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Originally published online as doi:10.1189/jlb.0804464 on January 24, 2006

Published online before print January 24, 2006
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(Journal of Leukocyte Biology. 2006;79:767-778.)
© 2006 by Society for Leukocyte Biology

CCL5-enhanced human immature dendritic cell migration through the basement membrane in vitro depends on matrix metalloproteinase-9

Valérie Chabot*,{dagger},{ddagger},1, Pascale Reverdiau{ddagger},§, Sophie Iochmann{ddagger},§, Angélique Rico*,{dagger},{ddagger}, Delphine Sénécal, Caroline Goupille{ddagger},||, Pierre-Yves Sizaret{ddagger},** and Luc Sensebé*,{ddagger}

* Etablissement Français du Sang Centre Atlantique, Tours, France;
§ Inserm U 618, Protéases et Vectorisation Pulmonaires,
** PPF Analyse des Systèmes Biologiques, Département des Microscopies,
{dagger} JE 2448 Cellules Dendritiques et Greffes,
|| Inserm EMI-HU 0211, Nutrition, Croissance et Cancer, and
{ddagger} IFR 135 Imagerie Fonctionnelle, Faculté de Médecine, Université de Tours, France; and
Service d’Oncologie Médicale et Maladies du sang, Centre Hospitalier Régional et Universitaire, Tours, France

1Correspondence: Etablissement Français du Sang Centre Atlantique, Service Recherche, 2 boulevard Tonnellé, 37020 Tours Cedex, France. E-mail: valerie.chabot{at}efs.sante.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The proinflammatory chemokine CC chemokine ligand 5 (CCL5) is a potent chemoattractant of immature dendritic cells (iDCs). It remains to be elucidated whether CCL5 may also enhance iDC migration through the basement membrane by affecting matrix metalloproteinase (MMP)-9 secretion. In this study, iDCs were differentiated in vitro from human monocytes of healthy donors. Zymographic analysis of cellular membranes of nontreated iDCs revealed a basal secretion of the pro- and active MMP-9, whereas only pro-MMP-9 was detected in conditioned media. Increasing concentrations of CCL5 significantly enhanced MMP-9 secretion by iDCs, peaking at 100 ng/ml, which optimally increased iDC migration through a reconstituted basement membrane (MatrigelTM) in vitro. The CCL5-enhanced secretion of MMP-9 occurred early (2 h) and was maintained at least for 10 h. A significant increase in MMP-9 mRNA synthesis was detected by reverse transcriptase-polymerase chain reaction, only at 6 h of CCL5 treatment, which suggests that the early effect of CCL5 (0–4 h) on MMP-9 secretion was independent of mRNA synthesis, whereas the more delayed effect (6–10 h) could be mediated through an increase in MMP-9 gene expression. In a Matrigel migration assay, the CCL5-enhanced iDC migration was reduced significantly by specific inhibitors of MMP-9, such as tissue inhibitor of metalloproteinase-1 or an anti-MMP-9 antibody, which indicates that iDC migration through the basement membrane depends on MMP-9. These results suggest that under inflammatory conditions, the chemokine CCL5 may enhance iDC migration through the basement membrane by rapidly increasing their MMP-9 secretion.

Key Words: antigen-presenting cell • RANTES • gelatinase B • cellular membranes • cell trafficking


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs), playing a pivotal role in the initiation of the primary immune response [1 , 2 ]. In vivo, immature DCs (iDCs) are distributed throughout many tissues and function as sentinels of the immune system. During pathogen invasion, iDCs are recruited at the site of inflammation. They undergo maturation after antigen capture and exposure to inflammatory stimuli and migrate to regional lymph nodes, where antigenic peptides are presented to T lymphocytes [3 ]. It is thus now established that the migratory pattern of DCs is integral to their function as APCs.

The migration of iDCs into tissues to reach an inflammatory site requires three major classes of molecules: chemokines [4 5 6 ], which direct iDCs to target sites; adhesion molecules [7 , 8 ]; and proteinases involved in the degradation of the extracellular matrix (ECM) [9 , 10 ].

Chemokines represent a family of secreted proteins of 8–10 kDa, which induces leukocyte accumulation at inflammatory sites or regulates leukocyte trafficking through lymphoid tissues [11 ]. CC chemokine ligand 5 (CCL5; regulated on activation, normal T cells expressed and secreted) is a proinflammatory CC chemokine, which plays an important role in multiple inflammatory conditions [12 13 14 15 16 ] and in tumor progression [17 18 19 20 21 ]. This molecule has been described as a potent chemoattractant of iDCs in vitro [22 ], which suggests that it may be responsible for the accumulation of iDCs at inflammatory sites in vivo. The chemotactic effect of CCL5 on iDCs is mediated through CC chemokine receptor (CCR)1 and CCR5 on the iDC cell surface [23 24 25 26 27 28 ].

To reach inflammatory sites, iDCs must move through connective tissues and cross basement membranes, which mainly consist of laminin, type IV collagen, and heparan sulfate proteoglycans [29 ]. The proteinases involved in the degradation of basement membrane components are gelatinases A and B, also known as matrix metalloproteinase (MMP)-2 and -9, respectively [30 31 32 ]. These molecules are secreted in a proenzyme form and are activated extracellularly [33 , 34 ]. The activity of pro-MMP-9 and pro-MMP-2 can be modulated in vivo by tissue inhibitors of metalloproteinase (TIMP)-1 and -2, respectively [35 , 36 ], and cell migration is controlled by the balance of MMPs and TIMPs [37 ]. Several studies have reported that monocyte-derived iDCs and Langerhans cells in humans produce MMP-9 and low levels of MMP-2 [9 , 38 39 40 41 42 43 ] and that CCL5 is able to increase the production of MMP-9 in monocytes [44 , 45 ] and lymphocytes [46 , 47 ]. It is interesting that results of a recent study of a mouse model of asthma [10 ] suggested that MMP-9 plays an important role in vivo in the recruitment of airway DCs during inflammation and participates in the in vitro trans-Matrigel migration of iDCs toward CCL5 and CCL20 (macrophage inflammatory protein-3{alpha}). If, as demonstrated in mice, MMP-9 has an important function in the migration of human iDCs through the basement membrane, such an increase of MMP-9 production by CCL5 may enhance their migration to a site of inflammation.

