migration. We conducted chemotaxis assays using the same Transwell system, but in the absence of Matrigel. Similar to migration across Matrigel (Fig. 2), chemotaxis of IFN-{alpha} DCs in response to CCL5 was increased relative to control DCs in the absence of Matrigel (Fig. 5A). Surprisingly, blocking MMP-9 with neutralizing Abs or TIMP-1 strongly suppressed CCL5-induced IFN-{alpha} DC chemotaxis even in the absence of an ECM barrier (Fig. 5B, left panel). In addition, basal motility of IFN-{alpha} DCs (in the absence of exogenous CCL5) was diminished by anti-MMP-9 Ab and TIMP-1 (Fig. 5B, middle panel). This finding prompted us to examine the regulation of basal motility, and we found that basal motility was strongly inhibited by blocking endogenous CCL5 produced by IFN-{alpha} DCs and by blocking CCR5, comparable to inhibition achieved using PTX, an inhibitor of G protein signaling (Fig. 5B, right panel). We conclude that MMP-9 regulates CCL5-mediated IFN-{alpha} DC motility and chemotaxis independently of ECM. The dependence of DC chemotaxis on MMP-9 even in the absence of ECM was confirmed using MMP-9-deficient DCs (Fig. 5C).


Figure 5
View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 5. MMP-9 is required for CCL5-induced chemotaxis of IFN-{alpha} DCs even in the absence of ECM. A, The migration of control DC and IFN-{alpha} DC through uncoated Transwell membranes (without Matrigel) was measured. Results are presented as the mean and SEM obtained from eight independent experiments. B, Left and middle panels, MMP-9 activity was inhibited by adding a neutralizing Ab (5 µg/ml) or TIMP-1 (5 nM) 30 min before chemotaxis assays that were performed using CCL5 (50 ng/ml) in the absence of Matrigel. One representative experiment of four is shown. Right panel, Activity of endogenous CCL5 was inhibited using a neutralizing CCL5 Abs (5 µg/ml), blocking CCR5 Abs, and PTX. C, Chemotaxis assays using CCL19 (200 ng/ml) in the absence of Matrigel were performed using murine BM-DCs from control and MMP-9-deficient mice; n = 3, p < 0.01 as determined by paired Student’s t test.

 
MMP-9 regulates chemokine receptor signaling and function

MMPs have been shown to cleave cytokines, chemokines, and cell surface proteins, and to activate cell surface receptors by proteolytic cleavage (59, 60, 61, 62, 63, 64, 65). However, cleavage products of CCL5 or CCR5 were not detected in IFN-{alpha} DCs by immunoblotting analysis, and purified active MMP-9 did not cleave CCL5 (data not shown). We then considered the possibility that MMP-9 regulated CCR1/CCR5 signaling, possibly by acting on a different receptor or a coreceptor. The seven transmembrane chemokine receptors such as CCR1 and CCR5 signal via the receptor-coupled G protein G{alpha}i. These receptors activate a rapid and transient calcium flux and downstream signaling molecules, including PI3K{gamma} and Akt, MAPKs (ERKs, JNK, p38), and Pyk2; some of these molecules can also be activated by parallel chemokine receptor-induced signaling pathways not dependent on G{alpha}i (29, 30, 66). PI3K{gamma}, JNK, and p38 are key mediators of migratory responses to chemokine stimulation, although their targets and mechanisms of action have not been fully clarified (67, 68, 69, 70, 71, 72, 73). Consistent with the literature, CCL5 induced a rapid and transient calcium flux in DCs (Fig. 6). There was a trend toward a higher peak level of intracellular calcium in IFN-{alpha} DCs (Fig. 6), but this was not consistently observed among different donors (data not shown). Anti-MMP-9 and TIMP-1 did not inhibit the CCL5-induced calcium flux (Fig. 6). In contrast, the CCL5-induced calcium flux was essentially completely inhibited by PTX, an inhibitor of G{alpha}i. These results indicate that MMP-9 does not regulate G{alpha}i-dependent calcium signaling downstream of CCR1/5.


Figure 6
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 6. MMP-9 does not regulate G{alpha}i-dependent calcium flux. Control or IFN-{alpha} DCs were loaded with Fluo-3, stimulated with CCL5 (100 ng/ml) and intracellular calcium levels were measured using flow cytometry. Isotype control or MMP-9 Abs (10 µg/ml), TIMP-1 (5 nM), and PTX (200 ng/ml) were added before CCL5.

