Fractalkine/CX3CL1 Via P38 MAPK Enhances the Adhesion Of

The CC Chemokine MCP-1 Stimulates Surface Expression of CX3CR1 and Enhances the Adhesion of Monocytes to Fractalkine/CX3CL1 via p38 MAPK This information is current as of September 25, 2021. Simone R. Green, Ki Hoon Han, Yiming Chen, Felicidad Almazan, Israel F. Charo, Yury I. Miller and Oswald Quehenberger J Immunol 2006; 176:7412-7420; ; doi: 10.4049/jimmunol.176.12.7412 Downloaded from http://www.jimmunol.org/content/176/12/7412

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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

Simone R. Green,2* Ki Hoon Han,2,3* Yiming Chen,* Felicidad Almazan,* Israel F. Charo,† Yury I. Miller,* and Oswald Quehenberger4*

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 Downloaded from 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 http://www.jimmunol.org/ 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. The Journal of Immunology, 2006, 176: 7412–7420.

he interaction of leukocytes with the vascular wall and the adhesion of leukocytes (8, 9). Chemokine signals also stimulate subsequent migration across endothelial junctions are the subsequent diapedesis of the adherent cells through the vascu- central to both immune surveillance and host defense. The lar wall and their migration to sites of inflammation. T by guest on September 25, 2021 molecular control of these trafficking processes is exquisitely reg- Recently, a novel integrin-independent pathway for leukocyte ulated and, in accordance with the classic multistep model of leu- capture has been described that involves the chemokine fractalkine kocyte recruitment, requires the coordinated actions of adhesion (or CX3CL15 in the standard chemokine nomenclature) (5). molecules and chemotactic factors (1–3). At least three distinct CX3CL1, the sole member of the CX3C family of chemokines families of adhesion molecules orchestrate leukocyte adhesion in- established on the basis of the arrangement of N-terminal con- cluding selectins, integrins, and members of the Ig superfamily (4). served cysteine residues, is structurally distinct from other chemo- Each is involved at different stages of leukocyte emigration, and kines and is encoded as a type I transmembrane protein containing the synchronization of their action is essential for functional im- multiple domains (10, 11). The chemokine domain is anchored to mune response. Immune surveillance under quiescent conditions the plasma membrane through an extended mucin-rich stalk, fused entails the continuous interaction of the circulating leukocytes with to a transmembrane helix and an intracellular domain (12, 13). the endothelium. However, in the absence of chemokines this in- CX3CL1 is highly expressed on vascular endothelial cells but only teraction is reversible and is mediated primarily by selectins (5–7). upon activation with inflammatory cytokines (10). Other cellular In the inflammatory event, locally produced chemokines lead to sources for CX3CL1 include neurons (14), epithelial cells (15, 16), the activation of integrins, which then allows shear-resistant firm smooth muscle cells (17), dendritic cells (18), and macrophages (19). The extended mucin-rich stalk of the endothelial-bound *Department of Medicine, University of California, San Diego, La Jolla, California 92093; and †Gladstone Institute of Cardiovascular Disease, San Francisco, California CX3CL1 allows the chemokine domain to be effectively presented 94141 to circulating leukocytes. Based on in vitro studies, the membrane- Received for publication September 27, 2005. Accepted for publication March bound configuration of CX3CL1 induces cell adhesion through 30, 2006. interaction with its cognate receptor CX3CR1, expressed on leu- The costs of publication of this article were defrayed in part by the payment of page kocytes including monocytes, T cells, and NK cells (20, 21). Like charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. other chemokine receptors, CX3CR1 is a seven-transmembrane 1 This work was supported by National Institutes of Health Grant HL56989 (La Jolla domain G protein-coupled receptor, but the adhesion does not re- Specialized Center of Research in Molecular Medicine and Atherosclerosis). quire transmembrane signaling and is independent of calcium 2 S.R.G. and K.H.H. contributed equally to this study. fluxes (22, 23). In addition to its function as an adhesion molecule, 3 Current address: College of Medicine, University of Ulsan, Seoul, South Korea. 4 Address correspondence and reprint requests to Dr. Oswald Quehenberger, Depart- 5 Abbreviations used in this paper: CX3CL1, fractalkine according to the new che- ment of Medicine, 0682, University of California, San Diego, 9500 Gilman Drive, La mokine nomenclature; his-CX3CL1, histidine-tagged secreted form of CX3CL1; Jolla, CA 92093-0682. E-mail address: [email protected] PTX, pertussis toxin.

