Costimulation of Chemokine Receptor Signaling by Matrix
Metalloproteinase-9 Mediates Enhanced Migration of IFN-?
Yang Hu* and Lionel B. Ivashkiv2†‡
Type I IFNs induce differentiation of dendritic cells (DCs) with potent Ag-presenting capacity, termed IFN-? DCs, that have been
implicated in the pathogenesis of systemic lupus erythematosus. In this study, we found that IFN-? DCs exhibit enhanced mi-
gration 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-? 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-? 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. The Journal of Immunology, 2006,
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 produc-
tion, and immunological memory (3). Type I IFNs have been im-
plicated in the development of autoimmunity and in the pathogen-
esis 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 produc-
tion. 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-? DCs. IFN-? DCs contribute to normal immune responses
and to mouse lupus (5–9), have been observed in human SLE (10),
and exhibit enhanced Ag uptake, cytokine production, and an in-
ype I IFNs (IFN-?/IFN-?/IFN-?) are pleiotropic cyto-
kines with antiviral and immunoregulatory properties (1,
2). Type I IFNs augment innate immune responses by
creased capacity to activate T cells (4, 10–15). 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 cy-
tokine 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 tis-
sues 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 re-
sponse to the chemokines CCL19 and CCL21 (16). In addition to
migration to lymph nodes, immature DCs and DC precursors cir-
culate in the blood and can be recruited to the spleen and periph-
eral tissues (17–23). This recruitment into peripheral tissues main-
tains DC homeostasis, and influx of DCs/DC precursors maintains
DC numbers at inflammatory sites, where DCs can produce cyto-
kines, 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?), CCL4 (MIP-
1?), 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 cap-
ture (auto)antigens, contributes to lupus nephritis (27). Inflamma-
tory DCs present in the joints of patients with rheumatoid arthritis
are also thought to arise at least in part from precursors that mi-
grate into the joint synovium (28).
Migratory cells like DCs move along concentration gradients of
chemokines that stimulate cells via seven-transmembrane, G pro-
tein-coupled chemokine receptors. The pertussis toxin (PTX)-sen-
sitive G?iprotein that regulates calcium influx is a key mediator of
*Graduate Program in Neuroscience, Weill Graduate School of Medical Sciences,
Cornell University, New York, NY 10021;†Arthritis and Tissue Degeneration Pro-
gram, Hospital for Special Surgery, New York, NY 10021; and‡Graduate Program in
Immunology and Microbial Pathogenesis, Weill Graduate School of Medical Sci-
ences, Cornell University, New York, NY 10021
Received for publication September 8, 2005. Accepted for publication March 2, 2006.
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.
1This 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 Fa-
cilities Improvement Program Grant Number C06-RR12538-01 from the National
Center for Research Resources, NIH.
2Address 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@HSS.edu
3Abbreviations 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, re-
combinant murine; TAPI, INF-? protease inhibitor.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc.0022-1767/06/$02.00
chemokine receptor function, and several signaling molecules ac-
tivated downstream of chemokine receptors, including Pyk2,
PI3K?, 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 mem-
branes and through tissues (31). Interaction of DCs with ECM is
mediated by two classes of molecules, adhesion receptors and pro-
teases. Adhesion receptors, such as integrins and the hyaluronan
receptor CD44, mediate direct contact of cells with ECM compo-
nents 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 com-
ponents. In addition, proteases can promote migration by activa-
tion of enzymatic cascades that effectively degrade ECM, and can
regulate chemotaxis by cleaving chemokines (32). Matrix metal-
loproteinase-9 (MMP-9) has been shown to play a key role in DC
migration in vivo and in migration through Matrigel, a model base-
ment membrane, in vitro (33–37).
We were interested in defining mechanisms by which IFN-?
could potentiate DC function and thereby contribute to enhanced
immune responses and potentially to autoimmunity. In this study,
we found that IFN-? DCs exhibit enhanced chemotaxis, and that
enhanced migration of IFN-? DCs was mediated by MMP-9. Sur-
prisingly, MMP-9 enhanced DC chemotaxis even in the absence of
ECM by a mechanism that involved regulation of chemokine re-
ceptor signaling and function. These results identify a new mech-
anism for regulating DC migration that may be operative in im-
mune responses characterized by high type I IFN or MMP-9
expression, including the human autoimmune diseases SLE and
rheumatoid arthritis (38–41).
Materials and Methods
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 metalloprotein-
ase-1 (TIMP-1), PTX, AKT inhibitor IV, SP600125, PD98059, SB203580
were obtained from Calbiochem. TNF? 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. ?-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-?A
(500 U/ml; BioSource International) to generate control DCs and IFN-?