 
Chemokine receptors can also activate signaling pathways independently of G{alpha}i (29, 30, 66), and we further analyzed the effects of blocking MMP-9 on CCL5-induced signaling. CCL5 rapidly and transiently activated Akt (downstream of PI3K), p38, ERK, and JNK in IFN-{alpha} DCs (Fig. 7A). We then used specific inhibitors to determine which of these kinases were important in IFN-{alpha} DC chemotactic responses to CCL5. Inhibition of Akt and JNK, but not MEK-ERK or p38, strongly suppressed CCL5-induced migration of IFN-{alpha} DCs (Fig. 7B, left panel). Consistent with the key role of endogenous CCL5 in basal IFN-{alpha} DC motility (Fig. 5B), inhibition of Akt and JNK also suppressed basal IFN-{alpha} DC motility (Fig. 7B, right panel). Interestingly, inhibition of JNK suppressed migration as potently as did inhibition of G{alpha}i using PTX (Fig. 7B), indicating a key nonredundant role for JNK in IFN-{alpha} DC chemotaxis.


Figure 7
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 7. MMP-9 regulates CCR5 signaling and function by costimulating JNK activation. A, CCL5-induced activation of AKT and MAPKs in IFN-{alpha} DCs was measured by immunoblot. B, CCL5-induced chemotaxis and basal motility of IFN-{alpha} DCs are dependent on AKT and JNK. Vehicle control (DMSO; 0.1%) or inhibitors of G{alpha}i (PTX; 200 ng/ml), AKT (AKT inhibitor IV; 1 µM), JNK (SP600125; 30 µM), MEK-ERK (PD98059; 50 µM), and p38 (SB203580; 20 µM) were added 30 min before migration assays. *, p < 0.05; **, p < 0.01 as determined by paired Student’s t test. C, Left panel, Human IFN-{alpha} DCs were treated with isotype control or MMP-9 Abs (5 µg/ml), TIMP-1 (5 nM), and PTX (200 ng/ml) for 4 h before stimulation with CCL5 (100 ng/ml) for 5 min. Right panel, Murine BM-DCs from control or MMP-9-deficient mice were treated with CCL19 (200 ng/ml) for indicated periods of time. Phospho-JNK and phospho-AKT levels were measured by immunoblot.

 
We then assessed the effects of blocking MMP-9 on CCL5-induced signaling in IFN-{alpha} DCs. Both MMP-9 Abs and TIMP-1 suppressed CCL5-induced activation of JNK but did not affect activation of Akt or p38 (Fig. 7C, left panel, and data not shown). In contrast, inhibition of the receptor-proximal G{alpha}i protein using PTX suppressed CCL5-induced activation of JNK, p38, ERK, and Akt (Fig. 7C and data not shown). The role of MMP-9 in modulating chemokine receptor-dependent activation of JNK, but not Akt, was confirmed using DCs from MMP-9-deficient mice (Fig. 7C, right panel). These results, together with the results showing inhibition of calcium fluxes by PTX but not anti-MMP-9 (Fig. 6), indicate that: 1) CCL5 activation of calcium fluxes and Akt is dependent on G{alpha}i signaling but not on MMP-9; and 2) CCL5 activation of JNK is dependent on both G{alpha}i and MMP-9. Thus, MMP-9 selectively regulates activation of the JNK-signaling pathway downstream of chemokine receptors and MMP-9 activity is required for full activation of JNK and chemotaxis.

