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The Journal of Immunology, 2006, 176: 7412-7420.
Copyright © 2006 by The American Association of Immunologists

The CC Chemokine MCP-1 Stimulates Surface Expression of CX3CR1 and Enhances the Adhesion of Monocytes to Fractalkine/CX3CL1 via p38 MAPK1

Simone R. Green2,*, Ki Hoon Han2,3,*, Yiming Chen*, Felicidad Almazan*, Israel F. Charo{dagger}, Yury I. Miller* and Oswald Quehenberger4,*

* Department of Medicine, University of California, San Diego, La Jolla, California 92093; and {dagger} Gladstone Institute of Cardiovascular Disease, San Francisco, California 94141


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The membrane-anchored form of CX3CL1 has been proposed as a novel adhesion protein for leukocytes. This functional property of CX3CL1 is mediated through CX3CR1, a chemokine receptor expressed predominantly on circulating white blood cells. Thus far, it is still uncertain at what stage of the trafficking process CX3CR1 becomes importantly involved and how the CX3CR1-dependent adhesion of leukocytes is regulated during inflammation. The objective of this study was to examine the functional effects of chemokine stimulation on CX3CR1-mediated adhesion of human monocytes. Consistent with previous reports, our data indicate that the activity of CX3CR1 on resting monocytes is sufficient to mediate cell adhesion to CX3CL1. However, the basal, nonstimulated adhesion activity is low, and we hypothesized that like the integrins, CX3CR1 may require a preceding activation step to trigger firm leukocyte adhesion. Compatible with this hypothesis, stimulation of monocytes with MCP-1 significantly increased their adhesion to immobilized CX3CL1, under both static and physiological flow conditions. The increase of the adhesion activity was mediated through CCR2-dependent signaling and obligatory activation of the p38 MAPK pathway. Stimulation with MCP-1 also induced a rapid increase of CX3CR1 protein on the cell surface. Inhibition of the p38 MAPK pathway prevented this increase of CX3CR1 surface expression and blunted the effect of MCP-1 on cell adhesion, indicating a causal link between receptor surface density and adhesion activity. Together, our data suggest that a chemokine signal is required for firm CX3CR1-dependent adhesion and demonstrate that CCR2 is an important regulator of CX3CL1-dependent leukocyte adhesion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The interaction of leukocytes with the vascular wall and the subsequent migration across endothelial junctions are central to both immune surveillance and host defense. The molecular control of these trafficking processes is exquisitely regulated and, in accordance with the classic multistep model of leukocyte recruitment, requires the coordinated actions of adhesion molecules and chemotactic factors (1, 2, 3). At least three distinct families of adhesion molecules orchestrate leukocyte adhesion including selectins, integrins, and members of the Ig superfamily (4). Each is involved at different stages of leukocyte emigration, and the synchronization of their action is essential for functional immune response. Immune surveillance under quiescent conditions entails the continuous interaction of the circulating leukocytes with the endothelium. However, in the absence of chemokines this interaction is reversible and is mediated primarily by selectins (5, 6, 7). In the inflammatory event, locally produced chemokines lead to the activation of integrins, which then allows shear-resistant firm adhesion of leukocytes (8, 9). Chemokine signals also stimulate the subsequent diapedesis of the adherent cells through the vascular wall and their migration to sites of inflammation.

Recently, a novel integrin-independent pathway for leukocyte capture has been described that involves the chemokine fractalkine (or CX3CL15 in the standard chemokine nomenclature) (5). CX3CL1, the sole member of the CX3C family of chemokines established on the basis of the arrangement of N-terminal conserved cysteine residues, is structurally distinct from other chemokines and is encoded as a type I transmembrane protein containing multiple domains (10, 11). The chemokine domain is anchored to the plasma membrane through an extended mucin-rich stalk, fused to a transmembrane helix and an intracellular domain (12, 13). CX3CL1 is highly expressed on vascular endothelial cells but only upon activation with inflammatory cytokines (10). Other cellular sources for CX3CL1 include neurons (14), epithelial cells (15, 16), smooth muscle cells (17), dendritic cells (18), and macrophages (19).

The extended mucin-rich stalk of the endothelial-bound CX3CL1 allows the chemokine domain to be effectively presented to circulating leukocytes. Based on in vitro studies, the membrane-bound configuration of CX3CL1 induces cell adhesion through interaction with its cognate receptor CX3CR1, expressed on leukocytes including monocytes, T cells, and NK cells (20, 21). Like other chemokine receptors, CX3CR1 is a seven-transmembrane domain G protein-coupled receptor, but the adhesion does not require transmembrane signaling and is independent of calcium fluxes (22, 23). In addition to its function as an adhesion molecule, CX3CL1 can be released from the cell surface through either stimulated or constitutive proteolytic cleavage to produce a soluble molecule (24, 25). In this form, CX3CL1 acts like a conventional chemokine exerting potent chemotactic activity (10, 11).

In this study, we examined the mechanism by which the CX3CR1-dependent adhesion of monocytes is regulated under conditions of inflammation in the presence of chemokines. We found that MCP-1 markedly enhanced the CX3CR1 expression on freshly isolated human peripheral blood monocytes or monocytic cell lines and stimulated the adhesion of these cells to immobilized CX3CL1. In contrast to the basal adhesion, the chemokine-stimulated adhesion activity required the activation of CCR2, was blocked by pertussis toxin (PTX) and involved MAPK-mediated signaling events. From our data, we concluded that chemokines stabilize the CX3CR1-CX3CL1 interaction in part by increasing receptor surface density, thus providing the basis for a more shear-resistant cell adhesion.


