In this study, we address the question of the cross-talk between two chemokines that are cosecreted during inflammation, namely monocyte chemoattractant protein-1 (MCP-1) and soluble fractalkine (s-FKN), toward monocyte migration. We found that s-FKN fails to induce MonoMac6 cell migration per se. Interestingly, this chemokine antagonizes transendothelial migration and chemotaxis of MonoMac6 cells and freshly isolated human monocytes induced by MCP-1, indicating a direct effect of s-FKN on monocytic cells. In this study, we found that stress-activated protein kinase (SAPK)1/c-Jun N-terminal kinase 1 and SAPK2/p38 are involved in the control of MCP-1-induced MonoMac6 cell migration. We demonstrated that s-FKN abrogates the MCP-1-induced SAPK2/p38 activation as well as the upstream Pyk2 activity. Furthermore, we observed that s-FKN also inhibits the activity of a major matrix metalloproteinase (MMP), namely MMP-2. Taken collectively, our results indicate that the s-FKN antagonizes the chemoattractant effect of MCP-1 on monocytes, likely by inhibiting crucial signaling pathways, like SAPK2/p38 and MMP-2 activities.

Chemokines are the major regulatory proteins for the recruitment and trafficking of leukocytes (1, 2, 3). Chemokines, which are divided into four subfamilies (C, CC, CXC, and CX3C chemokine) (4), exert their chemotactic effects through seven transmembrane-spanning, G protein-coupled receptors (5). Eighteen functional chemokine receptors are presently known and can bind multiple chemokines in a subclass-restricted manner (6).

Monocyte chemoattractant protein-1 (MCP-1),3 the first CC chemokine identified as a chemotactic factor for monocytes, is one of the most potent chemokines to induce migration of monocytes (7). Many other activities have been ascribed to MCP-1, including induction of migration of memory/activated T cells (8), NK cells (9), and myeloid dendritic cells (10). MCP-1 mediates its cellular effects primarily through CCR2 receptors, triggering a complex array of signaling pathways including a pertussis toxin-sensitive rise of intracellular calcium (11), inhibition of adenyl cyclase (11), phospholipase C activation (12), stimulation of Src (13), Syk (13), Pyk2 (14), and Janus kinase 2 (15) tyrosine kinase activities as well as extracellular signal-regulated kinase (ERK) and stress-activated protein kinase (SAPK) activities (13, 16, 17).

The CX3C fractalkine (FKN) is a unique membrane-bound chemokine with a transmembrane domain and a chemokine domain on top of a long mucin-rich stalk (18, 19). The membrane-anchored form of FKN can be cleaved from the cell membrane by proteolysis to yield chemoattractant soluble FKN (s-FKN) (18, 20, 21). Thus, FKN represents a new class of chemokine that behaves as a true chemokine in its soluble form, whereas it acts as an adhesion molecule in its membrane form (18). FKN binds specifically to the unique CX3CR1 receptor that, in the immune system, has been shown to be expressed on the majority of NK cells, monocytes, some T cell subpopulations (18, 22), and dendritic cells (23, 24). Curiously, s-FKN, which behaves as a potent chemottractant for NK cells, T cells, and dendritic cells, exerts poor or no effect on monocyte migration, albeit this lineage expresses the CX3CR1 receptor (19, 25). Although little information is yet available on the signaling pathways triggered by s-FKN, recent studies have pointed to the stimulation of SAPK, ERK, phosphatidylinositol 3-kinase, and Akt activities in response to this chemokine (26, 27, 28, 29).

MCP-1 and FKN are both produced in the context of inflammation, and the question thus arises as to how these chemokines can interfere with each other on monocyte recruitment because of their contrasting effects on this lineage. To address this question, we examined the effect of a simultaneous exposure to MCP-1 and s-FKN on the migration of MonoMac6 cells. We found that, in monocytic MonoMac6 cells, s-FKN not only fails to induce migration but abolishes the migratory response to MCP-1, following a process that impairs the MCP-1-induced activation of both the SAPK2/p38 pathway and matrix metalloproteinases (MMPs).

MonoMac6 cells (DSM ACC124) originally established from a patient with monoblastic leukemia were obtained from the German Collection of Microorganisms (Braunschweig, Germany). These cells were grown in suspension in RPMI 1640 (Life Technologies, Paisley Park, U. K.) supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT), l-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were passaged every 3–6 days using a split ratio of 1:3.

NK-L cells (30) derived from an aggressive human NK cell leukemia and obtained from J. Tabiasco (Institut National de la Santé et de la Recherche Médicale, Unité 395, Centre Hospitalier Universitaire Purpan, Toulouse, France) were grown in RPMI 1640 supplemented with 10% heat-inactivated AB human serum (BioWest, Chollet, France), l-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 μg/ml), and 100 IU/ml of human rIL-2 (Chiron, Amsterdam, The Netherlands). The culture medium was changed every 3 days.

Primary cultures of HUVECs purchased from BioWhittaker (Walkersville, MD) were grown in endothelial cell growth medium (EGM; BioWhittaker) supplemented with 10 pg/ml human epidermal growth factor (BioWhittaker), 1 μg/ml hydrocortisone (BioWhittaker), 50 μg/ml gentamicin (BioWhittaker), 3 μg/ml bovine brain extract (BioWhittaker), and 20% FBS (BioWhittaker). For transendothelial migration experiments, HUVECs were plated on polyethylene terephthalate (PET) inserts (pores, 8 μm) of 24-well chemotaxis chambers (BD Biosciences, San Jose, CA) and used 6–10 days after being plated.