MMP-9 was present in culture supernatants and also associated with IFN-{alpha} DC membranes (Fig. 4). Cell surface tethering of MMP-9 plays a key role in migration (56, 57, 74, 75) by focusing its enzymatic activity on adjacent substrates. IFN-{alpha} DCs express at least two receptors that bind MMP-9: the CD11b/CD11{alpha}M subunit of the {alpha}Mbeta2 integrin, and CD44, a receptor for hyaluronan. We tested the role of CD11b and CD44 in mediating CCL5-induced IFN-{alpha} DC chemotaxis. CD11b Abs suppressed CCL5-induced IFN-{alpha} DC migration, and Abs directed against the MMP-9-binding site (I domain) on CD11b were more effective (Fig. 8A, left panel). Abs against CD44 also suppressed IFN-{alpha} DC chemotaxis (Fig. 8A, right panel). Consistent with a role for CD11b in mediating MMP-9 regulation of chemotaxis, diminished DC chemotaxis in the absence of Matrigel was observed using CD11b-deficient DCs (Fig. 8B). Concomitant with inhibition of migration (Fig. 8A), CD11b and CD44 Abs suppressed CCL5-induced JNK activation (Fig. 8C, left panel). Diminished chemokine-induced JNK activation was also observed in CD11b-deficient DCs (Fig. 8C, right panel). These results implicate CD11b and CD44 in mediating CCL5-induced chemotaxis in an experimental system lacking ECM ligands for these receptors. The concordant regulation of CCR1/5 function and JNK activation by MMP-9 and CD11b and CD44 suggests that receptor complexes containing MMP-9 and CD11b/CD44 function to costimulate JNK activation by chemokine receptors. However, a role for soluble MMP-9 and for CD11b/CD44 other than binding MMP-9 has not been excluded.


Figure 8
View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 8. Role of CD11b and CD44 in CCL5-induced chemotaxis. A, CCL5-induced chemotaxis of IFN-{alpha} DCs is suppressed by CD11b and CD44 Abs. Isotype control, CD11b, and CD44 Abs (5 µg/ml) were added 30 min before chemotaxis assays. B, CCL5-induced chemotaxis is diminished in CD11b-deficient BM-DCs; n = 3, p < 0.01 as determined by paired Student’s t test. C, Isotype control, CD11b, and CD44 Abs were added to IFN-{alpha} DCs 4 h before stimulation with CCL5 for 5 min (left panel). BM-DC from control and CD11b-deficient mice were treated with CCL19 for indicated periods of time (right panel). Phospho-JNK levels were measured by immunoblot.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
IFN-{alpha} DCs have been implicated in enhancing humoral immunity, cross-priming of CD8+ T cells, and the pathogenesis of SLE. There is great interest in understanding mechanisms by which IFN-{alpha} DCs contribute to SLE pathogenesis and previous work has focused on augmented APC function and promotion of plasma cell differentiation (4). In this study, we found that IFN-{alpha} DCs exhibit enhanced chemotaxis to the inflammatory chemokines CCL3 and CCL5, but do not respond to a variety of other chemokines, including the CCR7 ligand CCL21 that recruits mature DCs to secondary lymphoid tissue. This migratory phenotype suggests a mechanism that allows recruitment of DCs with the high APC function exhibited by IFN-{alpha} DCs to inflammatory sites instead of to lymph nodes or spleen. Such recruited IFN-{alpha} DCs can contribute to inflammation by producing cytokines and presenting (auto)antigens locally, or after further maturation and subsequent migration to draining lymph nodes. CCL5-responsive DCs are present at elevated levels in the peripheral blood in the lupus mouse (26), and their recruitment to inflammatory sites where CCL3 and CCL5 are expressed, such as the kidney, offers a possible explanation for the infiltration of these tissues by DCs (A. Davidson, unpublished observations). The migratory properties of circulating DCs from active lupus patients that express elevated type I IFN levels will be investigated in future work. Enhanced responsiveness to inflammatory chemokines may also modulate DC trafficking during viral infections characterized by high IFN levels.

Another key finding of this study is that MMP-9 regulates chemokine receptor signal transduction and thereby modulates the amplitude of chemotactic responses and DC migration. Costimulation of CCR signaling by a MMP-9-dependent pathway extends our understanding of the regulation of CCR signaling and identifies a new way by which MMP-9 regulates DC migration. MMPs, and proteases in general, have been previously implicated in the regulation of cell migration by mechanisms that involve degradation of ECM, thus facilitating migration through tissues. MMPs also cleave cell surface receptors or their ligands, thereby promoting migration by releasing or activating ligands for receptors that regulate cell motility, or suppressing migration by inactivating chemokines (76). Serine proteases of the thrombin family have been long-appreciated to directly activate receptors, termed protease-activated receptors (PARs), by proteolytic cleavage (77). Similar to chemokine receptors, PARs are seven transmembrane receptors that activate G proteins and signaling pathways important in cell migration. A recent landmark study found that MMP-1 proteolytically activates PAR1 and thereby promotes tumor cell migration (65). This previous report (65) established the paradigm that MMPs can directly activate signal transduction by proteolytic cleavage of cell surface receptors, although such a function has not been described for MMP-9.