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 Com-
mittee. BM-derived DCs were cultured as described (43) with minor mod-
ifications. 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-?m pore size; BD Bio-
sciences) after vigorous pipetting and cultured in RPMI 1640 medium sup-
plemented with 100 U/ml penicillin and streptomycin, 2 mM L-glutamine,
50 ?M 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 nonad-
herent 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-?m pore size; Corning Costar). The
upper side of filters was coated with growth factor-reduced Matrigel (BD
Biosciences) diluted in HBSS (50 ?g/filter). DCs (0.5 ? 106in 100 ?l of
serum-free medium) were added to the upper chamber of the insert. The
lower chamber contained 500 ?l of serum-free medium with or without
chemokine. The plates were incubated at 37°C in 5% CO2for 4 h and cells
that had migrated into the lower chamber were counted using flow cytom-
etry with a fluorescent counting bead internal standard in each tube (Bangs
Laboratories); 10,000 singlet beads were acquired in each sample.
Total cell extracts were obtained as described (44). Cell extracts corre-
sponding to 3.3 ? 105cells were fractionated by 10% SDS-PAGE gel,
transferred to polyvinylidene fluoride membranes (Millipore), and incu-
bated 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 Ex-
traction kit (Calbiochem). Supernatant and membrane extracts were con-
centrated using Microcon centrifugal filters (Millipore) and proteins de-
rived from 2 ? 106cells 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 devel-
oping buffer overnight at 37°C. The gels were then stained with 0.5%
Coomassie blue and destained before imaging. MMP-9 levels were mea-
sured 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 ?M 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 rest-
ing 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 per-
formed 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 ?g 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). Rel-
ative expression was normalized relative to levels of GAPDH or ?-actin.
The generation of only the correct size amplification products was con-
firmed using agarose gel electrophoresis. Oligonucleotide primers were as
follows: GAPDH, 5?-GTGAAGGTCGGAGTCAAC-3? and 5?-TGGAAT
TTGCCATGGGTG-3?; ?-actin, 5?-GGATGCAGAAGGAGATCACTG-3?
and 5?-CGATCCACACGGAGTACTTG-3?; CCR1, 5?-GTGCCAGAAGGT
6023The Journal of Immunology
GAACGAGAGG-3? and 5?-TCCAACCAGGCCAATGACAAATAC-3?;
CCR5, 5?-TTCTCTTCTGGGCTCCCTACAACA-3? and 5?-CAGCAGT
GCGTCATCCCAAGAG-3?. MMP-9, 5?-TGCCCGGACCAAGGATACA
GT-3? and 5?-AGGCCGTGGCTCAGGTTCAGG-3?; CCR7 5?-TCCCACA
GACTCAAATGCTC-3? and 5?-TTCCTCACCAAGCCAAGAAG-3?.
Increased migration of IFN-? DCs mediated by CCR5 and
We wished to investigate the effects of IFN-? on the phenotype of
human myeloid DCs. Similar to the approaches taken in previous
reports (11–15), IFN-? DCs were generated from human CD14?
monocytes by culturing cells for 5 days with IFN-? and GM-CSF.
Control monocyte-derived DCs were obtained using the well-es-
tablished 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 mat-
uration after addition of LPS (Fig. 1, top panels, and Table I).
Consistent with previous reports (11–15), IFN-? 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-? DCs in the absence of any additional matu-
ration stimuli likely explains the enhanced Ag-presenting capacity
of these cells, as previously reported (10–15) and confirmed in our
laboratory (data not shown).
We then investigated whether IFN-? enhanced additional DC
functions that could contribute to immune responses. IFN-? 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-? on DC migratory ability was then
assessed using a Transwell system in which the membrane sepa-
rating 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 experimen-
tal 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 che-
mokine 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-? 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-? DCs to CCL5 was reproduced with DCs from 12 differ-
ent blood donors and was statistically significant (p ? 0.001,
paired Student t test). The additional CCR1/CCR5 ligand CCL3/
MIP-1? also induced IFN-? 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 pre-
vious reports (24, 49). The lack of chemotactic response of IFN-?
DCs to CCL21 could be explained by low CCR7 expression, com-
parable to that expressed by immature control DCs (Fig. 2C).
CCR7 expression increased comparably on control and IFN-? DCs
after maturation with LPS (Fig. 2C), and LPS-matured control and
IFN-? DCs had a robust chemotactic response to CCL21 (data not
shown). Low CCR7 expression by IFN-? DCs before further mat-
uration with LPS suggests that IFN-? DCs have different migra-
tory properties compared with DCs matured with activating stimuli
such as TLR ligands. Overall, the results demonstrate enhanced
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.