As previously described by Barleon et al (31), PBMC were obtained from buffy coats of blood from normal donors. Ten milliliters of blood was collected by standard venipuncture into heparinized tubes. Each tube was diluted with an equal volume of PBS, and 10 ml of blood preparation were overlaid onto 15 ml of Ficoll-Paque (Eurobio, Les Ulis, France). Tubes were centrifuged (700 × g); the buffy coat interface was removed, added to 10 ml of PBS, and centrifuged (300 × g); and the pellet was resuspended in α-MEM (Life Technologies) supplemented by 0.1% BSA. The number of PBMC was determined using a hemacytometer. A positive selection of CD14+ cells was performed by adding MACS colloidal superparamagnetic microbeads (Miltenyi Biotec, Paris, France) conjugated with monoclonal anti-human CD14 Abs to freshly prepared PBMC preparation in MACS buffer according to the manufacturer. Briefly, after incubation of cells and microbeads (15 min at 4°C), cells were washed with MACS buffer, resuspended, and loaded onto the top of the separation column. Trapped CD14+ PBMC were eluted with a 6-fold amount of cold MACS buffer, centrifuged, and resuspended in α-MEM plus 0.1% BSA. Using flow cytometry, the purity of the CD14+ cells was evaluated at 93.6%, corresponding to 4.3% of the total PBMC.

Human s-FKN and human MCP-1 were purchased from R&D Systems (Abingdon, U.K.) and TNF-α from PeproTech (Rocky Hill, NJ). BSA and fibronectin were obtained from Sigma-Aldrich (St. Louis, MO). Monoclonal anti-Pyk2 Ab was obtained from Transduction Laboratories (Lexington, KY), and polyclonal anti-Pyk2 phosphospecific Ab from BioSource International (Camarillo, CA). Polyclonal anti-phospho ERKs, anti-phospho-p38, and anti-phospho c-Jun N-terminal kinase (JNK)1 Abs were purchased from New England Biolabs (Beverly, MA), and polyclonal anti-ERK, anti-p38, and anti-JNK1 Abs from Santa Cruz Biotechnology (Santa Cruz, CA).

MonoMac6 and NK-L cells.

Migratory responses of MonoMac6 or NK-L cells were evaluated by using 24-well chemotaxis chambers and PET inserts (8-μm pore size) (BD Biosciences) coated with 6.5 μg/ml fibronectin on both sides for chemotaxis assays. For transendothelial migration assays, HUVECs cultured on PET inserts were incubated for 48 h before use, in EGM medium supplemented with 10 ng/ml TNF-α. MonoMac6 or NK-L cells were incubated for 16 h with [methyl-3H]thymidine (2.5 μCi/ml; ICN Pharmaceuticals, Costa Mesa, CA) and then placed in the upper well of migratory chambers (0.5 × 106 cells/100 μl), and EGM medium was added in the lower well in the absence or presence of 5 nM MCP-1, 20 nM s-FKN, or a mixture of 5 nM MCP-1 and 20 nM s-FKN. Plates were incubated for different time courses at 37°C in a 5% CO2 atmosphere, and the migrated cells collected in the lower well were evaluated by the measurement of incorporated [methyl-3H]thymidine by scintillation spectroscopy.

Freshly isolated human monocytes.

Migratory responses of human monocyte cells were evaluated by using 24-well chemotaxis chambers and polycarbonate inserts (5-μm pore size) (Costar; Corning, Corning, NY) without coating. CD14+ cell suspension were placed in the upper well of migratory chambers (1.5 × 106 cells/50 μl) and α-MEM plus 0.1% BSA was added in the lower well in the presence or absence of 5 nM MCP-1, 20 nM s-FKN, or a mixture of 5 nM MCP-1 and 20 nM s-FKN. Freshly isolated monocytes were unable to proliferate in culture conditions, and thus, the [3H]thymidine labeling of these cells is not possible. Quantification of chemotaxis process was performed as described by Barleon et al. (31). Briefly, after incubation of migratory chambers at 37°C in air with 5% CO2 for 60 min, filters were removed, and the cells remaining in the upper well were discarded. Then, the filter was fixed in PBS-formaldehyde (1%), and the lower side of the filter was stained for 30 s with hematoxylin, and five high-power oil immersion fields (1/100) were counted. Five fields per filter were counted (×100 magnification), and each experiment was performed in triplicate. The 1-h incubation time was chosen as the time where adherent human monocyte cells do not saturate the lower face of the filter, thus allowing an optimal quantification of the chemotaxis process.

MonoMac6 stimulations were conducted in six-well plates using PET inserts with 1-μm pores (BD Biosciences) coated with 6.5 μg/ml fibronectin on both sides. MonoMac6 cells (106 cells/ml) were starved 16 h in RPMI 1640 medium supplemented with 0.1% BSA and harvested by centrifugation for 5 min at 1000 × g before being resuspended in RPMI 1640-BSA at a concentration of 2 × 107 cells/ml. Cells (107) were placed in the upper well for the indicated time periods, and serum-free medium was added in the lower well in the absence or presence of 5 nM MCP-1, 20 nM s-FKN, or a mixture of 5 nM MCP-1 and 20 nM s-FKN. After incubation, 2× lysis buffer containing 300 mM NaCl, 1.6 mM MgCl2, 10 mM EGTA, 100 mM HEPES (pH 7.5), 2% Nonidet P-40, 1 μg/ml leupeptin, 1 μM pepstatin, 1 mM PMSF, 1 mM vanadate, 1 mM NaF, and 1 mM iodoacetamide was added in the upper well, and lysates were incubated 30 min at 4°C and centrifuged at 18,000 × g for 15 min at 4°C. Supernatants were collected, and the protein concentration was assayed using the Bradford method (Bio-Rad, Hercules, CA).