Experiments using MMP-9-deficient mice have clearly implicated MMP-9 in the migration of lymphocytes and DCs in vivo, and in modulation of stem cell mobilization, delayed type hypersensitivity, and the course of experimental allergic encephalitis (33, 34, 35, 36, 37, 55). Mechanisms by which MMP-9 promotes migration that have been previously identified include release of cell surface Kit ligand (55), cleavage and activation of latent TGFbeta by cell surface-localized MMP-9 (62), and degradation of ECM. We now provide evidence supporting a new mechanism whereby MMP-9 promotes IFN-{alpha} DC migration by enhancing chemokine receptor signaling. MMP-9 regulated CCR1/5 signaling in a selective manner, as MMP-9 expression and catalytic function were required for CCL5-induced activation of JNK, but not Akt or p38. Thus, JNK appears to be located at a nodal point that integrates signals emanating from chemokine receptors and a MMP-9-dependent pathway (Fig. 9). This is reminiscent of costimulation of T cells, where JNK integrates signals from the T cell Ag receptor and CD28 and mediates synergistic activation of T cells by these receptors (78).


Figure 9
View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 9. Model of costimulation of CCR signaling by MMP-9. MMP-9 cleaves and activates a receptor or ligand required for effective JNK activation. Activation of CCRs and the MMP-9-dependent pathway synergistically activate JNK, thereby augmenting chemotaxis. CD11b and CD44 can potentially trigger signaling pathways leading to activation of JNK independently of binding MMP-9.

 
MMP-9 is tethered to the cell surface by its receptors CD11b and CD44, and there is accumulating evidence that binding of proteases to cell surface receptors and incorporation into multireceptor complexes regulates protease function (56, 57, 76). Localization of MMP-9 to the cell surface allows directed focusing of MMP-9 activity on ECM degradation and is required for MMP-9-dependent cleavage of TGF-beta. CCL5-induced chemotaxis was suppressed by Abs against CD11b and CD44, including an Ab that binds to the MMP-9-binding site on CD11b (Fig. 8A). These results support a model whereby binding of MMP-9 to the surface of IFN-{alpha} DCs promotes its activation and/or facilitates cleavage of a substrate that is important for costimulating the JNK pathway (Fig. 9). It is possible that soluble MMP-9, which is present in greater concentrations in culture supernatants, contributes to activation of this pathway (Fig. 9). The most obvious substrate for MMP-9 would be either CCR1/5 or their ligands, but, consistent with the literature (32), we have not detected cleavage of these molecules in IFN-{alpha} DC supernatants or extracts, or by active MMP-9 in vitro (Y. Hu, unpublished data). Future work will determine whether cell MMP-9 costimulates CCR signaling by activating a receptor ligand (similar to TGFbeta) (62) or a signaling receptor (similar to activation of PAR1 by MMP-1) (65).

Overall, the data lead us to propose a model for MMP-9 regulation of CCR1/5 signaling that is illustrated in Fig. 9. In this model, cleavage of an MMP-9 substrate generates a signal leading to the activation of JNK. The interaction of MMP-9 with the relevant substrate can be facilitated by binding of MMP-9 to its cell surface receptors CD11b and CD44. CD11b and CD44 can also potentially signal independently of MMP-9 (Fig. 9, dotted line), but there are no known ligands for these receptors, other than MMP-9, in our experimental system. This MMP-9-mediated signal is insufficient to strongly activate JNK, but is required for synergistic activation of JNK together with chemokine receptors. This MMP-9-mediated signal may also contribute to basal JNK activity (Y. Hu, unpublished observation). A chemokine receptor signal alone is also insufficient to strongly activate JNK (and potentially other, as yet unidentified, pathways important for migration) and thus only relatively weakly activates migration. Simultaneous activation of chemokine receptors and the MMP-9-dependent pathway leads to strong activation of JNK together with activation of other chemokine receptor-dependent pathways important for migration (such as Akt) and results in enhanced migration. A recent report has described a similar function for the serine protease cathepsin G, which binds to neutrophils via an as yet unidentified receptor and regulates the signaling and function of integrins without directly cleaving the integrins or their ligands (79). Thus, proteases exhibit the unexpected role of regulating signal transduction by receptors important in cell migration.