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

Human MCP-1, mouse JE/MCP-1, the secreted forms of histidine-tagged full length human and mouse CX3CL1 (his-CX3CL1) and the anti-human MCP-1 mAb were purchased from R&D Systems. Ascitic fluid containing monoclonal anti-histidine Ab (mouse IgG2a isotype) derived from the HIS-1 hybridoma was from Sigma-Aldrich. Rabbit anti-human CX3CR1 IgG was obtained from Torrey Pines Biolabs. The anti-human CD11b/Mac-1 mAb (clone ICRF44) was from BD Bioscience, and the anti-human CD18 mAb (clone YFC118.3) was from Chemicon International. Recombinant human P-selectin/Fc chimera was purchase from R&D Systems. Rabbit anti-phospho-ERK1/2 IgG, anti-ERK1/2 IgG, anti-phospho-p38 MAPK IgG, and anti-p38 MAPK IgG were from Cell Signaling Technology. FITC-conjugated goat anti-rabbit IgG and alkaline phosphatase-conjugated goat anti-rabbit IgG were from Sigma-Aldrich. PTX and the MAPK inhibitors PD98059, U0126, and SB203580 were from Calbiochem. The CCR2-deficient mice were generated in the laboratory of Dr. Israel F. Charo at the Gladstone Institute of Cardiovascular Disease (26).

Cell culture and isolation of monocytes

THP-1 cells were obtained from American Type Culture Collection and maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS (Mediatech) as described (27). Human monocytes were isolated by magnetic separation of labeled cells using the depletion strategy to avoid cell stimulation during the isolation procedure. Whole blood was collected into 3 mM EDTA and centrifuged at 800 x g. The buffy coat from 10 ml of blood was mixed with 6 ml of PBS containing 0.2% EDTA, layered over 3 ml of Histopaque 1.077, and centrifuged at 400 x g for 30 min at room temperature. The mononuclear cells were washed twice with PBS containing 0.02% EDTA, and monocytes were isolated by negative selection with magnetic beads using the MiniMACS cell separation system according to protocol (Miltenyi Biotec). The purity of the monocytes was examined morphologically and by flow cytometry using FITC-labeled anti-CD14 Ab, as previously described (28), and was >85%.

Mouse monocytes were isolated by gradient centrifugation as described previously (28). Briefly, blood was drawn into 3 mM EDTA and centrifuged at 800 x g. The buffy coat was mixed with plasma, and 5 ml was layered onto 5 ml of Nycoprep 1.068 (VWR International) and centrifuged at 600 x g for 15 min at room temperature. The monocytes were collected and washed with PBS. The purity of the monocytes was examined morphologically and was >85%.

Analysis of surface expression of CX3CR1 by flow cytometry

THP-1 cells or freshly isolated human monocytes were resuspended at a concentration of 1 x 106 cells/ml in RPMI 1640 containing 1 mg/ml BSA and 10 mM HEPES, pH 7.4, and stimulated with various concentrations of MCP-1 for up to 45 min at 37°C. At the end of the incubation period, the cells were rapidly fixed with ice-cold 3% paraformaldehyde. The fixed cells were washed twice with ice-cold PBS and labeled with rabbit anti-human CX3CR1 IgG (2 µg/ml) followed by FITC-conjugated goat anti-rabbit IgG (1/100 dilution). The mean CX3CR1-specific fluorescence was corrected for background determined with nonspecific rabbit IgG isotypes (Jackson ImmunoResearch Laboratories). The corrected mean fluorescence of the untreated control cells was used to set the expression of CX3CR1 at 100%, and any changes in the mean fluorescence induced by the various treatments were normalized to that baseline. Flow cytometry was performed using a FACSCalibur instrument with CellQuest software (BD Bioscience).

Static adhesion assay

Wells of a 96-well plate (Corning) were coated overnight at 4°C with ascitic fluid containing monoclonal anti-polyhistidine Ab (1/1000 dilution in PBS). After two washes with PBS, the wells were blocked with adhesion buffer (RPMI 1640, 1 mg/ml BSA, 10 mM HEPES, pH 7.4) for 1 h at room temperature. The buffer was removed, the his-CX3CL1 was added in 50 µl of adhesion buffer at a final concentration of 30 nM and incubated for 1 h at 37°C. The wells were washed three times, and static adhesion assays were performed essentially as described (20).

Before the adhesion assay, THP-1 cells or freshly isolated human and murine monocytes were washed once with PBS, resuspended in adhesion buffer at 1 x 106 cells/ml, and stimulated with various concentrations of MCP-1. After the indicated stimulation periods, the chemokine was removed by centrifugation, the cells were resuspended in adhesion buffer at 20,000 cells/100 µl, added directly to each well, and incubated for 30 min at 37°C in a CO2 incubator. Nonadherent cells were removed by three washes, and the adherent cells were counted under an inverted microscope (x10). The cells of 10 randomly selected high power fields were averaged, and the data were expressed as the number of cells per field. The background binding was determined with wells that were coated with anti-histidine Ab only. Each condition was tested in three independent experiments. The specific involvement of CX3CR1 was determined in blocking experiments with anti-CX3CR1 IgG (2 µg/ml), anti-CD11b/Mac-1 mAb (10 µg/ml), and anti-CD18 mAb (10 µg/ml).