Pyk2, p38, and JNK1 activities.

Lysates from each condition containing equal amounts of protein (750 μg) were incubated with 1.25 μg of anti-Pyk2 or 2.5 μg of anti-p38 or anti-JNK1 Abs overnight at 4°C. Ab-Ag complexes were then immunoprecipitated by 1-h incubation at 4°C with protein A- or G-Sepharose and then washed twice with 1× lysis buffer supplemented with 0.25% sodium deoxycholate and twice with 1× lysis buffer. Immunocomplexes were resuspended in Laemmli buffer, subjected to SDS-PAGE on 10% gels, and transferred onto Immobilon-P membranes (Millipore, Bedford, MA).

ERK activities.

Cell lysates (100 μg) were boiled after addition of 9× Laemmli sample buffer before being separated by 12% SDS-PAGE and transferred onto Immobilon-P membranes.

The membranes were then blocked for 2 h in blocking buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 0.5% gelatin, and 3% BSA), incubated overnight at 4°C with anti-phospho-specific Pyk2, anti-phospho-specific p38, anti-phospho-specific ERK, or anti-phospho-specific JNK1 Abs, washed three times in washing buffer (100 mM Tris, 1.5 M NaCl, and 1% Nonidet P-40), incubated for 1 h at room temperature with peroxidase-conjugated secondary Ab, and then washed three times in wash buffer. The immunoreactive bands were visualized using ECL kit (Amersham, Arlington Heights, IL) and BioMax films (Kodak, Rochester, NY).

Transendothelial migration and chemotaxis assays of MonoMac6 cells were conducted in 24-well chemotaxis chambers, and at the end of the experiment, conditioned medium was collected and centrifuged for 5 min at 1000 × g. Supernatants were separated on SDS-polyacrylamide (11.5%) gels containing gelatin substrate to analyze MMP-2 and MMP-9 production. After electrophoresis, gels were washed in 2.5% Triton X-100 solution for 1 h and 30 min, and then incubated in 50 mM Tris (pH 7.4), 150 mM NaCl, and 5 mM CaCl2 buffer for 48 h, and stained for 15 min at 60°C with Coomassie blue supplemented by TCA. Gels were destained in distilled water containing 7% glacial acetic acid, and clear bands of protein degradation were visualized.

To test the possibility that s-FKN interferes with the chemotactic effects of other chemokines, we measured its influence on the migration elicited by a maximal concentration of the archetypical MCP-1 chemokine. To this end, [3H]thymidine-labeled MonoMac6 cells were induced to migrate through a monolayer of HUVEC in response to 5 nM MCP-1. In this model, s-FKN and MCP-1 were added simultaneously, in the lower reservoir of the migration chamber, 20 min before the loading of MonoMac6 cells to allow the establishment of a gradient for both chemokines. As shown in Fig. 1,A, under these conditions, s-FKN abrogated the transendothelial migratory effect of MCP-1. This inhibitory effect of s-FKN was dose dependent, with an IC50 value of 7 nM (Fig. 1 B).

FIGURE 1.

MonoMac6 cell migration across HUVEC-coated filters in response to MCP-1 and s-FKN. [methyl-3H]Thymidine-labeled MonoMac6 cells were washed and loaded in the upper wells of the chemotaxis chamber. The lower well contained serum-free medium (▵) or serum-free medium supplemented with 5 nM MCP-1 (▪), 5 nM MCP-1 and 20 nM s-FKN (•), or s-FKN (○) at 20 nM (A) or at various concentrations (B). Transmigration was assayed through a TNF-α-activated monolayer of HUVECs for indicated periods (A) or for 6 h (B) at 37°C. The amount of migrated cells, collected in the lower wells, was evaluated by liquid scintillation beta counts as described in Materials and Methods. Results are expressed as the mean ± SD of triplicate measurements and are representative of four (A) or two (B) separate experiments, respectively.

FIGURE 1.

MonoMac6 cell migration across HUVEC-coated filters in response to MCP-1 and s-FKN. [methyl-3H]Thymidine-labeled MonoMac6 cells were washed and loaded in the upper wells of the chemotaxis chamber. The lower well contained serum-free medium (▵) or serum-free medium supplemented with 5 nM MCP-1 (▪), 5 nM MCP-1 and 20 nM s-FKN (•), or s-FKN (○) at 20 nM (A) or at various concentrations (B). Transmigration was assayed through a TNF-α-activated monolayer of HUVECs for indicated periods (A) or for 6 h (B) at 37°C. The amount of migrated cells, collected in the lower wells, was evaluated by liquid scintillation beta counts as described in Materials and Methods. Results are expressed as the mean ± SD of triplicate measurements and are representative of four (A) or two (B) separate experiments, respectively.

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To better delineate which of the endothelial cells or monocytic cells was the target of the antagonizing effect of s-FKN, we investigated the effect of s-FKN on MonoMac6 MCP-1-induced chemotaxis through acellular filters coated with fibronectin. As shown in Fig. 2 A, s-FKN exhibited the same antagonistic effect on MonoMac6 migration, indicating that inhibition did result from a direct alteration of MonoMac6 locomotion and not from an indirect effect toward endothelial cells.

FIGURE 2.