Chemokine receptor signaling is subject to complex negative regulation that limits receptor function and thus fine tunes cell migration and controls localization of cells in response to chemokine gradients. Well-established mechanisms of negative regulation include down-modulation of cell surface chemokine receptor expression, desensitization mediated by phosphorylation, and induction of regulator of G protein signaling proteins (80). More recently, it has been demonstrated that chemokine receptors can be uncoupled from chemotactic responses by treatment with LPS + IL-10 (50) and that chemokine receptor signaling is negatively regulated by the Src family kinase Hck and Fgr via PIR-B inhibitory receptors and Src homology region 2 domain-containing phosphatase 1 (81). Understanding of the positive regulation of chemokine receptor signaling is less developed and is limited to induction of chemokine receptor expression by cytokines and amplification of chemokine receptor calcium signaling by CD38-dependent generation of cyclic adenosine diphosphate ribose (82). We have discovered that CCR1/5 signaling is also augmented by MMP-9 by a mechanism that involves costimulation of CCR-dependent signal transduction. The dependence of chemokine receptors on a costimulatory signal helps explain the well-known dissociation between chemokine receptor expression and function (83), and suggests that chemokine receptor function is subject to broader and more complex regulation than previously appreciated. For example, CCR signaling and function will be modulated by stimuli that regulate the expression and activation of MMP-9, CD11b, and CD44.

In summary, our results demonstrate costimulation of CCR1/5 signaling and downstream chemotaxis by MMP-9, CD11b, and CD44, molecules whose established functions are to mediate attachment to and focal degradation of ECM during cell migration. Signals from CCRs and adhesion receptors/MMP-9 are integrated at the level of JNK activation to determine the amplitude of the chemotactic response. Chemokine receptors and adhesion receptors/MMP-9 have been previously appreciated to control, respectively, motility and interactions with ECM. This study supports a model whereby adhesion receptors and MMP-9 also regulate the amplitude of DC responses to chemokines and thereby regulate cell motility in addition to mediating interactions with ECM.


   Acknowledgments
 
We thank Drs. C. Blobel, X. Hu, and T. Lu for critical review of the manuscript. We thank Dr. William Muller for his suggestion that MMP-9 may function in cell migration independent of cleavage of ECM. We also thank Dr. Joel Pardee for his continuing support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 work was supported by grants from the National Institutes of Health (NIH) (to L.B.I.) and was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06-RR12538-01 from the National Center for Research Resources, NIH. Back

2 Address correspondence and reprint requests to Dr. Lionel B. Ivashkiv at the current address: Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021. E-mail address: IvashkivL{at}HSS.edu Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; DC, dendritic cell; MMP-9, matrix metalloproteinase-9; ECM, extracellular matrix; PTX, pertussis toxin; BM-DC, bone marrow-derived DC; PAR, protease-activated receptor; rm, recombinant murine; TAPI, INF-{alpha} protease inhibitor. Back