The aim of this study was thus to determine whether CCL5 enhances the production and release of MMP-9 in human monocyte-derived iDCs and then to investigate the role of MMP-9 in the migration of human iDCs in response to a CCL5 gradient by using an in vitro Matrigel migration assay as a model of trans-basement membrane migration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of iDCs
iDCs were differentiated in vitro from human monocytes as follows: Peripheral blood cells were collected by cytapheresis from healthy donors after they gave written, informed consent. Mononuclear cells were isolated by Ficoll density gradient centrifugation (density=1.077, Eurobio, Les Ulis, France). Platelets were removed by two centrifugations of mononuclear cells at 120 g for 5 min, and then 1 x 108 cells were plated per 175 cm2 flask for 1 h at 37°C in 5% humidified CO2. Nonadherent cells were removed, the flasks were washed twice with 10 ml RPMI-1640 medium (Invitrogen, Cergy-Pontoise, France), and the adherent monocytes were cultured for 5 days in the presence of 1000 U/ml granulocyte macrophage-colony stimulating factor (GM-CSF; Leucomax Novartis, Huningue, France) and 25 ng/ml interleukin (IL)-4 (R&D Systems Europe, Abingdon, UK) in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (Invitrogen). Fresh medium containing GM-CSF and IL-4 was added on Day 3 (0.5 vol). After 5 days of culture, the cells were harvested, and the viability measured with Trypan blue exclusion test was greater than 95%. The purity of iDC preparation determined by flow cytometry analysis of CD1a expression on total cultured cells was greater than 95%. To induce maturation, 20 ng/ml tumor necrosis factor {alpha} (TNF-{alpha}; R&D Systems Europe) was added to iDCs on Day 5 of culture for 48 h.

Flow cytometric analyses
Monocytes and DCs were labeled with monoclonal antibodies (mAb) specific for human CD14 (clone RMO52), CD83 (clone HB15a), DC-specific intercellular adhesion molecule-grabbing nonintegrin (SIGN; clone AZND1; all from Beckman Coulter, Roissy, France), CD1a (clone HI149), CCR5 (clone 2D7; both from BD Biosciences, Le Pont de Claix, France), and CCR7 (clone 150503; R&D Systems Europe), conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Cells were also stained with the corresponding FITC- or PE-conjugated, isotype-matched control mAb. For CCR1 labeling, cells were incubated with rabbit anti-CCR1 antibody (Calbiochem, La Jolla, CA) or with its isotypic control (anti-glutathione S-transferase antibody; Oncogene Research, San Diego, CA) as described by Su et al. [48 ]. Subsequently, the cells were stained with a 50-fold diluted PE-conjugated donkey anti-rabbit immunoglobulin G (IgG) antibody (Jackson ImmunoResearch, West Grove, PA). For intracellular staining of DC-lysosome-associated membrane protein (LAMP; clone 104.G4; Beckman Coulter), cells were fixed and permeabilized with use of IntraPrep permeabilization reagent (Beckman Coulter), according to the manufacturer’s instructions. Acquisitions of at least 5000 cells were obtained with use of a 488-nm laser flow cytometer (FACSCalibur, BD Biosciences), and data were analyzed with use of CELLQuest® software (BD Biosciences). Results are expressed as the mean fluorescence intensity (MFI).

Scanning electron microscopy
The morphologic changes in monocytes during their differentiation into iDCs and mature DCs (mDCs) were observed by scanning electron microscopy. Adherent monocytes, 3 x 106 iDC or mDC in suspension were washed twice in phosphate-buffered saline (PBS) to remove FCS. Adherent monocytes were fixed with a solution of 4% paraformaldehyde and 1% glutaraldehyde for at least 1 h at room temperature according to the method of McDowell and Trump [49 ], and then preparations were stored at 4°C. The iDC and mDC suspensions were dropped onto a poly L-lysine-coated coverslip and then fixed in the same conditions as described above. Preparations were washed three times in PBS, post-fixed for 30 min with 1% osmium tetroxide in phosphate buffer, and washed three times in bidistilled water. Preparations were dehydrated in progressive concentrations of ethanol (50–100%), dried by the critical point method (CO2), and mounted on stubs, and a thin layer of platinum was deposited by use of a Magnetron sputtering device. Preparations were studied under an FEG Gemini 982 scanning electron microscope (Leo Microscopy, Germany).

Chemotaxis and trans-Matrigel assays
The chemotaxis of human iDCs to recombinant human (rh)CCL5 (R&D Systems Europe) was assayed in 24-well cell-culture plates with bare 8.0 µm pore polycarbonate cell culture inserts (BD Biosciences). To study the migration of iDCs through basement membrane components, cell culture inserts were coated with a thin layer (25 µg/insert) of MatrigelTM (BD Biosciences), according to the manufacturer’s instructions. iDCs were washed extensively in RPMI-1640 medium to remove FCS and resuspended in RPMI-1640 containing 2% (v/v) Nutridoma (Roche Diagnostics, Meylan, France) as described [50 ] at a concentration of 5 x 105 cells/400 µl. For optimal induction of iDC migration, 1 ml RPMI-1640 2% (v/v) Nutridoma alone (control iDCs) or with rhCCL5 at increasing concentrations (20, 100, 500 ng/ml) was added to wells. Each condition was set up in duplicate. Cell suspension (400 µl; 5x105 iDCs) in RPMI 2% Nutridoma was then added to the inserts, and the plates were incubated for 6 h at 37°C in 5% CO2. The cells remaining in the inserts were removed by aspiration and wiping with cotton swabs after the incubation, and migrated cells were harvested by washing the bottom side of the filter insert and the wells with 600 µl RPMI-1640. Migrated cells were counted by use of a Malassez chamber, and results are expressed as the mean number of migrating cells ± SEM.