Cell migration across fibronectin-coated filters in response to MCP-1 and s-FKN. Cell chemotaxis was tested by the ability of [methyl-3H]thymidine-labeled MonoMac6 cells (A) or NK-L cells (B) to migrate across a fibronectin-coated filter for different periods (A) or for 1 h (B) in the absence of chemokine (□; control) or in response to 5 nM MCP-1 (▪; MCP-1), 20 nM s-FKN (○; s-FKN), or a mixture of 5 nM MCP-1 and 20 nM s-FKN (▵; MCP-1 + s-FKN). Data are expressed as the mean ± SD of triplicate measurements and are representative of three separate experiments.

FIGURE 2.

Cell migration across fibronectin-coated filters in response to MCP-1 and s-FKN. Cell chemotaxis was tested by the ability of [methyl-3H]thymidine-labeled MonoMac6 cells (A) or NK-L cells (B) to migrate across a fibronectin-coated filter for different periods (A) or for 1 h (B) in the absence of chemokine (□; control) or in response to 5 nM MCP-1 (▪; MCP-1), 20 nM s-FKN (○; s-FKN), or a mixture of 5 nM MCP-1 and 20 nM s-FKN (▵; MCP-1 + s-FKN). Data are expressed as the mean ± SD of triplicate measurements and are representative of three separate experiments.

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To rule out the possibility that the inhibitory effect of s-FKN was dependent on the cell line used, we tested the ability of s-FKN to block MCP-1-induced chemotaxis of freshly isolated monocytes. Quantification of the chemotaxis process was performed after 1 h of exposure to chemokines for the reasons explained in Materials and Methods, and the results are presented in Table I. At this time, MCP-1 induced a significant increase (p < 0.01) of migration of freshly isolated monocytes compared with the control conditions. It is worthy to note that this extent of stimulation is comparable with that observed for MonoMac6 cells, after 1-h exposure to a gradient of MCP-1 in chemotaxis conditions (Fig. 2 A). Consistent with what we observed with MonoMac6 cells, s-FKN failed to induce monocytes chemotaxis, whereas it blocked MCP-1-induced monocyte chemotaxis.

Table I.

Human CD14+ cell migration across fibronectin-coated filters in response to MCP-1 and s-FKNa

ExperimentControlMCP-1s-FKNMCP-1 + s-FKN
Median95% CIMedian95% CIpMedian95% CIpMedian95% CIp
Mono 1 50.5 46.5–52.5 69 67–70.5 p < 0.01 49.5 47.5–52 NS 50.5 47.5–53.5 NS 
Mono 2 51 48–54 67 63–70.5 p < 0.01 50.5 45.5–54.5 NS 50.5 48.5–53 NS 
ExperimentControlMCP-1s-FKNMCP-1 + s-FKN
Median95% CIMedian95% CIpMedian95% CIpMedian95% CIp
Mono 1 50.5 46.5–52.5 69 67–70.5 p < 0.01 49.5 47.5–52 NS 50.5 47.5–53.5 NS 
Mono 2 51 48–54 67 63–70.5 p < 0.01 50.5 45.5–54.5 NS 50.5 48.5–53 NS 
a

Freshly isolated human CD14+ cells were loaded in the upper reservoir of the migration chamber and exposed for 60 min to control medium (control group), 5 nM MCP-1 (MCP-1 group), 20 nM s-FKN (s-FKN group), or a mixture of 5 nM MCP-1 and 20 nM s-FKN (MCP-1 + s-FKN group) in the lower reservoir. The values of migration for each group were compared to the value of the control group. Results are given as means with the SD and median with 95% confidence interval (CI). Due to the size of the samples (five fields counted per filter; three filters per group for each experiment), nonparametric tests were used (Mann-Whitney test) to test the difference between control values and those of the other groups. A value of 0.01 was chosen for α risk, due to the multiple comparisons, according to Bonferonni’s rules.

The inhibitory effect of s-FKN seems to be specific to the monocytic lineage, because, as shown in Fig. 2 B, s-FKN was able to promote chemotaxis of NK cells, as previously described (22, 32). Moreover, this effect was enhanced in the presence of MCP-1.

To gain information on the mechanisms through which s-FKN mediated its inhibitory action, we first analyzed by Western blot analysis or by flow cytometry that 1) MonoMac6 cells did express CCR2 and CX3CR1 (Fig. 3), specific receptors for MCP-1 and s-FKN, respectively described (22, 33), and 2) incubation of MonoMac6 cells with s-FKN did not modify the level of membrane-associated CCR2 (Fig. 4), ruling out the possibility that s-FKN impairs MonoMac6 migration through the down-regulation of the MCP-1R. Furthermore, we observed that the inhibitory effect of s-FKN on MCP-1-induced chemotaxis of MonoMac6 was abrogated in the presence of blocking anti-CX3CR1 Abs (data not shown), suggesting that MCP-1 response was blocked by signals resulting from the s-FKN/CX3CR pathway.

FIGURE 3.

Expression pattern of CCR2 and CX3CR in different hemopoietic cell lines. Protein (50 μg) extracted from lysates of Jurkat (lane 1), NK-L (lane 2), THP-1 (lane 3), or MonoMac6 (lane 4) human cells were resolved by SDS-PAGE and electrophoretically transferred to Immobilon-P membrane before being probed by immunoblotting with anti-CCR2 (upper panel) or anti-CX3CR (lower panel) Abs. The results are representative of two separate experiments.

FIGURE 3.