Received for publication September 8, 2005. Accepted for publication March 2, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Stark, G. R., I. M. Kerr, B. R. Williams, R. H. Silverman, R. D. Schreiber. 1998. How cells respond to interferons. Annu. Rev. Biochem. 67: 227-264. [Medline]
  2. Biron, C. A.. 2001. Interferons {alpha} and beta as immune regulators: a new look. Immunity 14: 661-664. [Medline]
  3. Theofilopoulos, A. N., R. Baccala, B. Beutler, D. H. Kono. 2005. Type I interferons ({alpha}/beta) in immunity and autoimmunity. Annu. Rev. Immunol. 23: 307-336. [Medline]
  4. Banchereau, J., V. Pascual, A. K. Palucka. 2004. Autoimmunity through cytokine-induced dendritic cell activation. Immunity 20: 539-550. [Medline]
  5. Gallucci, S., M. Lolkema, P. Matzinger. 1999. Natural adjuvants: endogenous activators of dendritic cells. Nat. Med. 5: 1249-1255. [Medline]
  6. Le Bon, A., G. Schiavoni, G. D’Agostino, I. Gresser, F. Belardelli, D. F. Tough. 2001. Type I interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo. Immunity 14: 461-470. [Medline]
  7. Le Bon, A., N. Etchart, C. Rossmann, M. Ashton, S. Hou, D. Gewert, P. Borrow, D. F. Tough. 2003. Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon. Nat. Immunol. 4: 1009-1015. [Medline]
  8. Asselin-Paturel, C., G. Brizard, K. Chemin, A. Boonstra, A. O’Garra, A. Vicari, G. Trinchieri. 2005. Type I interferon dependence of plasmacytoid dendritic cell activation and migration. J. Exp. Med. 201: 1157-1167. [Abstract/Free Full Text]
  9. Santiago-Raber, M. L., R. Baccala, K. M. Haraldsson, D. Choubey, T. A. Stewart, D. H. Kono, A. N. Theofilopoulos. 2003. Type-I interferon receptor deficiency reduces lupus-like disease in NZB mice. J. Exp. Med. 197: 777-788. [Abstract/Free Full Text]
  10. Blanco, P., A. K. Palucka, M. Gill, V. Pascual, J. Banchereau. 2001. Induction of dendritic cell differentiation by IFN-{alpha} in systemic lupus erythematosus. Science 294: 1540-1543. [Abstract/Free Full Text]
  11. Paquette, R. L., N. C. Hsu, S. M. Kiertscher, A. N. Park, L. Tran, M. D. Roth, J. A. Glaspy. 1998. Interferon-{alpha} and granulocyte-macrophage colony-stimulating factor differentiate peripheral blood monocytes into potent antigen-presenting cells. J. Leukocyte Biol. 64: 358-367. [Abstract]
  12. Santini, S. M., C. Lapenta, M. Logozzi, S. Parlato, M. Spada, T. Di Pucchio, F. Belardelli. 2000. Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice. J. Exp. Med. 191: 1777-1788. [Abstract/Free Full Text]
  13. Della Bella, S., S. Nicola, A. Riva, M. Biasin, M. Clerici, M. L. Villa. 2004. Functional repertoire of dendritic cells generated in granulocyte macrophage-colony stimulating factor and interferon-{alpha}. J. Leukocyte Biol. 75: 106-116. [Abstract/Free Full Text]
  14. Santodonato, L., G. D’Agostino, R. Nisini, S. Mariotti, D. M. Monque, M. Spada, L. Lattanzi, M. P. Perrone, M. Andreotti, F. Belardelli, M. Ferrantini. 2003. Monocyte-derived dendritic cells generated after a short-term culture with IFN-{alpha} and granulocyte-macrophage colony-stimulating factor stimulate a potent Epstein-Barr virus-specific CD8+ T cell response. J. Immunol. 170: 5195-5202. [Abstract/Free Full Text]
  15. Mohty, M., A. Vialle-Castellano, J. A. Nunes, D. Isnardon, D. Olive, B. Gaugler. 2003. IFN-{alpha} skews monocyte differentiation into Toll-like receptor 7-expressing dendritic cells with potent functional activities. J. Immunol. 171: 3385-3393. [Abstract/Free Full Text]
  16. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245-252. [Medline]
  17. Robert, C., R. C. Fuhlbrigge, J. D. Kieffer, S. Ayehunie, R. O. Hynes, G. Cheng, S. Grabbe, U. H. von Andrian, T. S. Kupper. 1999. Interaction of dendritic cells with skin endothelium: a new perspective on immunosurveillance. J. Exp. Med. 189: 627-636. [Abstract/Free Full Text]