Cell surface phenotype of human monocyte-derived control DCs and IFN-? DCs. DCs were generated as described in Materials and
Table I. DC surface markers
MFI% PositiveMFI% Positive MFI % PositiveMFI% Positive MFI % PositiveMFI% Positive
Control DCNo LPS
6024MMP-9 REGULATES CHEMOKINE RECEPTOR SIGNALING IN DCs
migration of IFN-? DCs in response to inflammatory chemokines
previously shown to recruit DCs to inflammatory sites.
We then investigated whether increased migration of IFN-?
DCs in response to CCR1/CCR3/CCR5 ligands could be ex-
plained, at least in part, by increased expression of these receptors.
IFN-? 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-? 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 sug-
gested that chemotaxis of IFN-? 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-?
DCs. We blocked CCR5 function using a CCR5 Ab and desensi-
tized CCR1 responses by using the CCR1-specific ligand CCL23/
MIP-3 to down-regulate CCR1, but not CCR5, cell surface ex-
pression (Fig. 3C). The CCR5-blocking Ab suppressed CCL5-
induced IFN-? 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-? DCs (Fig.
3D). Overall, these results demonstrate a role for CCR5 and CCR1
in mediating CCL5-induced migration of IFN-? DCs. Modestly
increased expression of these receptors may contribute to en-
hanced chemotaxis, but is unlikely to explain the dramatically in-
creased chemotaxis of IFN-? DCs. In addition, the results reveal
that the IFN-? 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 en-
hanced recruitment of highly functional DCs into inflammatory
MMP-9 expression is elevated in IFN-? DCs and promotes
To identify additional factors important for IFN-? DC migration
through ECM, we used microarray analysis. These experiments
confirmed increased expression of multiple canonical IFN-induc-
ible genes in IFN-? DCs relative to control DCs, including genes
whose expression was also elevated in blood cells of SLE patients
(51–53) (data not shown). A small number of genes involved in
cell migration were expressed at elevated levels in IFN-? 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 in-
vasion of tissues by tumor cells (32–37, 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 sur-
face 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-? DCs.
First, we confirmed the dramatic increase in MMP-9 expression
in IFN-? DCs in additional blood donors using real-time quanti-
tative 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-? 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-? 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 re-
vealed pro-MMP-9 and active MMP-9 enzymatic activity in mem-
branes of IFN-? 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-? 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-? DCs.
Next, we determined the effects of blocking MMP-9 function on
IFN-? DC migration using three different approaches. A neutral-
izing Ab specific for MMP-9 nearly completely suppressed CCL5-
induced IFN-? DC migration through Matrigel (Fig. 4D). In ad-
dition, migration also blocked by TIMP-1, a protein that binds to
and inactivates the catalytic function of several MMPs including
were added to the upper chambers of Transwell inserts that were coated
with Matrigel and cells that migrated into the lower chamber were enu-
merated 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-? 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-? DCs to various chemo-
kines. A representative experiment of three is shown. C, CCR7 expression
was measured using real-time PCR and normalized relative to GAPDH.
Increased migration of IFN-? DCs across Matrigel. DCs
6025The Journal of Immunology
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 ex-
pression of MMP-9 in IFN-? 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 (32–37, 58). The
results, taken together, support a role for increased expression and
proteolytic activity of MMP-9 in the enhanced migration of
MMP-9 increases DC chemotaxis even in the absence of ECM
We wished to investigate the mechanism by which MMP-9 in-
creases IFN-? 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
Table II. Examples of genes important for cell migration that are up-regulated in IFN-? DC
Gene Name Control DCIFN-? DC
Matrix metalloproteinase 9 (gelatinase B)
Ephrin receptor EphB2
Complement component 3a receptor 1
Lectin-like oxidized-LDL receptor 1;OLR1
Tyrosine kinase, receptor Axl, Alt. splice 2
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-? DC cell surface
CCR1 by CCL23. D, CCL5-induced migration of IFN-? 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
CCL5-induced IFN-? DC migration is mediated by CCR1 and CCR5. A, mRNA levels of CCR1 and CCR5 in control DC and IFN-? DC
6026MMP-9 REGULATES CHEMOKINE RECEPTOR SIGNALING IN DCs
same Transwell system, but in the absence of Matrigel. Similar to
migration across Matrigel (Fig. 2), chemotaxis of IFN-? 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-? DC chemotaxis even in the absence of an ECM barrier (Fig.