Flow chamber assay

The interaction between CX3CR1 and immobilized CX3CL1 under conditions of laminar flow was conducted by a modified method previously described (22). Plastic microscope slides (VWR International) were coated with monoclonal anti-histidine Ab as outlined above for the static adhesion assay. After blocking with BSA, a marked section on one side of the slide was incubated with his-CX3CL1 at 30 nM for 1 h at 37°C, and an equal section on the other side was left untreated and served as a control for background binding. For some experiments, slides were coated with a combination of CX3CL1 and MCP-1. To immobilize MCP-1, slides were coated overnight with monoclonal anti-MCP-1 IgG (1 µg/ml). In preliminary experiments using changes of intracellular calcium concentration as the readout, we confirmed that the anti-MCP-1 Ab did not interfere with the action of MCP-1. For coimmobilization of CX3CL1, the slides were coated simultaneously with monoclonal anti-histidine Ab. After washing and blocking, human his-CX3CL1 and/or MCP-1 were added to the slides at 30 nM and incubated as described above. For immobilization of P-selectin, slides were incubated overnight with P-selectin/Fc chimera (10 µg/ml). For coimmobilization, monoclonal anti-poly histidine Ab was present during the coating period, and his-CX3CL1 was then captured. As control, slides coated with anti-polyhistidine Ab only and without CX3CL1 or P-selectin were used. The slides were then inserted into a flow chamber with parallel plate geometry (Glycotech), mounted on an inverted microscope, and kept at 37°C.

Immediately before the start of the adhesion experiment, THP-1 cells or freshly isolated circulating monocytes were suspended at 1 x 106 cells/ml in adhesion buffer and stimulated with 20 nM MCP-1 for 15 min. The cell suspensions were perfused through the chamber for 3 min with a syringe pump (KD Scientific) set to give a flow rate that resulted in a shear force of 0.5 dyne/cm2. After the cells were loaded, the flow rate was adjusted, and the chamber was perfused with adhesion buffer at 2.5 dynes/cm2 for 3 min. In several experiments, we increased the shear stress to 5–10 dynes/cm2 but did not see a difference in adhesion (data not shown). Cells remaining firmly attached to the slide were counted in 10 randomly selected high power fields. Cells adhering to the region not coated with CX3CL1 were considered nonspecific background. Each condition was tested in three independent experiments.

Cell stimulation and MAPK assay

After serum starvation for 16 h in RPMI 1640, THP-1 cells (1.0 x 106 cells/ml) were stimulated with 20 nM MCP-1 for the indicated times. The various MAPK inhibitors or solvent vehicles were added 2 h before the chemokine stimulation. A protease inhibitor mixture (Sigma-Aldrich) and a phosphatase inhibitor mixture (Active Motif) were added according to specification, and the cells were lysed with 70 µl of Nupage LDS Sample Buffer (Invitrogen). The crude lysates were sonicated for 4 s and cleared by centrifugation at 13,000 x g for 15 min; then, equal amounts of cell lysates were analyzed by Western blot with anti-ERK1/2, anti-phospho-ERK1/2, anti-p38 MAPK, and anti-phospho-p38 MAPK Abs. Immunoreactive bands were visualized with alkaline phosphatase-conjugated secondary Ab.

Statistical analysis

All data are expressed as the mean ± SD. The two-tailed Student t test and the one-way ANOVA followed by Dunnett posttest were used for statistical analyses. p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
MCP-1 stimulates the adhesion of mononuclear cells to immobilized CX3CL1

Previous studies have demonstrated that immobilized CX3CL1 has the ability to mediate leukocyte adhesion under both static and flow conditions (20, 22). To examine whether, as with integrins, a chemokine signal enhances the basal adhesion activity, we stimulated leukocytes with MCP-1 and performed adhesion assays. Consistent with earlier reports, unstimulated THP-1 monocytes adhered to immobilized CX3CL1 under static conditions (Fig. 1A). Although the CX3CL1-specific adhesion was statistically significant, it was moderate, and the number of THP-1 cells adhering to CX3CL1 was only 2- to 3-fold higher than the number of cells adhering to the control slides, coated with the anti-histidine Ab only. However, the basal adhesion was greatly increased when the THP-1 cells were stimulated with physiological concentrations of MCP-1 before the adhesion assay (Fig. 1B). Optimal adhesion to CX3CL1 was achieved after prestimulation with MCP-1 at ~20 nM, which is also the concentration for optimal chemotaxis. A similar effect of MCP-1 was also observed with freshly isolated human peripheral blood monocytes (Fig. 2). As with THP-1 cells, MCP-1 stimulated the adhesion of human monocytes to CX3CL1-coated slides in a dose-dependent manner. Optimal adhesion was observed after stimulation with 20 nM MCP-1. The adhesion to immobilized CX3CL1 was completely blocked by soluble CX3CL1 added in excess to the adhesion buffer (data not shown).


Figure 1
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  		FIGURE 1. Adhesion of THP-1 cells to immobilized CX3CL1. THP-1 cells were applied to wells precoated with his-CX3CL1 and incubated for 1 h at 37°C as described in Materials and Methods. Nonadherent cells were removed by 3 rounds of washes, and the adherent cells were counted in 10 randomly selected high power fields and averaged. Data represent the mean ± SD of three independent experiments. A, Adhesion of unstimulated THP-1 cells to immobilized CX3CL1 ({blacksquare}). Background binding was determined with wells coated with anti-histidine Ab only ({square}).*, p < 0.01 compared with control. B, Adhesion of MCP-1-stimulated THP-1 cells. Before the adhesion assay, THP-1 cells were stimulated for 15 min with various concentrations of MCP-1. At the end of the stimulation period, the chemokine was removed by brief centrifugation, the cells were resuspended in adhesion buffer and applied to CX3CL1-coated wells (•) or to control wells coated with anti-histidine Ab but without CX3CL1 ({circ}). Adhesion values for MCP-1 above a concentration of 5 nM were significantly (p < 0.01) higher than that of the control (0 nM MCP-1). Adhesion to CX3CL1 was significantly (p < 0.001) higher at all MCP-1 concentrations than at background adhesion to control wells.