  18. Sallusto, F., A. Lanzavecchia. 1999. Mobilizing dendritic cells for tolerance, priming, and chronic inflammation. J. Exp. Med. 189: 611-614. [Free Full Text]
  19. Pendl, G. G., C. Robert, M. Steinert, R. Thanos, R. Eytner, E. Borges, M. K. Wild, J. B. Lowe, R. C. Fuhlbrigge, T. S. Kupper, et al 2002. Immature mouse dendritic cells enter inflamed tissue, a process that requires E- and P-selectin, but not P-selectin glycoprotein ligand 1. Blood 99: 946-956. [Abstract/Free Full Text]
  20. Dieu, M. C., B. Vanbervliet, A. Vicari, J. M. Bridon, E. Oldham, S. Ait-Yahia, F. Briere, A. Zlotnik, S. Lebecque, C. Caux. 1998. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J. Exp. Med. 188: 373-386. [Abstract/Free Full Text]
  21. McWilliam, A. S., D. Nelson, J. A. Thomas, P. G. Holt. 1994. Rapid dendritic cell recruitment is a hallmark of the acute inflammatory response at mucosal surfaces. J. Exp. Med. 179: 1331-1336. [Abstract/Free Full Text]
  22. McWilliam, A. S., S. Napoli, A. M. Marsh, F. L. Pemper, D. J. Nelson, C. L. Pimm, P. A. Stumbles, T. N. Wells, P. G. Holt. 1996. Dendritic cells are recruited into the airway epithelium during the inflammatory response to a broad spectrum of stimuli. J. Exp. Med. 184: 2429-2432. [Abstract/Free Full Text]
  23. Randolph, G. J., K. Inaba, D. F. Robbiani, R. M. Steinman, W. A. Muller. 1999. Differentiation of phagocytic monocytes into lymph node dendritic cells in vivo. Immunity 11: 753-761. [Medline]
  24. Sozzani, S., P. Allavena, A. Vecchi, A. Mantovani. 1999. The role of chemokines in the regulation of dendritic cell trafficking. J. Leukocyte Biol. 66: 1-9. [Abstract]
  25. Stumbles, P. A., D. H. Strickland, C. L. Pimm, S. F. Proksch, A. M. Marsh, A. S. McWilliam, A. Bosco, I. Tobagus, J. A. Thomas, S. Napoli, et al 2001. Regulation of dendritic cell recruitment into resting and inflamed airway epithelium: use of alternative chemokine receptors as a function of inducing stimulus. J. Immunol. 167: 228-234. [Abstract/Free Full Text]
  26. Ishikawa, S., S. Nagai, T. Sato, K. Akadegawa, H. Yoneyama, Y. Y. Zhang, N. Onai, K. Matsushima. 2002. Increased circulating CD11b+CD11c+ dendritic cells (DC) in aged BWF1 mice which can be matured by TNF-{alpha} into BLC/CXCL13-producing DC. Eur. J. Immunol. 32: 1881-1887. [Medline]
  27. Adachi, Y., S. Taketani, J. Toki, K. Ikebukuro, K. Sugiura, H. Oyaizu, R. Yasumizu, M. Tomita, H. Kaneda, Y. Amoh, et al 2002. Marked increase in number of dendritic cells in autoimmune-prone (NZW x BXSB)F1 mice with age. Stem Cells 20: 61-72. [Abstract/Free Full Text]
  28. Thomas, R., L. S. Davis, P. E. Lipsky. 1994. Rheumatoid synovium is enriched in mature antigen-presenting dendritic cells. J. Immunol. 152: 2613-2623. [Abstract]
  29. Mellado, M., J. M. Rodriguez-Frade, S. Manes, A. C. Martinez. 2001. Chemokine signaling and functional responses: the role of receptor dimerization and TK pathway activation. Annu. Rev. Immunol. 19: 397-421. [Medline]
  30. Wong, M. M., E. N. Fish. 2003. Chemokines: attractive mediators of the immune response. Semin. Immunol. 15: 5-14. [Medline]
  31. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, B. Pulendran, K. Palucka. 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18: 767-811. [Medline]
  32. Opdenakker, G., P. E. Van den Steen, J. Van Damme. 2001. Gelatinase B: a tuner and amplifier of immune functions. Trends Immunol. 22: 571-579. [Medline]
  33. Kouwenhoven, M., V. Ozenci, A. Tjernlund, M. Pashenkov, M. Homman, R. Press, H. Link. 2002. Monocyte-derived dendritic cells express and secrete matrix-degrading metalloproteinases and their inhibitors and are imbalanced in multiple sclerosis. J. Neuroimmunol. 126: 161-171. [Medline]