CX3CL1 can be released from the cell surface through either stim- 100%, and any changes in the mean fluorescence induced by the various ulated or constitutive proteolytic cleavage to produce a soluble treatments were normalized to that baseline. Flow cytometry was per- molecule (24, 25). In this form, CX3CL1 acts like a conventional formed using a FACSCalibur instrument with CellQuest software (BD Bioscience). chemokine exerting potent chemotactic activity (10, 11). In this study, we examined the mechanism by which the Static adhesion assay CX3CR1-dependent adhesion of monocytes is regulated under Wells of a 96-well plate (Corning) were coated overnight at 4°C with conditions of inflammation in the presence of chemokines. We ascitic fluid containing monoclonal anti-polyhistidine Ab (1/1000 dilution found that MCP-1 markedly enhanced the CX3CR1 expression on in PBS). After two washes with PBS, the wells were blocked with adhesion freshly isolated human peripheral blood monocytes or monocytic buffer (RPMI 1640, 1 mg/ml BSA, 10 mM HEPES, pH 7.4) for1hatroom temperature. The buffer was removed, the his-CX3CL1 was added in 50 ␮l cell lines and stimulated the adhesion of these cells to immobilized of adhesion buffer at a final concentration of 30 nM and incubated for 1 h CX3CL1. In contrast to the basal adhesion, the chemokine-stim- at 37°C. The wells were washed three times, and static adhesion assays ulated adhesion activity required the activation of CCR2, was were performed essentially as described (20). blocked by pertussis toxin (PTX) and involved MAPK-mediated Before the adhesion assay, THP-1 cells or freshly isolated human and murine monocytes were washed once with PBS, resuspended in adhesion signaling events. From our data, we concluded that chemokines buffer at 1 ϫ 106 cells/ml, and stimulated with various concentrations of stabilize the CX3CR1-CX3CL1 interaction in part by increasing MCP-1. After the indicated stimulation periods, the chemokine was re- receptor surface density, thus providing the basis for a more shear- moved by centrifugation, the cells were resuspended in adhesion buffer at resistant cell adhesion. 20,000 cells/100 ␮l, 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 Materials and Methods (ϫ10). The cells of 10 randomly selected high power fields were averaged, Downloaded from Abs and reagents and the data were expressed as the number of cells per field. The background binding was determined with wells that were coated with anti- Human MCP-1, mouse JE/MCP-1, the secreted forms of histidine-tagged histidine Ab only. Each condition was tested in three independent exper- full length human and mouse CX3CL1 (his-CX3CL1) and the anti-human iments. The specific involvement of CX3CR1 was determined in blocking MCP-1 mAb were purchased from R&D Systems. Ascitic fluid containing experiments with anti-CX3CR1 IgG (2 ␮g/ml), anti-CD11b/Mac-1 mAb monoclonal anti-histidine Ab (mouse IgG2a isotype) derived from the (10 ␮g/ml), and anti-CD18 mAb (10 ␮g/ml). HIS-1 hybridoma was from Sigma-Aldrich. Rabbit anti-human CX3CR1 http://www.jimmunol.org/ IgG was obtained from Torrey Pines Biolabs. The anti-human CD11b/ Flow chamber assay Mac-1 mAb (clone ICRF44) was from BD Bioscience, and the anti-human CD18 mAb (clone YFC118.3) was from Chemicon International. Recom- The interaction between CX3CR1 and immobilized CX3CL1 under con- binant human P-selectin/Fc chimera was purchase from R&D Systems. ditions of laminar flow was conducted by a modified method previously Rabbit anti-phospho-ERK1/2 IgG, anti-ERK1/2 IgG, anti-phospho-p38 described (22). Plastic microscope slides (VWR International) were coated MAPK IgG, and anti-p38 MAPK IgG were from Cell Signaling Technol- with monoclonal anti-histidine Ab as outlined above for the static adhesion ogy. FITC-conjugated goat anti-rabbit IgG and alkaline phosphatase-con- assay. After blocking with BSA, a marked section on one side of the slide jugated goat anti-rabbit IgG were from Sigma-Aldrich. PTX and the was incubated with his-CX3CL1 at 30 nM for1hat37°C, and an equal MAPK inhibitors PD98059, U0126, and SB203580 were from Calbio- section on the other side was left untreated and served as a control for chem. The CCR2-deficient mice were generated in the laboratory of Dr. background binding. For some experiments, slides were coated with a com-