5B, left panel). In addition, basal motility of IFN-? 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-? 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-? 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.
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 proteo-
lytic cleavage (59–65). However, cleavage products of CCL5 or
CCR5 were not detected in IFN-? DCs by immunoblotting anal-
ysis, 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?i. These receptors activate a rapid and transient calcium flux and
downstream signaling molecules, including PI3K? and Akt,
MAPKs (ERKs, JNK, p38), and Pyk2; some of these molecules
can also be activated by parallel chemokine receptor-induced sig-
naling pathways not dependent on G?i(29, 30, 66). PI3K?, JNK,
and p38 are key mediators of migratory responses to chemokine
stimulation, although their targets and mechanisms of action have
not been fully clarified (67–73). 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 cal-
cium in IFN-? 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 com-
pletely inhibited by PTX, an inhibitor of G?i. These results indicate
IFN-? 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 ?g/ml), TIMP-1 (5 nM), and TAPI-1 (100 ?M) 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.
MMP-9 expression is elevated in IFN-? DC and is required for migration through Matrigel. A, MMP-9 mRNA levels in control DC and
6027 The Journal of Immunology
that MMP-9 does not regulate G?i-dependent calcium signaling
downstream of CCR1/5.
Chemokine receptors can also activate signaling pathways in-
dependently of G?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-? DCs (Fig. 7A). We then used specific inhibitors
to determine which of these kinases were important in IFN-? DC
chemotactic responses to CCL5. Inhibition of Akt and JNK, but
not MEK-ERK or p38, strongly suppressed CCL5-induced migra-
tion of IFN-? DCs (Fig. 7B, left panel). Consistent with the key
role of endogenous CCL5 in basal IFN-? DC motility (Fig. 5B),
inhibition of Akt and JNK also suppressed basal IFN-? DC mo-
tility (Fig. 7B, right panel). Interestingly, inhibition of JNK sup-
pressed migration as potently as did inhibition of G?iusing PTX
(Fig. 7B), indicating a key nonredundant role for JNK in IFN-?
We then assessed the effects of blocking MMP-9 on CCL5-
induced signaling in IFN-? 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?iprotein using
PTX suppressed CCL5-induced activation of JNK, p38, ERK, and
Akt (Fig. 7C and data not shown). The role of MMP-9 in modu-
lating 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?isignaling but not on MMP-9; and 2) CCL5 ac-
tivation of JNK is dependent on both G?iand MMP-9. Thus,
MMP-9 selectively regulates activation of the JNK-signaling path-
way 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-? 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-? DCs express at
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 ?g/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 ?g/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 is required for CCL5-induced chemotaxis of IFN-? DCs even in the absence of ECM. A, The migration of control DC and IFN-?
6028 MMP-9 REGULATES CHEMOKINE RECEPTOR SIGNALING IN DCs
least two receptors that bind MMP-9: the CD11b/CD11?Msubunit
of the ?M?2integrin, and CD44, a receptor for hyaluronan. We
tested the role of CD11b and CD44 in mediating CCL5-induced
IFN-? DC chemotaxis. CD11b Abs suppressed CCL5-induced
IFN-? DC migration, and Abs directed against the MMP-9-bind-
ing site (I domain) on CD11b were more effective (Fig. 8A, left
was measured by immunoblot. B, CCL5-induced chemotaxis and basal motility of IFN-? DCs are dependent on AKT and JNK. Vehicle control (DMSO; 0.1%)
or inhibitors of G?i(PTX; 200 ng/ml), AKT (AKT inhibitor IV; 1 ?M), JNK (SP600125; 30 ?M), MEK-ERK (PD98059; 50 ?M), and p38 (SB203580; 20 ?M)
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-? DCs were treated
with isotype control or MMP-9 Abs (5 ?g/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.
MMP-9 regulates CCR5 signaling and function by costimulating JNK activation. A, CCL5-induced activation of AKT and MAPKs in IFN-? DCs
and intracellular calcium levels were measured using flow cytometry. Isotype control or MMP-9 Abs (10 ?g/ml), TIMP-1 (5 nM), and PTX (200 ng/ml)
were added before CCL5.