 

Figure 2
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  		FIGURE 2. Effect of soluble MCP-1 on CX3CL1-dependent adhesion of human monocytes. Freshly isolated human monocytes were stimulated for 15 min with the indicated concentrations of MCP-1 and adhesion assays were performed as described in Fig. 1. Specific adhesion was determined with CX3CL1-coated wells (•). Nonspecific background was estimated in the absence of his-CX3CL1 with wells coated only with anti-histidine Ab ({circ}). Adhesion values above 10 nM MCP-1 were significantly (p < 0.01) higher than those of control (0 nM MCP-1). Adhesion to CX3CL1 was significantly (p < 0.01) higher at all MCP-1 concentrations than at background adhesion to control wells.

 
Next, we investigated the effect of MCP-1 on monocyte adhesion to CX3CL1 under conditions of shear stress. Slides with immobilized CX3CL1 were placed into a parallel plate flow chamber, and THP-1 cells or freshly isolated human monocytes were perfused across under physiological shear stress. As shown in Fig. 3, MCP-1 significantly stimulated the adhesion of THP-1 cells under flow conditions. A similar increase in adhesion to immobilized CX3CL1 was also observed with freshly isolated human monocytes after stimulation with MCP-1.


Figure 3
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  		FIGURE 3. MCP-1-stimulated adhesion of monocytes to CX3CL1 under flow conditions. THP-1 cells or freshly isolated circulating human monocytes were stimulated with 20 nM MCP-1 for 15 min (MCP-1) and immediately used for flow adhesion analysis as described in Materials and Methods. Control cells were used without prior MCP-1 stimulation (control). The cells were perfused over immobilized his-CX3CL1 or anti-histidine Ab at a wall shear stress of 0.5 dyne/cm2 and then washed for 3 min at a wall shear stress of 2.5 dynes/cm2. Cells adhering to CX3CL1-coated slides were counted in 10 randomly selected high power fields and averaged ({blacksquare}). Nonspecific background adhesion was determined with slides coated with anti-histidine Ab only ({square}). Data represent the mean ± SD of three independent experiments. n.s., Not significant.

 
MCP-1-stimulated adhesion to CX3CL1 is transient and dependent on the engagement of CX3CR1

Analysis of the time course showed that the MCP-1-stimulated adhesion of monocytes to CX3CL1 was rapid and transient. As shown in Fig. 4, a significant increase in adhesion was observed as early as 2 min of stimulation with MCP-1. Maximal adhesion occurred within 15 min of MCP-1 stimulation but decreased thereafter. In these experiments, the cells were prestimulated with soluble MCP-1 before the adhesion assay. During the recruitment process in vivo, monocytes are exposed to chemokines presented by cells of the vessel wall only for a brief period as they move across the endothelium. To test whether short exposure to immobilized MCP-1 has similar effects on adhesion, we coated slides with a combination of MCP-1 and CX3CL1. As shown in Fig. 5, immobilized MCP-1 significantly increased the adhesion of THP-1 cells to CX3CL1 under flow without prior stimulation. Quantitatively, the adhesion was about one-half of that observed with cells that were prestimulated with MCP-1.


Figure 4
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  		FIGURE 4. Time course of MCP-1 stimulation of CX3CL1-dependent adhesion. Human monocytes were stimulated with 20 nM MCP-1 for the indicated times. At the end of the incubation periods, the chemokine was removed by centrifugation, and the cells were resuspended in adhesion buffer and used in static adhesion assays as described in Fig. 1. Shown is the number of cells specifically adhering to his-CX3CL1 after subtraction of nonspecific background adherence to wells coated with anti-histidine Ab only. Adherence was determined by averaging the number of cells in 10 randomly selected high power fields. Data represent the mean ± SD of three independent experiments. *, p < 0.01; **, p < 0.05 compared with control (0 min).

 

Figure 5
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  		FIGURE 5. Stimulation of CX3CL1-dependent flow adhesion by immobilized MCP-1. Unstimulated THP-1 cells were perfused over slides coated with either CX3CL1 or MCP-1 (imm.MCP-1) or a combination of both (CX3CL1/imm.MCP-1), and adhesion was assessed as described under Materials and Methods and Fig. 3. As a control, we included the adhesion to CX3CL1 of cells that were prestimulated with 20 nM soluble MCP-1 for 15 min (CX3CL1/sol.MCP1). Adherence was determined by averaging the number of cells in 10 randomly selected high power fields. Cell adhesion was corrected by subtracting nonspecific background adhesion, determined with slides coated with anti-histidine Ab only. Data represent the mean ± SD of three independent experiments.

 
As shown in Fig. 6, the adhesion to CX3CL1 was strictly dependent on the engagement of CX3CR1. A neutralizing anti-CX3CR1 Ab effectively inhibited both the basal as well as MCP-1-stimulated adhesion of THP-1 monocytes. In contrast, functional blockade of CD11b with antiCD11b/Mac-1 Ab (clone 44) had no affect on the adhesion. The functionality of the Ab was tested previously and shown to inhibit CD11b-dependent adhesion (29). Similarly, blockade of the common beta2 chain by anti-CD18 mAb (clone YFC118.3) had no measurable effects on the adhesion of THP-1 monocytes to immobilized CX3CL1 (data not shown).