Israel F. Charo at the Gladstone Institute of Cardiovascular Disease (26). bination of CX3CL1 and MCP-1. To immobilize MCP-1, slides were by guest on September 25, 2021 coated overnight with monoclonal anti-MCP-1 IgG (1 ␮g/ml). In prelim- Cell culture and isolation of monocytes inary experiments using changes of intracellular calcium concentration as the readout, we confirmed that the anti-MCP-1 Ab did not interfere with the THP-1 cells were obtained from American Type Culture Collection and action of MCP-1. For coimmobilization of CX3CL1, the slides were coated maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS simultaneously with monoclonal anti-histidine Ab. After washing and (Mediatech) as described (27). Human monocytes were isolated by mag- blocking, human his-CX3CL1 and/or MCP-1 were added to the slides at 30 netic separation of labeled cells using the depletion strategy to avoid cell nM and incubated as described above. For immobilization of P-selectin, stimulation during the isolation procedure. Whole blood was collected into slides were incubated overnight with P-selectin/Fc chimera (10 ␮g/ml). For 3 mM EDTA and centrifuged at 800 ϫ g. The buffy coat from 10 ml of coimmobilization, monoclonal anti-poly histidine Ab was present during blood was mixed with 6 ml of PBS containing 0.2% EDTA, layered over the coating period, and his-CX3CL1 was then captured. As control, slides 3 ml of Histopaque 1.077, and centrifuged at 400 ϫ g for 30 min at room coated with anti-polyhistidine Ab only and without CX3CL1 or P-selectin temperature. The mononuclear cells were washed twice with PBS contain- were used. The slides were then inserted into a flow chamber with parallel ing 0.02% EDTA, and monocytes were isolated by negative selection with plate geometry (Glycotech), mounted on an inverted microscope, and kept magnetic beads using the MiniMACS cell separation system according to at 37°C. protocol (Miltenyi Biotec). The purity of the monocytes was examined Immediately before the start of the adhesion experiment, THP-1 cells or morphologically and by flow cytometry using FITC-labeled anti-CD14 Ab, freshly isolated circulating monocytes were suspended at 1 ϫ 106 cells/ml as previously described (28), and was Ͼ85%. in adhesion buffer and stimulated with 20 nM MCP-1 for 15 min. The cell Mouse monocytes were isolated by gradient centrifugation as described suspensions were perfused through the chamber for 3 min with a syringe previously (28). Briefly, blood was drawn into 3 mM EDTA and centri- pump (KD Scientific) set to give a flow rate that resulted in a shear force fuged at 800 ϫ g. The buffy coat was mixed with plasma, and 5 ml was of 0.5 dyne/cm2. After the cells were loaded, the flow rate was adjusted, layered onto 5 ml of Nycoprep 1.068 (VWR International) and centrifuged and the chamber was perfused with adhesion buffer at 2.5 dynes/cm2 for 3 at 600 ϫ g for 15 min at room temperature. The monocytes were collected min. In several experiments, we increased the shear stress to 5–10 dynes/ and washed with PBS. The purity of the monocytes was examined mor- cm2 but did not see a difference in adhesion (data not shown). Cells re- phologically and was Ͼ85%. maining firmly attached to the slide were counted in 10 randomly selected high power fields. Cells adhering to the region not coated with CX3CL1 Analysis of surface expression of CX3CR1 by flow cytometry were considered nonspecific background. Each condition was tested in THP-1 cells or freshly isolated human monocytes were resuspended at a three independent experiments. 6 concentration of 1 ϫ 10 cells/ml in RPMI 1640 containing 1 mg/ml BSA Cell stimulation and MAPK assay 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 After serum starvation for 16 h in RPMI 1640, THP-1 cells (1.0 ϫ 106 cells were rapidly fixed with ice-cold 3% paraformaldehyde. The fixed cells/ml) were stimulated with 20 nM MCP-1 for the indicated times. The cells were washed twice with ice-cold PBS and labeled with rabbit anti- various MAPK inhibitors or solvent vehicles were added 2 h before the human CX3CR1 IgG (2 ␮g/ml) followed by FITC-conjugated goat anti- chemokine stimulation. A protease inhibitor mixture (Sigma-Aldrich) and rabbit IgG (1/100 dilution). The mean CX3CR1-specific fluorescence was a phosphatase inhibitor mixture (Active Motif) were added according to corrected for background determined with nonspecific rabbit IgG isotypes specification, and the cells were lysed with 70 ␮l of Nupage LDS Sample (Jackson ImmunoResearch Laboratories). The corrected mean fluorescence Buffer (Invitrogen). The crude lysates were sonicated for 4 s and cleared by of the untreated control cells was used to set the expression of CX3CR1 at centrifugation at 13,000 ϫ g for 15 min; then, equal amounts of cell lysates 7414 MCP-1 STIMULATES ADHESION OF MONOCYTES TO CX3CL1 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 Ϯ SD. The two-tailed Student t test and the one-way ANOVA followed by Dunnett posttest were used for statistical analyses. p Ͻ 0.05 was considered significant. Results 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 FIGURE 2. Effect of soluble MCP-1 on CX3CL1-dependent adhesion flow conditions (20, 22). To examine whether, as with integrins, a of human monocytes. Freshly isolated human monocytes were stimulated chemokine signal enhances the basal adhesion activity, we stimu- for 15 min with the indicated concentrations of MCP-1 and adhesion assays lated leukocytes with MCP-1 and performed adhesion assays. Con- were performed as described in Fig. 1. Specific adhesion was determined sistent with earlier reports, unstimulated THP-1 monocytes ad- with CX3CL1-coated wells (F). Nonspecific background was estimated in hered to immobilized CX3CL1 under static conditions (Fig. 1A). the absence of his-CX3CL1 with wells coated only with anti-histidine Ab Downloaded from Although the CX3CL1-specific adhesion was statistically signifi- (E). Adhesion values above 10 nM MCP-1 were significantly (p Ͻ 0.01) cant, it was moderate, and the number of THP-1 cells adhering to higher than those of control (0 nM MCP-1). Adhesion to CX3CL1 was Ͻ CX3CL1 was only 2- to 3-fold higher than the number of cells significantly (p 0.01) higher at all MCP-1 concentrations than at back- adhering to the control slides, coated with the anti-histidine Ab ground adhesion to control wells. 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 Next, we investigated the effect of MCP-1 on monocyte adhe- http://www.jimmunol.org/ CX3CL1 was achieved after prestimulation with MCP-1 at ϳ20 sion to CX3CL1 under conditions of shear stress. Slides with im- nM, which is also the concentration for optimal chemotaxis. A mobilized CX3CL1 were placed into a parallel plate flow chamber, similar effect of MCP-1 was also observed with freshly isolated and THP-1 cells or freshly isolated human monocytes were per- human peripheral blood monocytes (Fig. 2). As with THP-1 cells, fused across under physiological shear stress. As shown in Fig. 3, MCP-1 stimulated the adhesion of human monocytes to CX3CL1- MCP-1 significantly stimulated the adhesion of THP-1 cells under coated slides in a dose-dependent manner. Optimal adhesion was flow conditions. A similar increase in adhesion to immobilized observed after stimulation with 20 nM MCP-1. The adhesion to CX3CL1 was also observed with freshly isolated human monocytes after stimulation with MCP-1.