To study the role of MMPs in the CCL5-enhanced trans-Matrigel iDC migration, cells were preincubated for 30 min at 37°C with increasing concentrations of GM 6001 (Calbiochem), a potent, broad-spectrum inhibitor of MMPs, or its negative control (GM 6001 NC; Calbiochem) or for 1 h at 37°C with increasing concentrations of natural TIMPs, rhTIMP-1 or rhTIMP-2 (Oncogene Research). To assess the role of MMP-9 in trans-Matrigel migration of iDCs, cells were preincubated for 1 h with an optimal concentration (10 µg/ml) of a neutralizing anti-MMP-9 mAb (clone 6-6B; Oncogene Research) or its isotypic control (IgG1 NA/LE, BD Biosciences) before being added to bare or Matrigel-coated inserts. rhCCL5 (100 ng/ml) was added to wells, and the plates were incubated for 6 h at 37°C in 5% CO2. Migrated cells were counted by use of a Malassez chamber. The results were normalized to a proportion of CCL5 alone-enhanced migration, to which no inhibitors were added (CCL5 alone was set equal to 100%).

As controls, iDCs were preincubated with optimal concentrations of GM 6001 (10 µM), TIMP-1 (500 ng/condition), or the anti-MMP-9 mAb (10 µg/ml), and the trans-Matrigel assay was performed in the absence of rhCCL5.

iDC-conditioned media
For the CCL5 dose-dependent experiments, iDCs were incubated in serum-free conditions in 24-well plates with 0, 4, 20, 100, or 500 ng/ml rhCCL5 for 6 h, and for the time-dependence experiments, cells were incubated for 2, 4, 6, 8, and 10 h at 37°C in 5% CO2 in the absence or presence of 100 ng/ml rhCCL5. Each condition was run in duplicate. After incubation, the supernatants were collected and stored at –80°C until analysis by zymography. The cell pellets were used immediately for mRNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.

A conditioned medium of HT 1080 cell line (American Type Culture Collection, Manassas, VA), cultured in a serum-free condition with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO) for 18 h, was used as a positive control of MMP-9 and MMP-2 secretion.

Preparation of iDC cellular membranes
iDCs were incubated in a serum-free condition for 2, 4, 6, 8, and 10 h at 37°C in 5% CO2 in the absence or presence of 100 ng/ml rhCCL5. After incubation, the supernatants were collected and stored at –80°C until analysis by zymography. The cell pellets were used immediately for the preparation of iDC cellular membranes as described by Weissman [51 ] with minor modifications. Briefly, for each condition, iDCs (5x106) were washed twice with ice-cold PBS. The pellets were resuspended in ice-cold Dounce buffer [10 mM Tris-HCl, pH 7.6, 0.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 4 mM benzamidine (Sigma-Aldrich)] and homogenized by use of a Dounce homogenizer. The suspensions were centrifuged at 500 g at 4°C for 5 min to remove the nuclear fraction. The supernatants were collected and centrifuged at 100,000 g at 4°C for 1 h to pellet the membranes. The supernatants corresponding to cytosolic fractions were discarded, and the pellets containing the iDC cellular membranes were stored at –80°C until analysis by zymography.

Cellular membranes of HT 1080 cells treated (18 h, 37°C) with 100 ng/ml PMA were used as a positive control for MMP-9 and MMP-2 membrane association.

Total protein content was measured by use of the Coo protein assay determination kit (Uptima, Montluçon, France).

Gelatin zymography
MMP-9 and MMP-2 activity in conditioned media and cellular membranes of iDCs was determined by gelatin zymography with use of standard methodology [52 ]. Briefly, 7.5 µl serum-free conditioned media or 5 µg proteins from cellular membranes of iDCs were half-diluted in sample buffer and loaded onto 10% (v/v) sodium dodecyl sulfate (SDS) polyacrylamide gels containing 1 mg/ml gelatin (Prolabo, Fontenay-sous-Bois, France). rhpro- and active MMP-9 as well as pro- and active MMP-2 purchased from Calbiochem were used as standards. Electrophoresis was performed at 15 mA/gel constant current for 2 h. Gels were then incubated twice in 100 ml 2.5% (v/v) Triton X-100 (Merck-Eurolab, Fontenay-sous-Bois, France) for 15 min to remove SDS. The Triton solution was then removed and replaced with 100 ml developing buffer (50 mM Tris-HCl, pH 7.5, 5 mM CaCl2, and 0.02% sodium azide). After 40 h incubation at 37°C, the gels were stained for 3 h in 30% ethanol, 10% acetic acid, and 0.1% Coomassie brilliant blue R-350 (Amersham Biosciences, Chalfont St Giles, UK) and then destained in 45% ethanol and 10% acetic acid. Areas of gelatin digestion were visualized as nonstained regions of the gel, and gels were processed with use of a GS-690 imaging densitometer (Bio-Rad, Marnes la Coquette, France), and the intensity of the bands was quantified by use of Multi-Analyst computer software (Bio-Rad). A relative value of 1 was assigned to the bands of untreated iDCs (control), and results from CCL5-treated cells are expressed as mean increase (-fold) compared with control levels.