Expression pattern of CCR2 and CX3CR in different hemopoietic cell lines. Protein (50 μg) extracted from lysates of Jurkat (lane 1), NK-L (lane 2), THP-1 (lane 3), or MonoMac6 (lane 4) human cells were resolved by SDS-PAGE and electrophoretically transferred to Immobilon-P membrane before being probed by immunoblotting with anti-CCR2 (upper panel) or anti-CX3CR (lower panel) Abs. The results are representative of two separate experiments.

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FIGURE 4.

Surface expression of CCR2 on MonoMac6 cells. After a 4-h incubation on fibronectin-coated filters in the presence of 5 nM MCP-1 without (A) or with (B) 20 nM s-FKN, MonoMac6 cells were incubated with anti-CCR2 (continuous line) or control IgG of the same isotype (dotted line) Abs and then labeled with FITC-conjugated anti-mouse Abs. Surface expression of CCR2 was evaluated by flow cytometry. The results shown are representative of two separate experiments.

FIGURE 4.

Surface expression of CCR2 on MonoMac6 cells. After a 4-h incubation on fibronectin-coated filters in the presence of 5 nM MCP-1 without (A) or with (B) 20 nM s-FKN, MonoMac6 cells were incubated with anti-CCR2 (continuous line) or control IgG of the same isotype (dotted line) Abs and then labeled with FITC-conjugated anti-mouse Abs. Surface expression of CCR2 was evaluated by flow cytometry. The results shown are representative of two separate experiments.

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Mitogen-activated protein kinases (MAPKs) are known to play a pivotal role in the control of migration of many cells (34, 35, 36, 37). We investigated the role of the different MAPK pathways, namely ERKs, SAPK1/JNKs, and SAPK2/p38, on MonoMac6 migration in response to a MCP-1 gradient using specific pharmacological inhibitors. As shown in Fig. 5, U0126, an inhibitor of MAPK/ERK kinases (38, 39) failed to affect the MonoMac6 chemotaxis induced by MCP-1, indicating that this pathway was not involved in this process. Nonetheless, we observed that both SB203580, a specific SAPK2/p38 inhibitor (40), and SP600125, a SAPK1/JNK inhibitor (41), decreased the MCP-1-induced MonoMac6 migration by 58 and 37%, respectively.

FIGURE 5.

Effect of MAPK pharmacologic inhibitors on MCP-1-induced MonoMac6 cell chemotaxis. [methyl-3H]Thymidine-labeled MonoMac6 cells were pretreated for 1 h with 40 μM SB203580, or 10 μM U0126 or SP600125, before being tested for their ability to migrate across a fibronectin-coated filter toward 5 nM MCP-1 for 4 h. Migrated cells were counted by liquid scintillation as described in Materials and Methods. Results are presented as means ± SD of triplicate samples from three separate experiments.

FIGURE 5.

Effect of MAPK pharmacologic inhibitors on MCP-1-induced MonoMac6 cell chemotaxis. [methyl-3H]Thymidine-labeled MonoMac6 cells were pretreated for 1 h with 40 μM SB203580, or 10 μM U0126 or SP600125, before being tested for their ability to migrate across a fibronectin-coated filter toward 5 nM MCP-1 for 4 h. Migrated cells were counted by liquid scintillation as described in Materials and Methods. Results are presented as means ± SD of triplicate samples from three separate experiments.

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We thus explored the possibility that s-FKN exerts its inhibitory effect on MCP-1-induced MonoMac6 chemotaxis through a blockade of SAPK activities. To test this hypothesis, MAPK activities were measured, by Western blotting with specific anti-phosphorylated-MAPK Abs recognizing active forms of these kinases, in MonoMac6 cells incubated under MCP-1-induced chemotaxis conditions, on fibronectin-coated insert, in the presence or in the absence of the s-FKN. As shown in Fig. 6, MCP-1 markedly activated both SAPK1/JNK1 and SAPK2/p38 activities, whereas it had no effect on the ERK activities. We observed that s-FKN failed to activate SAPK2/p38 and ERKs, but stimulated SAPK1/JNK1 activity to an extent similar to that observed with MCP-1. Furthermore, s-FKN abrogated the SAPK2/p38 activation induced by exposing MonoMac6 cells to an MCP-1 gradient, whereas, under the same conditions, it had, respectively, no significant effect on ERK activities and a stimulatory action on SAPK1/JNK1 activity.

FIGURE 6.

Effect of s-FKN and MCP-1 on MAPK activities of MonoMac6 cells. Cells were incubated on fibronectin-coated filters in the absence (Fibro) or presence of 5 nM MCP-1 (Fibro + MCP-1) or 20 nM s-FKN (Fibro + s-FKN) or a mixture of 5 nM MCP-1 and 20 nM s-FKN (Fibro + MCP-1 + s-FKN) for the indicated times. p38 or JNK1 activities were immunoprecipitated with anti-p38 (IP p38) or anti-JNK1 (IP JNK1) Abs from cell lysates, resolved by SDS-PAGE, and electrophoretically transferred to Immobilon-P membrane before being detected by immunoblotting with anti-phosphorylated-p38 (WB pP38) or anti-phosphorylated-JNK1 (WB pJNK1) Abs. The amount of p38 or JNK1 in immunoprecipitates was assessed by Western blotting with specific anti-p38 (WB p38) or anti-JNK1 (WB JNK1) Abs. ERK1 and ERK2 activities were detected by immunoblotting with anti-active ERK (WB pERKs) Abs and the amount of ERKs by immunoblotting with anti-ERK (WB ERKs) Abs. The results are representative of two separate experiments. The fold increases in pp38 phosphorylation are shown below the blot.

FIGURE 6.