immobilized CX3CL1 was completely blocked by soluble by guest on September 25, 2021 CX3CL1 added in excess to the adhesion buffer (data not shown). 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

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 Ϯ SD of three independent experiments. A, Adhesion of unstimulated THP-1 cells to immobilized CX3CL1 (f). Background binding was FIGURE 3. MCP-1-stimulated adhesion of monocytes to CX3CL1 un- p Ͻ 0.01 der flow conditions. THP-1 cells or freshly isolated circulating human ,ء.(determined with wells coated with anti-histidine Ab only (Ⅺ compared with control. B, Adhesion of MCP-1-stimulated THP-1 cells. monocytes were stimulated with 20 nM MCP-1 for 15 min (MCP-1) and Before the adhesion assay, THP-1 cells were stimulated for 15 min with immediately used for flow adhesion analysis as described in Materials and various concentrations of MCP-1. At the end of the stimulation period, the Methods. Control cells were used without prior MCP-1 stimulation (con- chemokine was removed by brief centrifugation, the cells were resus- trol). The cells were perfused over immobilized his-CX3CL1 or anti- pended in adhesion buffer and applied to CX3CL1-coated wells (F)orto histidine Ab at a wall shear stress of 0.5 dyne/cm2 and then washed for 3 control wells coated with anti-histidine Ab but without CX3CL1 (E). Ad- min at a wall shear stress of 2.5 dynes/cm2. Cells adhering to CX3CL1- hesion values for MCP-1 above a concentration of 5 nM were significantly coated slides were counted in 10 randomly selected high power fields and (p Ͻ 0.01) higher than that of the control (0 nM MCP-1). Adhesion to averaged (f). Nonspecific background adhesion was determined with CX3CL1 was significantly (p Ͻ 0.001) higher at all MCP-1 concentrations slides coated with anti-histidine Ab only (Ⅺ). Data represent the mean Ϯ than at background adhesion to control wells. SD of three independent experiments. n.s., Not significant. The Journal of Immunology 7415

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. Quantita- tively, the adhesion was about one-half of that observed with cells that were prestimulated with MCP-1. 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 FIGURE 4. Time course of MCP-1 stimulation of CX3CL1-dependent previously and shown to inhibit CD11b-dependent adhesion (29). adhesion. Human monocytes were stimulated with 20 nM MCP-1 for the ␤ Similarly, blockade of the common 2 chain by anti-CD18 mAb indicated times. At the end of the incubation periods, the chemokine was (clone YFC118.3) had no measurable effects on the adhesion of removed by centrifugation, and the cells were resuspended in adhesion THP-1 monocytes to immobilized CX3CL1 (data not shown). buffer and used in static adhesion assays as described in Fig. 1. Shown is