Immunoblot analysis
A total of 5 µg proteins from cellular membranes of iDCs, treated or not with rhCCL5 for 10 h, was separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membranes during 1.30 h at 100 V. The nitrocellulose membranes were incubated with mAb against pro- and active MMP-9 (1 µg/ml; clone 6-6B, Oncogene Research) or rabbit polyclonal antibody (pAb) against pro-MMP-9 (Sigma-Aldrich), diluted 1/1000e for 1 h at room temperature. After being washed, the nitrocellulose membranes were incubated with peroxidase-conjugated goat anti-mouse Igs (1/15000e, Bio-Rad) or peroxidase-conjugated goat anti-rabbit Igs (1/15000e, Bio-Rad). The amplified Opti4-CN revelation kit (Bio-Rad) was used to detect MMP-9, according to the manufacturer’s instructions. The ability of the two anti-MMP-9 antibodies to recognize latent or active MMP-9 specifically was confirmed by use of pro- and active MMP-9 standards (Calbiochem).

RT-PCR
Total mRNA was isolated from 5 x 105 iDCs, incubated for 2, 4, 6, 8, and 10 h at 37°C in 5% CO2 in the absence or presence of 100 ng/ml rhCCL5 or 100 ng/ml PMA (Sigma-Aldrich) as a positive control of MMP-9 stimulation, by use of the Dynabeads mRNA direct kit (Dynal France SA, Compiègne, France), according to the manufacturer’s instructions. Total mRNA was then reverse-transcribed for 1 h at 42°C in 1x incubation buffer containing 250 µM each deoxynucleotide triphosphate, 5 µM oligo (dT)20, 12 units RNase inhibitor, and 10 units avian myeloblastosis virus RT (Roche Diagnostics).

Standard PCR was then performed as described previously [53 ] with cDNA obtained from 5 x 104 cells in a total reaction volume of 50 µl containing 10 mM Tris-Hcl, pH 9.0, 50 mM KCl, 0.01% (w/v) gelatin, 1.5 mM MgCl2, 0.1% Triton X-100, 50 µM each deoxynucleoside triphosphate, 1 µM each forward- and reverse-synthesized oligonucleotide primer (Genset SA, Paris, France), and 1 unit Super Taq® DNA polymerase (A.T.G.C. Biotechnologie, Noisy-le-Grand, France). Primers used for specific PCR to MMP-9 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control gene were as follows: forward, 5'-ACCACCACAACATCACCTATTGGATCC-3', and reverse, 5'-TGTAGAGTCTCTCGCTGGGGCAGAAG-3'; and forward, 5'-ACAGTCCATGCCATCACTGCC-3', and reverse, 5'-GCCTGCTTCACCACCTTCTTG-3', respectively. PCR involved use of the GeneAmp PCR System 2400 (Applied Biosystems, Courtaboeuf, France) programmed for an initial denaturation step of 3 min at 94°C, followed by 35 (MMP-9) or 25 (GAPDH) cycles at 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s, and the final extension step performed at 72°C for 7 min. PCR products were then analyzed by electrophoresis with 1.6% agarose gel in Tris-boric acid-EDTA buffer (90 mM Tris-HCl, 90 mM borate acid, 2.5 mM EDTA) containing 1 µg/ml ethidium bromide and visualized by ultraviolet transillumination (Gel Doc 1000 System®, Bio-Rad). Band intensities of PCR products were measured by use of Multi-Analyst Macintosh software. RT-PCR products specific for MMP-9 and GADPH were sequenced with use of the dideoxynucleotide chain termination method on a Perkin Elmer Abi Perkin 377 automat (INSERM U619, France). Sequencing was performed on both strands with the reverse and forward primers were specific for MMP-9 and GAPDH used for RT-PCR.

Statistical analysis
The effect of rhCCL5 on MMP-9 secretion and mRNA synthesis and MMP inhibitors on iDC migration was analyzed by the Wilcoxon signed rank test. Statistical analysis involved use of Statview software (SAS Institute, Berkeley, CA), and data were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of iDCs
DCs were generated from human monocytes selected by adherence. After 5 days’ culture with GM-CSF and IL-4, cells exhibited typical iDC morphological features, with membrane veils and numerous squat protrusions, in contrast to numerous needle-like dendrites shown by TNF-{alpha} mDCs (Fig. 1A ). Flow cytometric analysis revealed that DCs at Day 5 of differentiation were strongly positive for CD1a and DC-SIGN, did not express markers of monocyte lineage (CD14) or of maturation such as DC-LAMP, CD83, or CCR7 (Fig. 1B) , and displayed high endocytic activity (data not shown). iDCs exhibited a strong, dose-dependent, chemotactic response to rhCCL5 (Fig. 2A ), which was related to the high expression of the CCL5 receptors, CCR5, and CCR1 (Fig. 2B) . Treatment with 100 ng/ml rhCCL5 for 6 h down-regulated the expression of CCR5 but maintained the expression of CCR1 at the iDC cell surface (Fig. 2C) . Control iDCs showed a basal migration through a reconstituted basement membrane (Matrigel), which was enhanced significantly (fourfold that of control iDCs) by treatment with an optimal concentration of 100 ng/ml rhCCL5 (Fig. 2A) .


Figure 1
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  		Figure 1. Morphological and phenotypic characterization of iDCs compared with monocytes and mDCs. iDCs were obtained after 5 days’ culture of human monocytes with GM-CSF and IL-4. Maturation was obtained after an additional 48-h culture with 20 ng/ml TNF-{alpha}. (A) Adherent monocytes, iDCs, and mDCs were analyzed by scanning electron microscopy; original scale bar = 5 µm. (B) The phenotype of monocytes, iDCs, and mDCs was determined by flow cytometry. Cells were harvested and stained for PE- or FITC-labeled antibodies against CD14, CD1a, DC-SIGN, CD83, or CCR7. For DC-LAMP intracellular labeling, cells were fixed and permeabilized prior to staining with DC-LAMP antibody. In each plot, thin lines represent the background staining with isotype-matched control antibodies, and bold lines show the specific staining. The number in each histogram indicates the MFI. Results are representative of three independent experiments.