Effect of s-FKN and MCP-1 on MAPK activities of MonoMac6 cells. Cells were incubated on fibronectin-coated filters in the absence (Fibro) or presence of 5 nM MCP-1 (Fibro + MCP-1) or 20 nM s-FKN (Fibro + s-FKN) or a mixture of 5 nM MCP-1 and 20 nM s-FKN (Fibro + MCP-1 + s-FKN) for the indicated times. p38 or JNK1 activities were immunoprecipitated with anti-p38 (IP p38) or anti-JNK1 (IP JNK1) Abs from cell lysates, resolved by SDS-PAGE, and electrophoretically transferred to Immobilon-P membrane before being detected by immunoblotting with anti-phosphorylated-p38 (WB pP38) or anti-phosphorylated-JNK1 (WB pJNK1) Abs. The amount of p38 or JNK1 in immunoprecipitates was assessed by Western blotting with specific anti-p38 (WB p38) or anti-JNK1 (WB JNK1) Abs. ERK1 and ERK2 activities were detected by immunoblotting with anti-active ERK (WB pERKs) Abs and the amount of ERKs by immunoblotting with anti-ERK (WB ERKs) Abs. The results are representative of two separate experiments. The fold increases in pp38 phosphorylation are shown below the blot.

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It is now well established that the focal adhesion kinase (FAK) tyrosine kinase family plays a crucial role in the signaling pathways implicated during the cellular adherence and migration (42, 43, 44). We first observed that, as demonstrated in freshly isolated human monocytes (45, 46) and in THP1 cells (14), the human monocytic MonoMac cells express only the related FAK tyrosine kinase Pyk2 and not p125FAK (Fig. 7).

FIGURE 7.

Expression pattern of FAK tyrosine kinase family in different human cell lines. Proteins (50 μg) extracted from lysates of human osteoblastic MG-63 cells (lane 1), freshly isolated monocytes (lane 2), and MonoMac6 (lane 3) cells were resolved by SDS-PAGE and electrophoretically transferred to Immobilon-P membrane before being detected by immunoblotting with anti-FAK or anti-Pyk2 Abs. MG-63 cells are used as positive control for FAK tyrosine kinase expression and freshly isolated monocytes as a positive control for Pyk2 tyrosine kinase expression. The results are representative of two separate experiments.

FIGURE 7.

Expression pattern of FAK tyrosine kinase family in different human cell lines. Proteins (50 μg) extracted from lysates of human osteoblastic MG-63 cells (lane 1), freshly isolated monocytes (lane 2), and MonoMac6 (lane 3) cells were resolved by SDS-PAGE and electrophoretically transferred to Immobilon-P membrane before being detected by immunoblotting with anti-FAK or anti-Pyk2 Abs. MG-63 cells are used as positive control for FAK tyrosine kinase expression and freshly isolated monocytes as a positive control for Pyk2 tyrosine kinase expression. The results are representative of two separate experiments.

Close modal

In monocytes, activation of Pyk2 appears as a critical step for monocyte motility, because inhibition of this kinase prevents cell migration (46). Several reports have shown that activation of Pyk2 was necessary for the activation of ERKs and/or SAPKs in different cell lines and in response to various stimuli (47, 48, 49, 50, 51, 52). Thus, it was of interest to determine whether the inhibition by s-FKN of MCP-1-induced p38/SAPK2 activity, which leads to the abrogation of MCP-1-induced chemotaxis of MonoMac6 cells, was associated with an inhibition of Pyk2 activity.

To analyze the state of tyrosine phosphorylation of Pyk2 under chemotaxis conditions, MonoMac6 cells were incubated on fibronectin-coated insert in the presence or in the absence of MCP-1 and/or s-FKN for the indicated durations. Cell lysates were immunoprecipitated with anti-Pyk2 Ab and subjected to immunoblot analysis with anti-phospho-Pyk2 Abs. As shown in Fig. 8, adherence to fibronectin under chemotaxis conditions in the presence of MCP-1 resulted in an enhanced Pyk2 tyrosine phosphorylation, which is completely abolished when s-FKN was added along with MCP-1.

FIGURE 8.

Inhibition of MCP-1-induced phosphorylation of Pyk2 in MonoMac6 cells. MonoMac6 cells were incubated on fibronectin-coated filters in the absence (Fibro) or presence of 5 nM MCP-1 (Fibro + MCP-1), or 20 nM s-FKN (Fibro + s-FKN), or a mixture of 5 nM MCP-1 and 20 nM s-FKN (Fibro + MCP-1 + s-FKN) for the indicated times. Pyk2 tyrosine kinases were immunoprecipitated with specific anti-Pyk2 Abs (IP Pyk2) from cell lysates, resolved by SDS-PAGE, and electrophoretically transferred to Immobilon-P membrane before being detected by immunoblotting with anti-phosphorylated Pyk2 Abs (WB pPyk2). The amount of Pyk2 in immunoprecipitates was assessed by Western blotting with specific anti-Pyk2 Abs (WB Pyk2). The results are representative of three separate experiments. The fold increases in Pyk2 phosphorylation are shown below the blot.

FIGURE 8.