To examine whether CX3CL1 acts alone or whether it operates Downloaded from the number of cells specifically adhering to his-CX3CL1 after subtraction of nonspecific background adherence to wells coated with anti-histidine Ab in conjunction with other cell adhesion molecules in the recruit- only. Adherence was determined by averaging the number of cells in 10 ment of monocytes under shear force, slides were coated with a randomly selected high power fields. Data represent the mean Ϯ SD of combination of CX3CL1 and P-selectin and used for flow adhesion p Ͻ 0.05 compared with of THP-1 cells. As shown in Fig. 7, the adhesion of THP-1 cells to ,ءء ;p Ͻ 0.01 ,ء .three independent experiments control (0 min). slides coated with CX3CL1/P-selectin was significantly higher

than the adhesion to slides coated with CX3CL1 only. This was http://www.jimmunol.org/ true for both nonstimulated and MCP-1-stimulated THP-1 cells. early as 2 min of stimulation with MCP-1. Maximal adhesion oc- Nevertheless, the increase in adhesion by P-selectin was particu- curred within 15 min of MCP-1 stimulation but decreased there- after. In these experiments, the cells were prestimulated with sol- larly pronounced after stimulation of the cells with MCP-1. Co- uble MCP-1 before the adhesion assay. During the recruitment immobilization of CX3CL1 and P-selectin induced the adhesion of ϳ 2 process in vivo, monocytes are exposed to chemokines presented an additional 80 cells/ﬁeld at 2.5 dynes/cm that was not ob- by cells of the vessel wall only for a brief period as they move served 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. by guest on September 25, 2021

FIGURE 5. Stimulation of CX3CL1-dependent flow adhesion by im- FIGURE 6. Adhesion to CX3CL1 requires the engagement of mobilized MCP-1. Unstimulated THP-1 cells were perfused over slides CX3CR1. The specific involvement of CX3CR1 in the CX3CL1-depen- coated with either CX3CL1 or MCP-1 (imm.MCP-1) or a combination of dent adhesion was determined with blocking Abs. THP-1 monocytes were both (CX3CL1/imm.MCP-1), and adhesion was assessed as described unstimulated for 15 min with 20 nM MCP-1, and adhesion analyses were der Materials and Methods and Fig. 3. As a control, we included the ad- performed in the presence of neutralizing anti-CX3CR1 Ab (anti- hesion to CX3CL1 of cells that were prestimulated with 20 nM soluble CX3CR1), anti-CD11b Ab (anti-CD11b), or nonspecific IgG (none). In MCP-1 for 15 min (CX3CL1/sol.MCP1). Adherence was determined by control experiments, cell adhesion was determined without prior MCP-1 averaging the number of cells in 10 randomly selected high power fields. stimulation (control). Shown is the specific adhesion as described in Fig. 4. p Ͻ ,ء .Cell adhesion was corrected by subtracting nonspecific background adhe- Data represent the mean Ϯ SD of three independent experiments sion, determined with slides coated with anti-histidine Ab only. Data rep- 0.01 compared with the same treatment in the control group; n.s., not resent the mean Ϯ SD of three independent experiments. significant. 7416 MCP-1 STIMULATES ADHESION OF MONOCYTES TO CX3CL1

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 Downloaded from after subtraction of the nonspeciﬁc background adhesion, determined as described in Fig. 3. Data represent the mean Ϯ SD of three independent p Ͻ 0.01 compared with the same treatment (ϮMCP-1) in ,ء .experiments the CX3CL1 group.