 

Figure 2
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  		Figure 2. Migratory capacities of iDCs in response to rhCCL5. (A) The chemotactic and trans-Matrigel migration of iDCs in response to increasing concentrations of rhCCL5 (20, 100, or 500 ng/ml) for 6 h was assayed in vitro with use of 8 µm cell culture inserts. Control shows the migration to the serum-free medium alone. Data are mean number ± SEM of migrated iDCs of three separate experiments. *, P < 0.05, compared with control. (B, C) Flow cytometry analysis of the cell-surface expression of CCR5 and CCR1 in control iDCs (B) and iDCs treated with 100 ng/ml rhCCL5 for 6 h (C). For CCR1 labeling, cells were stained with purified anti-CCR1 antibody and then incubated with PE-conjugated donkey anti-rabbit IgG antibody. The thin lines represent the background staining with isotype-matched antibodies, and bold lines show the specific staining. Numbers indicate the MFI. Results are representative of three independent experiments.

 
CCL5 increases pro-MMP-9 release by iDCs in a dose- and time-dependent manner
As MMP-9 and MMP-2 were the major MMPs involved in the degradation of basement membrane components, we determined whether CCL5 enhances MMP-9 and MMP-2 release in iDCs. We examined the effect of CCL5 on MMP production in serum-free medium, as FCS contained high levels of both MMPs. The proenzyme form of MMP-9 was detected by zymography in conditioned media of untreated (control) iDCs, shown by a band at 92 kDa (Fig. 3A ). A band at 72 kDa revealed low levels of pro-MMP-2 secreted by iDCs. Both proenzyme forms were secreted specifically by iDCs, as no gelatinolytic activity was observed in medium alone (Fig. 3A) . Treatment of iDCs with increasing concentrations of rhCCL5 for 6 h significantly enhanced pro-MMP-9 release (P<0.05, for all CCL5 concentrations tested), as assessed by densitometric analysis of the bands from zymograms (Fig. 3B) , and the maximal effect (twofold increase as compared with control) was 100 ng/ml. By contrast, no effect of rhCCL5 on pro-MMP-2 release was detected. No band at 83 kDa was detected in control or treated conditions, which suggests that the active form of MMP-9 was not released in conditioned media. Similar results were obtained by zymographic analysis of media from trans-Matrigel migration of control and treated iDCs (data not shown).


Figure 3
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  		Figure 3. CCL5 increases pro-MMP-9 release by iDCs in a dose- and time-dependent manner. iDCs were cultured in serum-free condition, and conditioned media were collected and analyzed by gelatin zymography to detect MMP-9 and MMP-2 enzymatic activity. (A, B) For dose-dependent experiments, iDCs were not treated [control (C)] or treated with increasing concentrations (4, 20, 100, or 500 ng/ml) of rhCCL5 for 6 h. (A) One representative zymogram of nine is presented. (B) The intensity of gelatin lysis bands was obtained after scanning densitometry, and a relative value of one was assigned to the bands from the conditioned media of control, untreated iDCs. Results from CCL5-treated iDCs are expressed as mean increase (-fold) compared with control levels ± SEM from nine zymograms. *, P < 0.05, compared with control. (C, D) For time-dependent experiments, cells were treated (+) or not (–) with 100 ng/ml rhCCL5 for 2, 4, 6, 8, and 10 h. Conditioned medium from HT 1080 cells treated with 100 ng/ml PMA was used as a control, and 75 pg-purified pro- and active MMP-9 and MMP-2 were used as a standard (ST). (C) One representative zymogram of three is presented. (D) Quantification of gelatin lysis bands was performed as in B, and results from CCL5-treated iDCs are expressed as a mean increase (-fold) compared with control levels ± SEM from three zymograms. *, P < 0.05, compared with control at each time.

 
Zymographic analysis of media from time-dependent experiments performed between 0 and 10 h revealed a basal release of pro-MMP-9 by iDCs beginning between 0 and 2 h of culture (Fig. 3C) . The addition of 100 ng rhCCL5 significantly increased pro-MMP-9 release by iDCs beginning at 4 h (P<0.05), which was maintained at each time-point tested (Fig. 3D) . The 83-kDa active form of MMP-9 was never detected in iDC-conditioned media, even at 10 h of CCL5 treatment.

CCL5 increases the level of the pro- and active forms of MMP-9 in iDC cellular membranes
Analysis of iDC cellular membranes by zymography revealed two major bands at 92 and 83 kDa, which may correspond to the pro- and active forms, respectively, of MMP-9 (Fig. 4A ). A slight band at 79 kDa, which may represent nonglycosylated MMP-9, was detected in all samples. MMP-2, in its proenzyme or active form, was not detected in iDC cellular membranes, whereas three bands at 72, 64, and 62 kDa, corresponding to the pro-, intermediate, and active forms of MMP-2, respectively, were detected in HT 1080 cell membranes. Densitometric analysis of the bands from zymograms revealed that CCL5 treatment significantly increased the level of the pro (Fig. 4B) - and active (Fig. 4C) MMP-9 in iDC cellular membranes at each time-point tested (up to 2.8-fold for pro-MMP-9 and 2.2-fold for active MMP-9 compared with controls; P<0.05).