MMP-9 does not regulate G?i-dependent calcium flux. Control or IFN-? DCs were loaded with Fluo-3, stimulated with CCL5 (100 ng/ml)
6029 The Journal of Immunology
panel). Abs against CD44 also suppressed IFN-? DC chemotaxis
(Fig. 8A, right panel). Consistent with a role for CD11b in medi-
ating MMP-9 regulation of chemotaxis, diminished DC chemo-
taxis in the absence of Matrigel was observed using CD11b-defi-
cient 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 me-
diating CCL5-induced chemotaxis in an experimental system lack-
ing 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 chemo-
kine receptors. However, a role for soluble MMP-9 and for
CD11b/CD44 other than binding MMP-9 has not been excluded.
IFN-? 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-? 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-? DCs exhibit
enhanced chemotaxis to the inflammatory chemokines CCL3 and
CCL5, but do not respond to a variety of other chemokines, in-
cluding the CCR7 ligand CCL21 that recruits mature DCs to sec-
ondary lymphoid tissue. This migratory phenotype suggests a
mechanism that allows recruitment of DCs with the high APC
function exhibited by IFN-? DCs to inflammatory sites instead of
to lymph nodes or spleen. Such recruited IFN-? DCs can contrib-
ute to inflammation by producing cytokines and presenting (au-
to)antigens locally, or after further maturation and subsequent mi-
gration 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 pos-
sible 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
Another key finding of this study is that MMP-9 regulates che-
mokine receptor signal transduction and thereby modulates the
amplitude of chemotactic responses and DC migration. Costimu-
lation of CCR signaling by a MMP-9-dependent pathway extends
our understanding of the regulation of CCR signaling and identi-
fies 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 degrada-
tion of ECM, thus facilitating migration through tissues. MMPs
also cleave cell surface receptors or their ligands, thereby promot-
ing 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 pro-
tease-activated receptors (PARs), by proteolytic cleavage (77).
Similar to chemokine receptors, PARs are seven transmembrane
receptors that activate G proteins and signaling pathways impor-
tant 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 impli-
cated MMP-9 in the migration of lymphocytes and DCs in vivo,
Abs. Isotype control, CD11b, and CD44 Abs (5 ?g/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-? 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.
Role of CD11b and CD44 in CCL5-induced chemotaxis. A, CCL5-induced chemotaxis of IFN-? DCs is suppressed by CD11b and CD44
6030 MMP-9 REGULATES CHEMOKINE RECEPTOR SIGNALING IN DCs
and in modulation of stem cell mobilization, delayed type hyper-
sensitivity, and the course of experimental allergic encephalitis
(33–37, 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 TGF? by cell
surface-localized MMP-9 (62), and degradation of ECM. We now
provide evidence supporting a new mechanism whereby MMP-9
promotes IFN-? DC migration by enhancing chemokine receptor
signaling. MMP-9 regulated CCR1/5 signaling in a selective man-
ner, 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 em-
anating from chemokine receptors and a MMP-9-dependent path-
way (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).
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 multire-
ceptor complexes regulates protease function (56, 57, 76). Local-
ization 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-?. 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 re-
sults support a model whereby binding of MMP-9 to the surface of
IFN-? 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-? 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 TGF?) (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 regu-
lation 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 rel-
evant 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 syner-
gistic 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 ac-
tivation of chemokine receptors and the MMP-9-dependent path-
way leads to strong activation of JNK together with activation of
other chemokine receptor-dependent pathways important for mi-
gration (such as Akt) and results in enhanced migration. A recent
report has described a similar function for the serine protease ca-
thepsin G, which binds to neutrophils via an as yet unidentified
receptor and regulates the signaling and function of integrins with-
out directly cleaving the integrins or their ligands (79). Thus, pro-
teases 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 chemo-
kine gradients. Well-established mechanisms of negative regula-
tion include down-modulation of cell surface chemokine receptor
expression, desensitization mediated by phosphorylation, and in-
duction 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 inhib-
itory 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 am-
plification of chemokine receptor calcium signaling by CD38-de-
pendent generation of cyclic adenosine diphosphate ribose (82).
We have discovered that CCR1/5 signaling is also augmented by
of CCR signaling by MMP-9. MMP-9
cleaves and activates a receptor or li-
gand required for effective JNK activa-
9-dependent pathway synergistically
activate JNK, thereby augmenting che-
motaxis. CD11b and CD44 can poten-
to activation of JNK independently of
Model of costimulation
6031The Journal of Immunology
MMP-9 by a mechanism that involves costimulation of CCR-de-
pendent signal transduction. The dependence of chemokine recep-
tors on a costimulatory signal helps explain the well-known dis-
sociation 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 at-
tachment 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 recep-
tors/MMP-9 have been previously appreciated to control, respec-
tively, 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.
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
ECM. We also thank Dr. Joel Pardee for his continuing support.
The authors have no financial conflict of interest.
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