Figure 6
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  		FIGURE 6. Adhesion to CX3CL1 requires the engagement of CX3CR1. The specific involvement of CX3CR1 in the CX3CL1-dependent adhesion was determined with blocking Abs. THP-1 monocytes were stimulated for 15 min with 20 nM MCP-1, and adhesion analyses were performed in the presence of neutralizing anti-CX3CR1 Ab (anti-CX3CR1), anti-CD11b Ab (anti-CD11b), or nonspecific IgG (none). In control experiments, cell adhesion was determined without prior MCP-1 stimulation (control). Shown is the specific adhesion as described in Fig. 4. Data represent the mean ± SD of three independent experiments. *, p < 0.01 compared with the same treatment in the control group; n.s., not significant.

 
To examine whether CX3CL1 acts alone or whether it operates in conjunction with other cell adhesion molecules in the recruitment of monocytes under shear force, slides were coated with a combination of CX3CL1 and P-selectin and used for flow adhesion of THP-1 cells. As shown in Fig. 7, the adhesion of THP-1 cells to slides coated with CX3CL1/P-selectin was significantly higher than the adhesion to slides coated with CX3CL1 only. This was true for both nonstimulated and MCP-1-stimulated THP-1 cells. Nevertheless, the increase in adhesion by P-selectin was particularly pronounced after stimulation of the cells with MCP-1. Coimmobilization of CX3CL1 and P-selectin induced the adhesion of an additional ~80 cells/field at 2.5 dynes/cm2 that was not observed with CX3CL1 alone. These results suggest that in vivo CX3CL1 may work in conjunction with P-selectin to mediate the recruitment of leukocytes from the circulation.


Figure 7
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  		FIGURE 7. P-selectin cooperates with CX3CL1 to mediate adhesion under flow conditions. THP-1 cells were stimulated with 20 nM MCP-1 for 15 min (MCP-1) or used without stimulation (unstimulated) for flow adhesion analysis as described under Materials and Methods. The flow chamber slides were coated with either CX3CL1 or P-selectin alone or with a combination of both (CX3CL1/P-selectin). Shown is the specific adhesion after subtraction of the nonspecific background adhesion, determined as described in Fig. 3. Data represent the mean ± SD of three independent experiments. *, p < 0.01 compared with the same treatment (±MCP-1) in the CX3CL1 group.

 
MCP-1 stimulates transient surface expression of CX3CR1

To examine whether the increase of adhesion was due to changes in receptor surface density, we examined by flow cytometry CX3CR1 expression on intact, nonpermeabilized monocytes. As shown in Fig. 8, A and B, treatment of THP-1 monocytes with MCP-1 induced a time- and dose-dependent expression of CX3CR1 on the cell surface. Maximal stimulation was seen with 10–20 nM MCP-1, the concentration range that was also optimal for CX3CL1-dependent adhesion. The response to MCP-1 was transient, and maximal surface expression of CX3CR1 was observed after 15 min. The fast kinetic of the surface expression excluded de novo synthesis of CX3CR1 protein and suggested mechanisms involving translocation of preformed receptors. To examine whether intracellular receptor pools are available for translocation, we analyzed total expression and surface expression by flow cytometry using permeabilized and intact cells, respectively. As shown in Fig. 8C, a major fraction of CX3CR1 localizes to cytoplasmic pools and may be available for translocation to the plasma membrane.


Figure 8
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  		FIGURE 8. Stimulation of CX3CR1 surface expression by MCP-1. Freshly isolated human monocytes were stimulated for various times with 20 nM MCP-1 or for 15 min with various chemokine concentrations and then rapidly fixed with paraformaldehyde. A, CX3CR1 surface expression was determined by flow cytometry using rabbit anti-human CX3CR1 IgG followed by the FITC-conjugated secondary Ab. The mean CX3CR1-specific fluorescence was corrected for background, determined with nonspecific rabbit IgG as the primary Ab. The surface expression of CX3CR1 was normalized to that of the untreated control cells. Data are derived from three independent experiments and expressed as the mean ± SD. *, p < 0.05; **, p < 0.01 compared with control (0 min or 0 nM MCP-1); n.s., not significant. B, Representative FACS histograms of the kinetics of CX3CR1 surface expression shown in A. The fine line represents nonspecific isotype control IgG. C, Analysis of total and surface expression of CX3CR1 in nonstimulated cells. Total expression was determined with paraformaldehyde-fixed cells after permeabilization with saponin (0.2%) for 15 min at room temperature. Surface expression was determined with fixed intact cells without permeabilization. Background fluorescence was determined with nonspecific control IgG.

 
The MCP-1-induced but not the basal adhesion to CX3CL1 requires functional CCR2 and is sensitive to PTX

Next we determined whether transmembrane-signaling events associated with CCR2, the receptor for MCP-1 (30), were required to stimulate CX3CL1-dependent adhesion. Monocytes were isolated from wild-type and CCR2-deficient mice (26) and stimulated with JE/MCP-1; the adhesion to immobilized mouse CX3CL1 was determined under flow conditions. The chemokine stimulated adhesion of monocytes from normal mice but not of monocytes from the CCR2-deficient animals (Fig. 9A). CCR2 has been shown to signal mainly through a PTX-sensitive heterotrimeric G protein of the Gi family (31, 32). As shown in Fig. 9B, PTX completely abrogated the MCP-1-stimulated adhesion of the THP-1 monocytes to human CX3CL1 under flow conditions. These results indicated that MCP-1 stimulates adhesion through a signaling pathway that is coupled to a PTX-sensitive Gi protein. Interestingly, PTX did not inhibit the nonstimulated basal adhesion of the cells to CX3CL1. This is consistent with a previous report showing that the basal CX3CL1-dependent adhesion of CX3CR1-expressing cells is independent of G protein activation (22, 23). Only the chemokine-stimulated adhesion of monocytes to CX3CL1 requires functional signaling.