Figure 4
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  		Figure 4. CCL5 enhances the levels of the pro- and active MMP-9 in iDC cellular membranes. (A–C) Cells were treated (+) or not (–) with 100 ng/ml rhCCL5 for 2, 4, 6, 8, and 10 h, and cellular membranes were prepared as described in Materials and Methods; 5 µg each was analyzed by gelatin zymography. Cellular membranes from HT 1080 cells treated with 100 ng/ml PMA were used as a control, and 75 pg-purified pro- and active MMP-9 and MMP-2 were used as a standard. (A) One representative zymogram of three is presented. (B, C) The intensity of gelatin lysis bands corresponding to the pro (B)- and active (C) MMP-9 was obtained after scanning densitometry, and a relative value of one was assigned to the bands from the membrane fractions of control, untreated iDCs. Results from CCL5-treated iDCs are expressed as mean increase (-fold) compared with control levels ± SEM from three zymograms. *, P < 0.05, compared with control at each time. (D, E) Immunoblot analysis of MMP-9 expression in cellular membrane preparations from nontreated (–) or CCL5-treated (+) iDCs cultured for 10 h at 37°C. Samples were subjected to 10% SDS-PAGE and blotted to nitrocellulose membrane. The blots were incubated with mAb 6-6b (D), which recognizes the pro- and active forms of MMP-9, or rabbit pAb (E), which recognizes only the pro-MMP-9. Purified pro- and active MMP-9 (8 ng) was used as a standard.

 
To determine whether the 83-kDa band represented the truly active form of MMP-9 and not an underglycosylated form of the latent enzyme, the cellular membranes of iDCs, treated or not with rhCCL5 for 10 h, underwent immunoblotting with use of two anti-MMP-9 antibodies specific for the pro- and active MMP-9 (Fig. 4D) or the pro-MMP-9 (Fig. 4E) . Two bands at 92 and 83 kDa were highlighted by the antibody directed against both forms of the enzyme, and two bands at 92 and 79 kDa were revealed by the antibody specific for the latent enzyme, which demonstrates that only the 92- and 79-kDa bands contained the complete NH2-terminal prodomain of MMP-9 (Fig. 4E) . These results demonstrate that the 92- and 83-kDa bands detected on isolated cellular membranes of control and CCL5-treated iDCs were the pro- and active forms, respectively, of MMP-9.

CCL5 increases MMP-9 mRNA synthesis in iDCs
As rhCCL5 increases MMP-9 secretion, we investigated whether this effect was detectable at the mRNA level. RT-PCR analysis revealed that control iDCs synthesized low levels of MMP-9 mRNA until 6 h of culture, which then doubled at 8 and 10 h (Fig. 5A and 5B ). The addition of 100 ng/ml rhCCL5 to the culture media significantly enhanced the synthesis of MMP-9 mRNA only at 6 h of treatment (up to 2.4-fold compared with control; P<0.05; Fig . 5A and 5B ), which was decreased at 8 and 10 h, as MMP-9 mRNA synthesis increased in control iDCs.


Figure 5
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  		Figure 5. CCL5 increases MMP-9 mRNA synthesis by iDCs at 6 h of culture. Cells were treated (+) or not (–) with 100 ng/ml rhCCL5 or PMA (used as positive control of stimulation) for 2, 4, 6, 8, or 10 h. (A) MMP-9 and GAPDH gene expression was analyzed by RT-PCR, performed from 5 x 104 iDCs. (B) Densitometric analysis of the bands with use of Multi-Analyst software. Data are expressed as the ratio of MMP-9 and GAPDH, and results are means ± SEM from three experiments. *, P < 0.05, compared with controls at each time.

 
MMP inhibitors reduce the CCL5-enhanced trans-Matrigel migration of iDCs
To determine whether MMPs were involved in trans-Matrigel migration of iDCs toward a CCL5 gradient, cells were first incubated with a broad-spectrum hydroxamate inhibitor of MMPs (GM 6001) before being assayed. Results in Figure 6 6A revealed 54% inhibition of CCL5-enhanced iDC migration through Matrigel (55,720 migrated cells±5047 vs. 25,429±4386, P<0.05) at 10 µM GM 6001. This effect was specific, as the GM 6001 NC used at the same concentration did not modify the migration of iDCs through Matrigel. Furthermore, GM 6001 (10 µM) significantly reduced the trans-Matrigel migration of control iDCs by 47% (11,070 migrated cells±2550 vs. 5850±3556, P<0.05). To rule out a cytotoxic effect of GM 6001 on iDCs, we evaluated the morphology, viability, and functionality of cells cultured with the highest concentration of GM 6001 (10 µM). GM 6001-treated iDCs appeared normal on contrast-phase microscopy and viable as assessed by Trypan blue exclusion after 6 h culture. Flow cytometric analysis revealed that the cell surface expression of CD1a, CD14, CD32, and CCR5 and the functionality of iDCs, as evaluated by endocytosis of FITC-Dextran, did not change (data not shown).


Figure 6
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  		Figure 6. GM 6001 and TIMP-1 but not TIMP-2 decrease the CCL5-enhanced iDC migration through Matrigel. (A) iDCs were preincubated with increasing concentrations of GM 6001 or its NC, (B) rhTIMP-1, or (C) rhTIMP-2 before being loaded onto the insert. rhCCL5 (100 ng/ml) was added to wells, and the plates were incubated for 6 h at 37°C. The CCL5-enhanced migration of iDCs without inhibitors was set at 100% (CCL5), and the proportion of migration in the presence of each inhibitor compared with CCL5 is represented as the mean ± SEM from three to five separate experiments. Percentages of inhibition are indicated above histogram bars. *, P < 0.05, compared with CCL5 alone.