  34. Kobayashi, Y., M. Matsumoto, M. Kotani, T. Makino. 1999. Possible involvement of matrix metalloproteinase-9 in Langerhans cell migration and maturation. J. Immunol. 163: 5989-5993. [Abstract/Free Full Text]
  35. Ratzinger, G., P. Stoitzner, S. Ebner, M. B. Lutz, G. T. Layton, C. Rainer, R. M. Senior, J. M. Shipley, P. Fritsch, G. Schuler, N. Romani. 2002. Matrix metalloproteinases 9 and 2 are necessary for the migration of Langerhans cells and dermal dendritic cells from human and murine skin. J. Immunol. 168: 4361-4371. [Abstract/Free Full Text]
  36. Vermaelen, K. Y., D. Cataldo, K. Tournoy, T. Maes, A. Dhulst, R. Louis, J. M. Foidart, A. Noel, R. Pauwels. 2003. Matrix metalloproteinase-9-mediated dendritic cell recruitment into the airways is a critical step in a mouse model of asthma. J. Immunol. 171: 1016-1022. [Abstract/Free Full Text]
  37. Ichiyasu, H., J. M. McCormack, K. M. McCarthy, D. Dombkowski, F. I. Preffer, E. E. Schneeberger. 2004. Matrix metalloproteinase-9-deficient dendritic cells have impaired migration through tracheal epithelial tight junctions. Am. J. Respir. Cell Mol. Biol. 30: 761-770. [Abstract/Free Full Text]
  38. Faber-Elmann, A., Z. Sthoeger, A. Tcherniack, M. Dayan, E. Mozes. 2002. Activity of matrix metalloproteinase-9 is elevated in sera of patients with systemic lupus erythematosus. Clin. Exp. Immunol. 127: 393-398. [Medline]
  39. Ainiala, H., A. Hietaharju, P. Dastidar, J. Loukkola, T. Lehtimaki, J. Peltola, M. Korpela, T. Heinonen, S. T. Nikkari. 2004. Increased serum matrix metalloproteinase 9 levels in systemic lupus erythematosus patients with neuropsychiatric manifestations and brain magnetic resonance imaging abnormalities. Arthritis Rheum. 50: 858-865. [Medline]
  40. Jovanovic, D. V., J. Martel-Pelletier, J. A. Di Battista, F. Mineau, F. C. Jolicoeur, M. Benderdour, J. P. Pelletier. 2000. Stimulation of 92-kd gelatinase (matrix metalloproteinase 9) production by interleukin-17 in human monocyte/macrophages: a possible role in rheumatoid arthritis. Arthritis Rheum. 43: 1134-1144. [Medline]
  41. Ahrens, D., A. E. Koch, R. M. Pope, M. Stein-Picarella, M. J. Niedbala. 1996. Expression of matrix metalloproteinase 9 (96-kd gelatinase B) in human rheumatoid arthritis. Arthritis Rheum. 39: 1576-1587. [Medline]
  42. Granelli-Piperno, A., A. Golebiowska, C. Trumpfheller, F. P. Siegal, R. M. Steinman. 2004. HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation. Proc. Natl. Acad. Sci. USA 101: 7669-7674. [Abstract/Free Full Text]
  43. Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rossner, F. Koch, N. Romani, G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223: 77-92. [Medline]
  44. Lee, I. H., W. P. Li, K. B. Hisert, L. B. Ivashkiv. 1999. Inhibition of interleukin 2 signaling and signal transducer and activator of transcription (STAT)5 activation during T cell receptor-mediated feedback inhibition of T cell expansion. J. Exp. Med. 190: 1263-1274. [Abstract/Free Full Text]
  45. Sengupta, T. K., A. Chen, Z. Zhong, J. E. Darnell, Jr, L. B. Ivashkiv. 1995. Activation of monocyte effector genes and STAT family transcription factors by inflammatory synovial fluid is independent of interferon {gamma}. J. Exp. Med. 181: 1015-1025. [Abstract/Free Full Text]
  46. Hu, X., K. H. Park-Min, H. H. Ho, L. B. Ivashkiv. 2005. IFN-{gamma}-primed macrophages exhibit increased CCR2-dependent migration and altered IFN-{gamma} responses mediated by Stat1. J. Immunol. 175: 3637-3647. [Abstract/Free Full Text]
  47. Hu, X., C. Herrero, W. P. Li, T. T. Antoniv, E. Falck-Pedersen, A. E. Koch, J. M. Woods, G. K. Haines, L. B. Ivashkiv. 2002. Sensitization of IFN-{gamma} Jak-STAT signaling during macrophage activation. Nat. Immunol. 3: 859-866. [Medline]
  48. Penna, G., S. Sozzani, L. Ador