MCP-1 stimulates transient surface expression of CX3CR1 http://www.jimmunol.org/ 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 FIGURE 8. Stimulation of CX3CR1 surface expression by MCP-1. for CX3CL1-dependent adhesion. The response to MCP-1 was Freshly isolated human monocytes were stimulated for various times with by guest on September 25, 2021 transient, and maximal surface expression of CX3CR1 was ob- 20 nM MCP-1 or for 15 min with various chemokine concentrations and served after 15 min. The fast kinetic of the surface expression then rapidly fixed with paraformaldehyde. A, CX3CR1 surface expression excluded de novo synthesis of CX3CR1 protein and suggested was determined by flow cytometry using rabbit anti-human CX3CR1 IgG followed by the FITC-conjugated secondary Ab. The mean CX3CR1-spe- mechanisms involving translocation of preformed receptors. To cific fluorescence was corrected for background, determined with nonspe- examine whether intracellular receptor pools are available for cific rabbit IgG as the primary Ab. The surface expression of CX3CR1 was translocation, we analyzed total expression and surface expression normalized to that of the untreated control cells. Data are derived from p Ͻ ,ء .by flow cytometry using permeabilized and intact cells, respec- three independent experiments and expressed as the mean Ϯ SD p Ͻ 0.01 compared with control (0 min or 0 nM MCP-1); n.s., not ,ءء ;tively. As shown in Fig. 8C, a major fraction of CX3CR1 localizes 0.05 to cytoplasmic pools and may be available for translocation to the significant. B, Representative FACS histograms of the kinetics of CX3CR1 plasma membrane. surface expression shown in A. The fine line represents nonspecific isotype control IgG. C, Analysis of total and surface expression of CX3CR1 in The MCP-1-induced but not the basal adhesion to CX3CL1 nonstimulated cells. Total expression was determined with paraformalde- requires functional CCR2 and is sensitive to PTX hyde-fixed cells after permeabilization with saponin (0.2%) for 15 min at room temperature. Surface expression was determined with fixed intact Next we determined whether transmembrane-signaling events as- cells without permeabilization. Background fluorescence was determined sociated with CCR2, the receptor for MCP-1 (30), were required to with nonspecific control IgG. 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 de- cells is independent of G protein activation (22, 23). Only the termined under flow conditions. The chemokine stimulated adhe- chemokine-stimulated adhesion of monocytes to CX3CL1 requires sion of monocytes from normal mice but not of monocytes from functional signaling. the CCR2-deficient animals (Fig. 9A). CCR2 has been shown to signal mainly through a PTX-sensitive heterotrimeric G protein of MCP-1 stimulates the MAPK signaling cascade the Gi family (31, 32). As shown in Fig. 9B, PTX completely The MAPK cascade is a common signaling pathway by which G abrogated the MCP-1-stimulated adhesion of the THP-1 mono- protein-coupled receptors, including CCR2, initiate functional cel- cytes to human CX3CL1 under flow conditions. These results in- lular responses (33, 34). In concordance with these reports, MCP-1 dicated that MCP-1 stimulates adhesion through a signaling path- rapidly and robustly stimulated ERK1/2 activities in THP-1 mono- way that is coupled to a PTX-sensitive Gi protein. Interestingly, cytes, with maximal phosphorylation observed at 3–10 min (Fig. PTX did not inhibit the nonstimulated basal adhesion of the cells 10A). MCP-1 also stimulated transient p38 MAPK phosphoryla- to CX3CL1. This is consistent with a previous report showing that tion, which peaked at ϳ3 min (Fig. 10B). To ascertain that the the basal CX3CL1-dependent adhesion of CX3CR1-expressing various pharmacological inhibitors were fully active in intact cells, The Journal of Immunology 7417

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 FIGURE 10. MCP-1 stimulates ERK1/2 and p38 MAPK activities in mouse CX3CL1 (f). As a control, cells were used without chemokine THP-1 cells. Serum starved (16 h) THP-1 cells were stimulated with Ⅺ stimulation ( ). B, THP-1 cells were treated with 100 ng/ml PTX for 16 h Downloaded from MCP-1 (20 nM) for the indicated time periods with or without prior treat- or maintained in normal culture medium (control) and then stimulated with ment for 2 h with either the ERK1/2 specific inhibitor U0126 (25 ␮M) or 20 nM MCP-1 for 15 min (f) or used without chemokine stimulation (Ⅺ). the p38 MAPK-specific inhibitor SB20380 (25 ␮M). All cells that were not The cells were perfused over immobilized his-CX3CL1 or anti-histidine treated with the inhibitors received the same amount of solvent vehicle. Ab under physiological flow conditions as described in Fig. 3. Shown is the Cell lysates were directly subjected to immunoblotting with Abs against specific adhesion to CX3CL1 after subtraction of nonspecific background phosphorylated ERK1/2 (top) or total ERK1/2 (bottom)(A) and phosphor- adhesion. Data represent the mean Ϯ SD of three independent experiments; ylated p38 MAPK (top) or total p38 MAPK (bottom)(B). Representative n.s., not significant. http://www.jimmunol.org/ Western blots from three experiments are shown.