 
For better identification of MMPs involved in the trans-Matrigel migration of iDCs in response to a CCL5 gradient, cells were incubated with rhTIMP-1 and rhTIMP-2, the natural inhibitors of MMPs. rhTIMP-1, at 500 ng/condition, significantly reduced the CCL5-enhanced migration of iDCs through Matrigel by 47% (P<0.05; Fig. 6B ) but did not significantly modify the trans-Matrigel migration of control iDCs (10,463 migrated cells±2067 vs. 14,288±2046). rhTIMP-2 had no effect on CCL5-enhanced iDC migration through Matrigel for all of the concentrations tested (Fig. 6C) .

A specific anti-MMP-9 antibody decreases the CCL5-enhanced migration of iDCs through Matrigel
rhTIMP-1 has been described as an inhibitor of pro-MMP-9 [36 ]. To confirm that MMP-9 was involved in iDC trans-Matrigel migration toward a CCL5 gradient, cells were incubated with an anti-MMP-9 mAb (10 µg/ml) before being assayed. Results in Figure 7 reveal 34% inhibition (P<0.05) of CCL5-enhanced iDC migration through Matrigel with the antibody. This inhibitory effect was specific, as incubation of cells with the isotypic control IgG1, used at the same concentration, had no effect. By contrast, the anti-MMP-9 antibody did not significantly modify the trans-Matrigel migration of control iDCs (11,070 migrated cells±2550 vs. 12,780±820), and importantly, did not inhibit CCL5-enhanced iDC migration through bare inserts (Fig. 7) .


Figure 7
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  		Figure 7. The anti-MMP-9 mAb decreases the CCL5-enhanced iDC trans-Matrigel migration. iDCs were preincubated with 10 µg/ml anti-MMP-9 mAb or mouse IgG1 isotypic control for 1 h at 37°C before being loaded onto Matrigel-coated or bare inserts. rhCCL5 (100 ng/ml) was added to wells, and the plates were incubated for 6 h at 37°C. The CCL5-enhanced migration of iDCs without inhibitor was set at 100% (CCL5) and the proportion of migration in the presence of the anti-MMP-9 mAb or its isotypic control compared with CCL5 is represented as the mean ± SEM from three to five separate experiments. Percentage of inhibition is indicated above the histogram bar. *, P < 0.05, compared with CCL5 alone.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study reveals for the first time that the chemokine CCL5 increases the synthesis and secretion of the pro- and active forms of MMP-9 by human iDCs. Our data, showing that MMP-9 is involved in trans-Matrigel iDC migration in response to a CCL5 gradient, strongly suggest that this chemokine may enhance the migration of iDCs across the basement membrane in vitro through its effect on MMP-9 secretion.

As shown by other studies [22 , 24 , 54 ], DCs obtained after 5 days’ culture of monocytes with GM-CSF and IL-4 exhibited morphological, phenotypic, and functional features typical of iDCs. They were able to degrade basement membrane components and thus migrate through Matrigel in response to a CCL5 gradient. The main proteinases involved in the degradation of basement membrane components are MMP-9 and MMP-2. Our data revealed the presence of two major forms of MMP-9, of 92 and 83 kDa, in cellular membranes of iDCs but not MMP-2. Pro-MMP-9 was released as well in the iDC-conditioned media, and pro-MMP-2 was weakly detected. Immunoblot analysis with an antibody against the NH2-terminal propeptide of MMP-9 demonstrated that the 83-kDa form resulted from proenzyme activation and did not correspond to a latent enzyme with immature glycosylation, as was demonstrated in another cell type [55 ]. As most MMPs are secreted as latent enzymes and activated extracellularly [33 , 34 ], the detection of enzymes with mature glycosylation and the active form of MMP-9 in iDC cellular membranes strongly suggests that they may be localized near or at the cell surface of monocyte-derived iDCs.

Our findings are the first detection of active MMP-9 in iDCs. Indeed, recent works revealed the presence of membrane-associated MMP-9 and MMP-2 in human-activated Langerhans cells by flow cytometry analysis [43 , 56 ] but not in monocyte-derived iDC or mDC [56 ]. In another study, zymographic analysis of membrane extracts of monocyte-derived iDCs revealed the presence of membrane-bound pro-MMP-9 but not the active form of MMP-9, which was detected only in mDCs [41 ]. In these studies, the presence of active MMP-9 bound to the membrane of DCs was related to their ability to migrate through the ECM.

It is interesting that our results revealed that the inflammatory chemokine CCL5, normally used to attract iDCs in vitro, enhanced the release of pro-MMP-9 by iDCs and increased the level of pro- and active MMP-9 in iDC cellular membranes, which were maintained at least for 10 h. This effect was specific, as pro-MMP-2 release in iDC-conditioned media was not modified by CCL5 treatment. These results suggest that in iDCs, CCL5 increases MMP-9 activity at the level of secretion, membrane binding, and activation. The effect of CCL5 on pro-MMP-9 release was maximal at 100 ng/ml, which optimally enhanced trans-Matrigel migration of iDCs in vitro. Such a stimulating effect of CCL5 on pro-MMP-9 secretion has been reported in lymphocytes [46 , 47 ] and monocytes [44 , 45 ] and was related to their migratory potencies. Thus, the strong relation among the dose of CCL5, level of secreted MMP-9, and number of migrated cells found in our study strongly suggests that this chemokine may be involved in trans-Matrigel iDC migration through its effect on MMP-9.