Figure 9
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  		FIGURE 9. Stimulation of CX3CL1-dependent flow adhesion by MCP-1 requires CCR2-dependent transmembrane signaling. A, Monocytes were isolated from normal C57BL/6 mice (wild type) or from CCR2-deficient mice (CCR2 KO) on a C57BL/6 background, stimulated with 20 nM JE/MCP-1 for 15 min and then immediately used for flow adhesion to mouse CX3CL1 ({blacksquare}). As a control, cells were used without chemokine stimulation ({square}). B, THP-1 cells were treated with 100 ng/ml PTX for 16 h or maintained in normal culture medium (control) and then stimulated with 20 nM MCP-1 for 15 min ({blacksquare}) or used without chemokine stimulation ({square}). The cells were perfused over immobilized his-CX3CL1 or anti-histidine Ab under physiological flow conditions as described in Fig. 3. Shown is the specific adhesion to CX3CL1 after subtraction of nonspecific background adhesion. Data represent the mean ± SD of three independent experiments; n.s., not significant.

 
MCP-1 stimulates the MAPK signaling cascade

The MAPK cascade is a common signaling pathway by which G protein-coupled receptors, including CCR2, initiate functional cellular responses (33, 34). In concordance with these reports, MCP-1 rapidly and robustly stimulated ERK1/2 activities in THP-1 monocytes, with maximal phosphorylation observed at 3–10 min (Fig. 10A). MCP-1 also stimulated transient p38 MAPK phosphorylation, which peaked at ~3 min (Fig. 10B). To ascertain that the various pharmacological inhibitors were fully active in intact cells, THP-1 cells were pretreated with the MEK inhibitor U0126 and with the p38-MAPK inhibitor SB20380. Both ERK1/2 and p38 MAPK phosphorylation were blocked effectively by the respective inhibitors (Fig. 10). These results suggested that the MAP kinases are specifically activated by kinases upstream in the signaling pathway and indicated that the inhibitors are fully active in intact cells at the concentrations used.


Figure 10
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  		FIGURE 10. MCP-1 stimulates ERK1/2 and p38 MAPK activities in THP-1 cells. Serum starved (16 h) THP-1 cells were stimulated with MCP-1 (20 nM) for the indicated time periods with or without prior treatment for 2 h with either the ERK1/2 specific inhibitor U0126 (25 µM) or the p38 MAPK-specific inhibitor SB20380 (25 µM). All cells that were not treated with the inhibitors received the same amount of solvent vehicle. Cell lysates were directly subjected to immunoblotting with Abs against phosphorylated ERK1/2 (top) or total ERK1/2 (bottom) (A) and phosphorylated p38 MAPK (top) or total p38 MAPK (bottom) (B). Representative Western blots from three experiments are shown.

 
Inhibition of p38 MAPK but not ERK abrogated the MCP-1-stimulated CX3CR1 surface expression and adhesion to CX3CL1

To examine whether the stimulation of CX3CR1 expression and adhesive activity involves MAPK signaling pathways, we pretreated THP-1 cells and human peripheral blood monocytes with p38 MAPK or ERK inhibitors and then analyzed CX3CR1 surface expression as well as cell adhesion after stimulation with MCP-1. Pretreating THP-1 cells with 25 µM U0126, the selective inhibitor of MEK1/2, affected neither the basal nor the MCP-1-stimulated adhesion to immobilized CX3CL1 (Fig. 11A). Similarly, PD98059, another specific inhibitor of MEK1/2, had no effect on basal or stimulated adhesion. In contrast, SB203580, a specific inhibitor of p38{alpha} and p38beta MAPK, strongly suppressed the MCP-1-stimulated adhesion to immobilized CX3CL1 (Fig. 11A). Analysis of fixed but nonpermeabilized intact cells by flow cytometry demonstrated that inhibition of the p38 MAPK pathway blocked the increase of surface CX3CR1 (Fig. 11, B and C). PTX, which abrogated the MCP-1-stimulated adhesion (Fig. 9), had similar effects on CX3CR1 surface expression (Fig. 11, B and C). In contrast, inhibition of the ERK1/2 pathway affected neither adhesion nor CX3CR1 expression. Under identical experimental conditions, freshly isolated human monocytes provided similar results. Like with THP-1 cells, inhibition of the p38 MAPK pathway, but not the ERK1/2 pathway, blocked the MCP-1-dependent surface expression of CX3CR1 as well as adhesion of blood monocytes to immobilized CX3CL1 (Fig. 12). These data indicate the involvement of p38 MAPK in the MCP-1-induced expression of CX3CR1 and establish a mechanism by which MCP-1 promotes CX3CL1-dependent monocyte adhesion.


Figure 11
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  		FIGURE 11. Inhibition of p38 MAPK blocks MCP-1-dependent CX3CR1 surface expression and adhesion of THP-1 cells to CX3CL1. THP-1 monocytes were pretreated for 2 h with 25 µM concentrations of the ERK1/2 inhibitors U0126 or PD98059 and with the p38 MAPK inhibitor SB203580. DMSO was used to deliver the inhibitors and was tested separately, as well as cells that did not receive any addition (none). PTX was used at a concentration of 100 ng/ml for 16 h. Cells were subsequently stimulated with 20 nM MCP-1 for 15 min or used without stimulation (control). A, Static adhesion assays were performed as described in Fig. 1. Shown is the specific CX3CL1-dependent adhesion after subtraction of the nonspecific adhesion determined with wells coated with anti-histidine Ab only. The results represent the mean ± SD of three independent experiments. *, p < 0.001 compared with the same treatment in the control group; n.s., not significant. B, CX3CR1 surface expression on fixed cells was determined by flow cytometry with anti-human CX3CR1 IgG as described in Fig. 8. The CX3CR1 surface expression was normalized to that of untreated control cells in the absence of inhibitors and without stimulation, which was set at 100%. The results represent the mean ± SD of three independent experiments. *, p < 0.01 compared with the same treatment in the control group; n.s., not significant. C, Representative FACS histograms of the effect of the various inhibitors on the MCP-1-stimulated CX3CR1 surface expression. The mean fluorescence at 15 min was used for the presentation in B. Background fluorescence was determined with FITC-conjugated secondary Ab only and is shown in fine line. Untreated control cells are shown in Fig. 8B.