Inhibition of MCP-1-induced phosphorylation of Pyk2 in MonoMac6 cells. MonoMac6 cells were incubated on fibronectin-coated filters in the absence (Fibro) or presence of 5 nM MCP-1 (Fibro + MCP-1), or 20 nM s-FKN (Fibro + s-FKN), or a mixture of 5 nM MCP-1 and 20 nM s-FKN (Fibro + MCP-1 + s-FKN) for the indicated times. Pyk2 tyrosine kinases were immunoprecipitated with specific anti-Pyk2 Abs (IP Pyk2) from cell lysates, resolved by SDS-PAGE, and electrophoretically transferred to Immobilon-P membrane before being detected by immunoblotting with anti-phosphorylated Pyk2 Abs (WB pPyk2). The amount of Pyk2 in immunoprecipitates was assessed by Western blotting with specific anti-Pyk2 Abs (WB Pyk2). The results are representative of three separate experiments. The fold increases in Pyk2 phosphorylation are shown below the blot.

Close modal

MMPs are involved in extracellular matrix degradation required for migration of monocytes (53, 54), as well as other leukocytes (55), and many reports suggest that chemokines play a key role in the regulation of MMPs during transmigration (54, 56, 57, 58). In our system, we found (Fig. 9) that the MCP-1-induced migration of MonoMac6 was abrogated in the presence of batimastat (59), a broad inhibitor of MMPs, suggesting that those enzymatic activities were mandatory for the progression of MonoMac6 cells through the endothelial monolayer.

FIGURE 9.

Effect of the MMP inhibitor batimastat on MonoMac6 transmigration. [methyl-3H]Thymidine-labeled MonoMac6 cells were subjected to migration through a TNF-α-activated monolayer of HUVECs for the indicated periods in the absence of chemokine (▴, ○) or in response to 5 nM MCP-1 (▪, •), in the absence (▴, ▪) or presence (○, •) of 1 μM batimastat. Data are expressed as the mean ± SD of triplicate measurements and are representative of two separate experiments.

FIGURE 9.

Effect of the MMP inhibitor batimastat on MonoMac6 transmigration. [methyl-3H]Thymidine-labeled MonoMac6 cells were subjected to migration through a TNF-α-activated monolayer of HUVECs for the indicated periods in the absence of chemokine (▴, ○) or in response to 5 nM MCP-1 (▪, •), in the absence (▴, ▪) or presence (○, •) of 1 μM batimastat. Data are expressed as the mean ± SD of triplicate measurements and are representative of two separate experiments.

Close modal

Gelatin zymography was then performed to evaluate MMP-2 and MMP-9 activities in the conditioned medium obtained from MonoMac6 cells subjected to transendothelial migration in the absence or presence of MCP-1, s-FKN, or both. In absence of chemokine, only latent proforms of MMP-2 (72 kDa) and MMP-9 (92 kDa) were detected in supernatants of cells (Fig. 10,A). After 16 and 24 h of migration in presence of MCP-1, we detected the presence of the two active forms of MMP-2 of 62 and 59 kDa. Interestingly, s-FKN, which blocked MCP-1-induced transendothelial migration as presented in Fig. 10 B, abrogated the apparition of the MCP-1-induced MMP-2 active forms.

FIGURE 10.

Effect of s-FKN on secreted MMP-2 and MMP-9 during MCP-1-induced MonoMac6 cell migration and chemotaxis. A and B, MonoMac6 cells were loaded in the upper well to migrate through a TNF-α-activated monolayer of HUVECs for 1 h (lanes 1 and □), 16 h (lanes 2 and ▧), or 24 h (lanes 3 and ▦) in the absence of chemokine or in response to 20 nM s-FKN, 5 nM MCP-1, or a mixture of 5 nM MCP-1 and 20 nM s-FKN. Conditioned medium was harvested and subjected to gelatin zymogram (A), and the amount of migrated cells collected in the lower wells was evaluated by liquid scintillation beta counts (B). The results shown are representative of two separate experiments. C, MonoMac6 cells were loaded in the upper well to migrate across fibronectin-coated filters for 24 h in the absence of chemokine (lane 1) or in response to 5 nM MCP-1 (lane 2), 20 nM s-FKN (lane 3), or a mixture of 5 nM MCP-1 and 20 nM s-FKN (lane 4). Conditioned medium was harvested and subjected to gelatin zymogram. The gel shown is representative of two separate experiments.

FIGURE 10.

Effect of s-FKN on secreted MMP-2 and MMP-9 during MCP-1-induced MonoMac6 cell migration and chemotaxis. A and B, MonoMac6 cells were loaded in the upper well to migrate through a TNF-α-activated monolayer of HUVECs for 1 h (lanes 1 and □), 16 h (lanes 2 and ▧), or 24 h (lanes 3 and ▦) in the absence of chemokine or in response to 20 nM s-FKN, 5 nM MCP-1, or a mixture of 5 nM MCP-1 and 20 nM s-FKN. Conditioned medium was harvested and subjected to gelatin zymogram (A), and the amount of migrated cells collected in the lower wells was evaluated by liquid scintillation beta counts (B). The results shown are representative of two separate experiments. C, MonoMac6 cells were loaded in the upper well to migrate across fibronectin-coated filters for 24 h in the absence of chemokine (lane 1) or in response to 5 nM MCP-1 (lane 2), 20 nM s-FKN (lane 3), or a mixture of 5 nM MCP-1 and 20 nM s-FKN (lane 4). Conditioned medium was harvested and subjected to gelatin zymogram. The gel shown is representative of two separate experiments.

Close modal

Endothelial cells and MonoMac6 cells are both potentially able to secrete MMPs in response to chemokines (60, 61). Thus, it was important to determine whether the MMPs of MonoMac6 cells could be involved in the inhibitory effect of s-FKN. To this purpose, we investigated the effect of s-FKN on MonoMac6 cells alone in the context of chemotaxis conditions through acellular filters. The data presented in Fig. 10 C suggest that MonoMac6 cells can secrete active MMP-2 in response to MCP-1, and that s-FKN can abrogate this secretion.