Several hypotheses can explain the mechanism(s) by which CCL5 increases the level of MMP-9 in iDCs. Our data showed that CCL5 differentially regulated the cell-surface expression of its own receptors, leading to the rapid down-regulation of CCR5 and the maintenance of CCR1 expression. This result suggests that the long-term effects of CCL5 on MMP-9 production by iDCs may be mediated more likely through CCR1. This hypothesis was reinforced by results in monocytes, in which the use of a CCR1 antagonist demonstrated the implication of this receptor in the CCL5-enhanced secretion of MMP-9 [44 ]. Furthermore, the CCL5-enhanced levels of MMP-9 in cellular membranes described in our study may result from the increased secretion of MMP-9 but also from an increase in the level of membrane molecules involved in the binding of MMP-9 to the iDC cellular membranes. In other cell types, the localization of MMP-9 to the membrane is mediated through the cell surface hyaluronan receptor CD44 [57 , 58 ] or {alpha}2 (IV) chain of collagen IV [59 ]. iDCs express the hyaluronan receptor [60 , 61 ], and CCL5 enhances the CD44 mRNA synthesis in monocytes [62 ]. Such an effect in iDCs may explain the increase in MMP-9 levels detected in cellular membranes, which then would become available for activation. The low level of pro-MMP-2 detected in iDC-conditioned media and its absence in cellular membranes ruled out the involvement of MMP-2 in pro-MMP-9 activation in monocyte-derived iDCs, as was suggested in other cell types [63 , 64 ].

MMP-9 production is known to be regulated at different levels in many cell types [34 ]. In our study, CCL5 treatment significantly increased the synthesis of MMP-9 mRNA by iDCs only at 6 h of culture. As zymography showed that CCL5 enhanced MMP-9 secretion throughout the 10-h culture, RT-PCR results strongly suggest that the early effect (0–4 h) of CCL5 on MMP-9 secretion occurred through a post-transcriptional mechanism, as no increase in mRNA level was observed, whereas the delayed effect (6–10 h) may be mediated through stimulation of MMP-9 gene expression. In human monocytes, the synthesis of MMP-9 mRNA was increased at 8 h CCL5 treatment [45 , 62 ], and the increase in MMP-9 production was mediated through an effect of CCL5 on TNF-{alpha} secretion [45 ]. Furthermore, in murine bone marrow DCs, CCL5 induced a rapid (3 h) up-regulation of TNF-{alpha} transcripts and stimulated production of TNF-{alpha} proteins [65 ]. This mechanism cannot be ruled out for human iDCs to explain the later increase in MMP-9 production. Thus, our results showed that in iDCs, the increase in MMP-9 levels detected after CCL5 treatment may occur first at the level of secretion, a mechanism that will provide a rapid mobilization of cells at the site of inflammation.

ECM proteolysis and consequently, traffic of cells depend on a balance between MMPs and TIMPs. A unique property of latent MMP-9 is its ability to form a noncovalent complex with TIMP-1 [36 ]. In iDCs, CCL5 did not significantly modify the release of TIMP-1 (unpublished data), which suggests that MMP-9 activity may not be counteracted by a concomitant stimulation of TIMP-1. The balance between MMP-9 and TIMP-1 in CCL5-treated cells was thus in favor of basement membrane degradation and consequently, iDC migration.

The inhibition of CCL5-enhanced trans-Matrigel iDC migration by rhTIMP-1 or a specific anti-MMP-9 mAb revealed that MMP-9 is involved in the migration of human iDCs through the reconstituted basement membrane, as was demonstrated in mice [10 ]. These findings and the fact that TIMP-1 and the anti-MMP-9 antibody did not affect the Matrigel migration of control iDCs support the view that increased MMP-9 levels in CCL5-treated iDCs may explain their enhanced migration into tissues under inflammatory conditions. The inhibitory effects of rhTIMP-1 and the anti-MMP-9 antibody did not completely block the iDC migration through Matrigel in response to a CCL5 gradient, which suggests that other MMPs or other groups of proteinases may be involved in this process. Another gelatinase, MMP-2, reported to be involved in activated Langerhans cell migration [56 ], was weakly detected in our study and in other studies [9 , 40 , 41 ]. Furthermore, the absence of a stimulatory effect of CCL5 on pro-MMP-2 release and MMP-2 in cellular membranes and an inhibitory effect of rhTIMP-2 on CCL5-enhanced trans-Matrigel migration of iDCs strongly suggest that MMP-2 is not involved in monocyte-derived iDC migration through the basement membrane in vitro. Other MMPs, such as MMP-1 and MMP-3, are secreted by monocyte-derived iDCs [42 ], but their respective roles in iDC migration have not been investigated. The use of GM 6001, a broad-spectrum inhibitor of MMPs, did not completely inhibit the iDC trans-Matrigel migration in response to a CCL5 gradient, which indicates that other groups of proteinases may be involved in this process [9 , 66 ]. Serine proteinase, as elastase in neutrophils [67 , 68 ], can degrade type IV collagen in vitro and may be involved in their migration through the basement membrane. The role of serine proteinases in iDC migration has not been studied to date.

In conclusion, our results reveal that under inflammatory conditions, the chemokine CCL5 directs iDC migration through its chemotactic effect and also may enhance iDC traffic through the basement membrane by increasing MMP-9 secretion. Furthermore, the detection of active MMP-9 in iDC cellular membranes strongly suggests that it was localized near or at the cell surface of iDCs and may be crucial for proteolysis of ECM in areas of cell-matrix contacts and thus for iDC migration to a site of inflammation. Further studies are necessary to understand the mechanism(s) by which CCL5 increases MMP-9 secretion in iDCs, to characterize more exactly the binding of MMP-9 to the membrane, and identify the molecules involved in its activation.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a grant from La Ligue contre le Cancer. The authors thank Dr. J. L. Gatty (INRA, Nouzilly, France) for kindly providing gelatin and Dr. I. Dimier-Poisson (UFR des Sciences Pharmaceutiques, Tours, France) and Dr. F. Velge-Roussel (UFR de Médecine, Tours, France) for assistance in preparing membrane fractions. We give many thanks to the staff of the Etablissement Français du Sang (Tours, France) for performing cytapheresis on healthy donors.

Received August 23, 2004; revised November 4, 2005; accepted November 21, 2005.


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