THP-1 cells were pretreated with the MEK inhibitor U0126 and with the p38-MAPK inhibitor SB20380. Both ERK1/2 and p38 Discussion MAPK phosphorylation were blocked effectively by the respective In this study, we identified CCR2 as a key regulator of the adhesive inhibitors (Fig. 10). These results suggested that the MAP kinases properties of monocytes that are unrelated to the classic adhesion are specifically activated by kinases upstream in the signaling cascade involving the integrins. Recent studies have revealed a pathway and indicated that the inhibitors are fully active in intact role of CX3CL1 and its cognate receptor CX3CR1 in leukocyte cells at the concentrations used. recruitment to the inflamed vasculature (35). Although some in- by guest on September 25, 2021 vestigators favor a role in all stages of recruitment from the initial Inhibition of p38 MAPK but not ERK abrogated the MCP-1- capture to firm adhesion, others prefer a model in which the stimulated CX3CR1 surface expression and adhesion to CX3CL1 CX3CR1/CX3CL1 system cooperates with the integrin system to To examine whether the stimulation of CX3CR1 expression and mediate firm adhesion but not capture of monocytes (36, 37). Most adhesive activity involves MAPK signaling pathways, we pre- of these studies, however, were performed with nonstimulated leu- treated THP-1 cells and human peripheral blood monocytes with kocytes. Our data show that brief stimulation of monocytes with p38 MAPK or ERK inhibitors and then analyzed CX3CR1 surface physiological concentrations of MCP-1 greatly enhanced their ad- expression as well as cell adhesion after stimulation with MCP-1. hesion to immobilized CX3CL1, both under static and flow con- Pretreating THP-1 cells with 25 ␮M U0126, the selective inhibitor ditions. We hypothesize that in vivo CX3CL1 and MCP-1 act con- of MEK1/2, affected neither the basal nor the MCP-1-stimulated currently as well as sequentially to support the recruitment of adhesion to immobilized CX3CL1 (Fig. 11A). Similarly, monocytes from the circulation to inflamed vascular tissues. PD98059, another specific inhibitor of MEK1/2, had no effect on Despite evidence from in vitro studies for an important role of basal or stimulated adhesion. In contrast, SB203580, a specific CX3CL1 and CX3CR1 in the recruitment of leukocytes, the in inhibitor of p38␣ and p38␤ MAPK, strongly suppressed the MCP- vivo function and relative contribution of this chemokine system in 1-stimulated adhesion to immobilized CX3CL1 (Fig. 11A). Anal- immunity remains uncertain. The targeted disruption of the mouse ysis of fixed but nonpermeabilized intact cells by flow cytometry CX3CL1 gene did not produce an apparent phenotype (38). The demonstrated that inhibition of the p38 MAPK pathway blocked animals developed without histological abnormalities in any major the increase of surface CX3CR1 (Fig. 11, B and C). PTX, which organs, showed the expected leukocyte distribution, and responded abrogated the MCP-1-stimulated adhesion (Fig. 9), had similar ef- normally to a variety of inflammatory stimuli. Similarly, CX3CR1 fects on CX3CR1 surface expression (Fig. 11, B and C). In con- deficiency did not interfere with monocyte extravasation in a trast, inhibition of the ERK1/2 pathway affected neither adhesion mouse peritonitis model, and dendritic cell migration and differ- nor CX3CR1 expression. Under identical experimental conditions, entiation as well as the microglial response to peripheral nerve freshly isolated human monocytes provided similar results. Like injury were normal (39). However, more recent phenotypic studies with THP-1 cells, inhibition of the p38 MAPK pathway, but not with mouse models of atherosclerosis demonstrated that CX3CR1 the ERK1/2 pathway, blocked the MCP-1-dependent surface ex- deficiency caused a significant reduction of monocyte infiltration pression of CX3CR1 as well as adhesion of blood monocytes to into the vessel wall and decreased atherosclerotic lesion formation immobilized CX3CL1 (Fig. 12). These data indicate the involve- (40, 41). Increasing evidence indicates that atherosclerosis is an ment of p38 MAPK in the MCP-1-induced expression of CX3CR1 inflammatory disease characterized by chronic monocyte infiltra- and establish a mechanism by which MCP-1 promotes CX3CL1- tion (42). Chemokines, including MCP-1, play an important role in dependent monocyte adhesion. disease initiation and progression; CX3CR1 deficiency produced 7418 MCP-1 STIMULATES ADHESION OF MONOCYTES TO CX3CL1 Downloaded from

FIGURE 11. Inhibition of p38 MAPK blocks MCP-1-dependent CX3CR1 surface expression and adhesion of THP-1 cells to CX3CL1. THP-1 mono-

cytes were pretreated for 2 h with 25 ␮M concentrations of the ERK1/2 inhibitors U0126 or PD98059 and with the p38 MAPK inhibitor SB203580. DMSO http://www.jimmunol.org/ 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 p Ͻ 0.001 compared with the same treatment ,ء .coated with anti-histidine Ab only. The results represent the mean Ϯ SD of three independent experiments 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, p Ͻ 0.01 compared with the same treatment in the control ,ء .which was set at 100%. The results represent the mean Ϯ SD of three independent experiments 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. by guest on September 25, 2021