 

Figure 12
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  		FIGURE 12. Role of p38 MAPK in the MCP-1-induced CX3CR1 surface expression and adhesion of human blood monocytes to CX3CL1. Freshly isolated human monocytes were pretreated for 2 h with either 25 µM concentrations of the ERK1/2 inhibitor U0126 or with 25 µM p38 MAPK inhibitor SB203580 or received solvent only (DMSO). Cells were subsequently stimulated with 20 nM MCP-1 for 15 min (MCP-1) or analyzed without stimulation (control). A, Static adhesion assays were performed as described in Fig. 1. Shown is the specific CX3CL1-dependent adhesion after subtraction of the nonspecific adhesion. Results represent the mean ± SD of three independent experiments.*, p < 0.01 compared with the same treatment in the control group; n.s., not significant. B, CX3CR1 surface expression was determined by flow cytometry as described in Fig. 8. The surface expression was normalized to that of untreated control cells in the absence of inhibitors and without stimulation, which was set at 100%. The results represent the mean ± SD of three independent experiments. *, p < 0.01 compared with the same treatment in the control group; n.s., not significant. C, Representative FACS histograms of the MCP-1-stimulated CX3CR1 surface expression on cells that received solvent vehicle (control) or p38 MAPK or ERK inhibitors. The mean fluorescence at 15 min was used for the presentation in B. Background fluorescence was determined with FITC-conjugated secondary Ab only and is shown as a fine line.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we identified CCR2 as a key regulator of the adhesive properties of monocytes that are unrelated to the classic adhesion cascade involving the integrins. Recent studies have revealed a role of CX3CL1 and its cognate receptor CX3CR1 in leukocyte recruitment to the inflamed vasculature (35). Although some investigators favor a role in all stages of recruitment from the initial capture to firm adhesion, others prefer a model in which the CX3CR1/CX3CL1 system cooperates with the integrin system to mediate firm adhesion but not capture of monocytes (36, 37). Most of these studies, however, were performed with nonstimulated leukocytes. Our data show that brief stimulation of monocytes with physiological concentrations of MCP-1 greatly enhanced their adhesion to immobilized CX3CL1, both under static and flow conditions. We hypothesize that in vivo CX3CL1 and MCP-1 act concurrently as well as sequentially to support the recruitment of monocytes from the circulation to inflamed vascular tissues.

Despite evidence from in vitro studies for an important role of CX3CL1 and CX3CR1 in the recruitment of leukocytes, the in vivo function and relative contribution of this chemokine system in immunity remains uncertain. The targeted disruption of the mouse CX3CL1 gene did not produce an apparent phenotype (38). The animals developed without histological abnormalities in any major organs, showed the expected leukocyte distribution, and responded normally to a variety of inflammatory stimuli. Similarly, CX3CR1 deficiency did not interfere with monocyte extravasation in a mouse peritonitis model, and dendritic cell migration and differentiation as well as the microglial response to peripheral nerve injury were normal (39). However, more recent phenotypic studies with mouse models of atherosclerosis demonstrated that CX3CR1 deficiency caused a significant reduction of monocyte infiltration into the vessel wall and decreased atherosclerotic lesion formation (40, 41). Increasing evidence indicates that atherosclerosis is an inflammatory disease characterized by chronic monocyte infiltration (42). Chemokines, including MCP-1, play an important role in disease initiation and progression; CX3CR1 deficiency produced the most prominent phenotype in a disease model that is characterized by the critical involvement of MCP-1 (43, 44). This may establish a role for CCR2 as an activating switch for CX3CR1 and supports the model in which both MCP-1 and CX3CL1 act in unison to recruit monocytes from the circulation.

Chemokines are involved in every step of leukocyte trafficking; in addition to chemotaxis they have been shown to elicit integrin-dependent leukocyte-endothelial cell interactions (29, 45, 46). In this study, we present evidence that MCP-1 promotes the adhesion of monocytes also in an integrin-independent mechanism that involves CX3CR1. Brief activation of monocytes with MCP-1 significantly increased the adhesiveness of the cells to CX3CL1 both under static and flow conditions, suggesting that the chemokine-to-CX3CR1 signal may be important to trigger CX3CL1-dependent cell adhesion in inflammation. Analysis of the underlying mechanism showed that MCP-1 induced a transient increase of CX3CR1 expression on the plasma membrane. The kinetics of the change in receptor surface density is indicative of mobilization from intracellular pools to the plasma membrane rather than de novo synthesis of receptor protein. Treatment with MCP-1 also stimulated adhesion that followed a similar transient pattern and, like receptor expression, responded to the same pharmacological inhibitors, strongly suggesting a causal link between CX3CR1 surface density and the observed increase in adhesion.