A great deal of effort has been devoted to decipher the molecular events intervening in the mode of action of chemokines. However, most of these in vitro studies have been conducted using a unique chemokine, which is far from the physiologic conditions, where a mixture of chemokines and cytokines are cosecreted at the inflammation site. This situation generates a complex array of events that are integrated at the cell level to determine the cellular activation/migratory status. To date, little information is available regarding possible reciprocal interferences between these effectors, when they are used in a combined manner. Most of the studies aimed at investigating cross talk between chemokines have focused on synergistic or down-regulating effects among chemokines of the same family (e.g., CC or CXC chemokines (62)).

Monocytes are key actors of the inflammatory process. It is now well documented that their extravasation is promoted by a panel of chemokines of the CC family, among which MCP-1 appears as the archetype (7). FKN, the unique member of the CX3C family, is markedly induced at the surface of endothelial cells in response to IL-1 and TNF-α (18). In its membrane form, FKN behaves essentially as an adhesion molecule that interacts with its coligand CX3CR1 expressed at the surface of T lymphocytes, NK cells, monocytes (18, 22), dendritic cells (23, 24), microglial cells (63), and neurons (27). The G-coupled CX3CR1 also transduces, in addition to the intrinsic adhesive function of FKN, signals that augment the integrin avidity for their ligands (64). The membrane-anchored form of FKN can be cleaved from the cell membrane by proteolysis to yield s-FKN (18, 20, 21). The resulting shedded FKN contains the chemokine domain and the mucin-like stalk domain and, conversely to the membrane form of FKN, behaves as a potent chemoattractant for almost all NK cells (65), T lymphocytes (18, 22, 66), and dendritic cells (24, 67). Migration of monocytes in response to s-FKN is still under debate. Indeed, many groups have reported that s-FKN can mediate both chemotaxis and adhesion of monocytes and THP-1 cells, whereas Pan et al. (19) have reported that neither the chemokine-like domain nor the entire extracellular domain of FKN have chemotactic effects on human monocytes and THP1 cells. In our present study, we show that s-FKN, which as we have previously reported, increases adherence of monocytic MonoMac6 cells (28), fails to induce the migration of MonoMac6 cells and monocytes. Imai et al. (22), who had initially reported a chemotactic effect of s-FKN, which they qualified as marginal compared with MCP-1, presented evidence in a following report (25), that, in monocytes, FKN functioned as an adhesion molecule, strengthening the interaction between monocytes and endothelial cells, rather than as a chemotactic factor. This data implies that, depending on the cell type, the CX3CR1/s-FKN couple induces different signaling pathways that result either in cell migration or, on the contrary, in an enhanced cell adhesion. In this context, we sought to determine how MCP-1 and s-FKN, which are coinduced by inflammatory cytokines (18, 68, 69) and which trigger opposite effects on monocytes, inducing, respectively, monocyte migration and increased stickiness, could interfere each other. Interestingly, we could demonstrate that, even in its soluble form, FKN abrogated the chemoattracting effect that MCP-1 exerts on freshly isolated monocytes as well as MonoMac6 cells, whereas it efficiently attracted NK-L cells. It is thus noteworthy that, depending on the nature of the cell lineage used, s-FKN was able either to potentiate or, the opposite, to antagonize the MCP-1 chemotactic effect.

MAPK activities have been shown to exert a crucial role in cellular migration, and their implication in this process has been highlighted by the use of specific inhibitors. Indeed, several reports have shown that SAPK2/p38 is involved in chemokine-induced chemotaxis of cells (16, 34, 70, 71). Our study also presents evidence that SAPK2/p38 and SAPK1/JNK1, but not ERKs, intervene in the control of MCP-1-induced MonoMac6 migration. Moreover, in this report, we show that, under chemotaxis conditions, SAPK2/p38 activity that is stimulated by MCP-1 is totally abrogated in the presence of s-FKN, highly suggesting that the s-FKN inhibitory effect on MCP-1-induced chemotaxis of MonoMac6 cells is mediated through the regulation of SAPK2/p38 activity.

Members of the FAK family have been shown to play a key role in the control of the adhesion/migration process (42, 43, 44) and also to intervene in the control of MAPK pathways (47, 49, 50, 51). We found that FAK is not expressed in MonoMac6 cells, whereas Pyk2, which is highly expressed in this cell line, was rapidly stimulated in response to MCP-1, in accord with previous data obtained on THP-1 cells (14) and on freshly isolated monocytes with RANTES (45). Remarkably, s-FKN abrogated the activation of Pyk2 as induced by MCP-1, consistent with a possible upstream regulation of SAPK2/p38 activity by Pyk2.

It has long been known that MMPs play an important role in the extracellular matrix remodeling that is required to allow monocyte extravasation and migration into the tissues (72).

Interestingly, s-FKN inhibits one of the major MMPs secreted by monocytes, namely MMP-2. The mechanism underlying the inhibition by s-FKN of activation of pro-MMP-2 remains to be elucidated. The observation that s-FKN can limit monocyte migration in response to MCP-1 shed a new light on the implication of this unique member of CX3C chemokine in the control of the inflammation process.

1

This work was supported by the Institut National de la Santé et de la Recherche Médicale, Association pour la Recherche sur le Cancer (Grant 5417).

3

Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein-1; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; FKN, fractalkine; s-FKN, soluble FKN; MMP, matrix metalloproteinase; EGM, endothelial cell growth medium; PET, polyethylene terephthalate; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; FAK, focal adhesion kinase.

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