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 ␮M concentrations of the ERK1/2 inhibitor U0126 or with 25 ␮M 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 Ϯ SD p Ͻ 0.01 compared ,ء.of three independent experiments 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 p Ͻ ,ء .the mean Ϯ SD of three independent experiments 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. The Journal of Immunology 7419 the most prominent phenotype in a disease model that is charac- which may then stimulate CX3CR1 surface trafficking and sus- terized by the critical involvement of MCP-1 (43, 44). This may tained adhesion. establish a role for CCR2 as an activating switch for CX3CR1 and Although an increase in surface density of CX3CR1 may be supports the model in which both MCP-1 and CX3CL1 act in necessary for maximal and sustained adhesion, it may not be suf- unison to recruit monocytes from the circulation. ficient and additional mechanisms related to receptor activation Chemokines are involved in every step of leukocyte trafficking; may become functionally important. MCP-1 stimulated adhesion in addition to chemotaxis they have been shown to elicit integrin- at a faster rate than expected from the change in surface expres- dependent leukocyte-endothelial cell interactions (29, 45, 46). In sion. MCP-1 induced adhesion as early as 1–2 min of stimulation, this study, we present evidence that MCP-1 promotes the adhesion whereas CX3CR1 surface density increased after 10–15 min, of monocytes also in an integrin-independent mechanism that in- which coincided with maximal adhesion activity. Additionally, volves CX3CR1. Brief activation of monocytes with MCP-1 sig- short chemokine stimulation achieved by perfusing monocytes nificantly increased the adhesiveness of the cells to CX3CL1 both across a combination of immobilized MCP-1 and CX3CL1 signif- under static and flow conditions, suggesting that the chemokine- icantly increased adhesion, albeit not to the maximal extent to-CX3CR1 signal may be important to trigger CX3CL1-depen- achieved by prestimulating cells with MCP-1 before adhesion. dent cell adhesion in inflammation. Analysis of the underlying These data invite the speculation that MCP-1 stimulates adhesion mechanism showed that MCP-1 induced a transient increase of via two discrete p38 MAPK-dependent pathways, one leading to CX3CR1 expression on the plasma membrane. The kinetics of the enhanced surface trafficking of CX3CR1 and a separate process change in receptor surface density is indicative of mobilization that may augment the intrinsic activity of CX3CR1 present on the from intracellular pools to the plasma membrane rather than de cell surface. Such mechanisms have been described as a model of Downloaded from novo synthesis of receptor protein. Treatment with MCP-1 also the integrin-dependent adhesion, where chemokines induce inte- stimulated adhesion that followed a similar transient pattern and, grin mobilization from intracellular pools and also trigger the ac- like receptor expression, responded to the same pharmacological tivation of pre-existing membrane integrins (49, 50). Some of inhibitors, strongly suggesting a causal link between CX3CR1 sur- these mechanisms may also be functional during CX3CR1 activa- face density and the observed increase in adhesion. tion. Recently, the concept that G protein-coupled receptors func-

Agonist activation of CCR2 can stimulate a number of signaling tion as monomeric entities has been unsettled, and ligand-induced http://www.jimmunol.org/ pathways that are functionally linked to discrete monocyte re- homo- and heterodimerization/oligomerization has emerged as a sponses pertinent to inflammation. It is evident from our data that mechanism for receptor activation (51). Alternatively, MCP-1 functional signaling via CCR2 is required for the enhancement of could trigger CX3CR1 clustering and receptor trafficking to spe- the adhesion activity. First, the MCP-1-stimulated adhesion is seen cialized membrane compartments, which would change CX3CR1 only with monocytes from normal but not CCR2-deficient mice. avidity. These conformational and spatial rearrangements are Second, the MCP-1-stimulated adhesion of monocytes to CX3CL1 thought to be fast processes and may work in conjunction with is PTX sensitive, suggesting signaling mechanisms that involve CX3CR1 surface trafficking to mediate fast and sustained cell ad- Gi␣. In contrast, the basal, unstimulated adhesion occurs without hesion. Studies to address these issues are currently under way. G protein activation (22, 23). As expected, in the absence of In summary, we provide evidence demonstrating that the basal by guest on September 25, 2021 MCP-1, neither treatment with PTX nor CCR2 deficiency affected CX3CL1-dependent adhesion of monocytes is greatly enhanced by monocyte adhesion to CX3CL1. MCP-1. The signaling pathway by which MCP-1 activates Consistent with previous reports, MCP-1 activated members of CX3CR1/CX3CL1-dependent adhesion involves the p38 MAPK the MAPK family including ERK1/2 and p38 MAPK. Using phar- signaling cascade. This pathway is also involved in MCP-1-medi- macological inhibitors, we demonstrated that the CX3CL1-depen- ated chemotaxis but is distinct from the pathway that mediates dent adhesion of THP-1 cells and freshly isolated human mono- integrin-dependent adhesion (52). Both pathways may therefore cytes was stimulated by MCP-1 through the p38 MAPK but not the work independently and cooperatively to support the firm adhesion ERK1/2 signaling cascade. A substantial body of evidence indi- of monocytes in vivo. cates that p38 MAPK is critical for many cellular processes asso- ciated with immunity and participates in the functional responses Disclosures of monocytes and macrophages to proinflammatory stimuli (47). A The authors have no financial conflict of interest. recent report described the involvement of the p38 MAPK in the stimulation of serotonin transporter trafficking to the cell surface References by the adenosine receptor (48). MCP-1 may activate a similar p38 1. Butcher, E. 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