Agonist activation of CCR2 can stimulate a number of signaling pathways that are functionally linked to discrete monocyte responses pertinent to inflammation. It is evident from our data that functional signaling via CCR2 is required for the enhancement of the adhesion activity. First, the MCP-1-stimulated adhesion is seen only with monocytes from normal but not CCR2-deficient mice. Second, the MCP-1-stimulated adhesion of monocytes to CX3CL1 is PTX sensitive, suggesting signaling mechanisms that involve Gi{alpha}. In contrast, the basal, unstimulated adhesion occurs without G protein activation (22, 23). As expected, in the absence of MCP-1, neither treatment with PTX nor CCR2 deficiency affected monocyte adhesion to CX3CL1.

Consistent with previous reports, MCP-1 activated members of the MAPK family including ERK1/2 and p38 MAPK. Using pharmacological inhibitors, we demonstrated that the CX3CL1-dependent adhesion of THP-1 cells and freshly isolated human monocytes was stimulated by MCP-1 through the p38 MAPK but not the ERK1/2 signaling cascade. A substantial body of evidence indicates that p38 MAPK is critical for many cellular processes associated with immunity and participates in the functional responses of monocytes and macrophages to proinflammatory stimuli (47). A recent report described the involvement of the p38 MAPK in the stimulation of serotonin transporter trafficking to the cell surface by the adenosine receptor (48). MCP-1 may activate a similar p38 MAPK-dependent mechanism to stimulate the translocation of CX3XR1 to the cell surface. Consistent with this, our data demonstrated that the disruption of the p38 MAPK module effectively blocked CX3CR1 surface expression during treatment with MCP-1. The fact that inhibition of p38 MAPK also abrogated the increase in adhesion suggests that translocation of CX3CR1 to the plasma membrane may be a crucial step of the mechanism by which MCP-1 modulates the adhesion activity to CX3CL1.

The adhesion of monocytes to CX3CL1 was significantly increased by the presence of P-selectins, indicating that CX3CL1 may work in conjunction with other adhesion molecules. We concluded that after P-selectins establish the initial interaction of the monocytes with the vascular wall, CX3CR1 may then become engaged to preserve adhesion in an inflammatory event. The initial tethering and rolling of monocytes along the endothelial lining result in enhanced exposure to MCP-1 (and other chemokines), which may then stimulate CX3CR1 surface trafficking and sustained adhesion.

Although an increase in surface density of CX3CR1 may be necessary for maximal and sustained adhesion, it may not be sufficient and additional mechanisms related to receptor activation may become functionally important. MCP-1 stimulated adhesion at a faster rate than expected from the change in surface expression. MCP-1 induced adhesion as early as 1–2 min of stimulation, whereas CX3CR1 surface density increased after 10–15 min, which coincided with maximal adhesion activity. Additionally, short chemokine stimulation achieved by perfusing monocytes across a combination of immobilized MCP-1 and CX3CL1 significantly increased adhesion, albeit not to the maximal extent achieved by prestimulating cells with MCP-1 before adhesion. These data invite the speculation that MCP-1 stimulates adhesion via two discrete p38 MAPK-dependent pathways, one leading to enhanced surface trafficking of CX3CR1 and a separate process that may augment the intrinsic activity of CX3CR1 present on the cell surface. Such mechanisms have been described as a model of the integrin-dependent adhesion, where chemokines induce integrin mobilization from intracellular pools and also trigger the activation of pre-existing membrane integrins (49, 50). Some of these mechanisms may also be functional during CX3CR1 activation. Recently, the concept that G protein-coupled receptors function as monomeric entities has been unsettled, and ligand-induced homo- and heterodimerization/oligomerization has emerged as a mechanism for receptor activation (51). Alternatively, MCP-1 could trigger CX3CR1 clustering and receptor trafficking to specialized membrane compartments, which would change CX3CR1 avidity. These conformational and spatial rearrangements are thought to be fast processes and may work in conjunction with CX3CR1 surface trafficking to mediate fast and sustained cell adhesion. Studies to address these issues are currently under way.

In summary, we provide evidence demonstrating that the basal CX3CL1-dependent adhesion of monocytes is greatly enhanced by MCP-1. The signaling pathway by which MCP-1 activates CX3CR1/CX3CL1-dependent adhesion involves the p38 MAPK signaling cascade. This pathway is also involved in MCP-1-mediated chemotaxis but is distinct from the pathway that mediates integrin-dependent adhesion (52). Both pathways may therefore work independently and cooperatively to support the firm adhesion of monocytes in vivo.


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


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant HL56989 (La Jolla Specialized Center of Research in Molecular Medicine and Atherosclerosis). Back

2 S.R.G. and K.H.H. contributed equally to this study. Back

3 Current address: College of Medicine, University of Ulsan, Seoul, South Korea. Back

4 Address correspondence and reprint requests to Dr. Oswald Quehenberger, Department of Medicine, 0682, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0682. E-mail address: oquehenberger{at}ucsd.edu Back

5 Abbreviations used in this paper: CX3CL1, fractalkine according to the new chemokine nomenclature; his-CX3CL1, histidine-tagged secreted form of CX3CL1; PTX, pertussis toxin. Back

Received for publication September 27, 2005. Accepted for publication March 30, 2006.


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C. Combadiere, S. Potteaux, M. Rodero, T. Simon, A. Pezard, B. Esposito, R. Merval, A. Proudfoot, A. Tedgui, and Z. Mallat
Combined Inhibition of CCL2, CX3CR1, and CCR5 Abrogates Ly6Chi and Ly6Clo Monocytosis and Almost Abolishes Atherosclerosis in Hypercholesterolemic Mice
Circulation, April 1, 2008; 117(13): 1649 - 1657.
[Abstract] [Full Text] [PDF]

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