Chemotaxis to CX3CL1 (Fractalkine) Syk Is Required for Monocyte/Macrophage

Syk Is Required for Monocyte/Macrophage Chemotaxis to CX3CL1 (Fractalkine) Jean-Claude Gevrey, Beth M. Isaac and Dianne Cox This information is current as J Immunol 2005; 175:3737-3745; ; of September 26, 2021. doi: 10.4049/jimmunol.175.6.3737 http://www.jimmunol.org/content/175/6/3737 Downloaded from References This article cites 59 articles, 36 of which you can access for free at: http://www.jimmunol.org/content/175/6/3737.full#ref-list-1

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Syk Is Required for Monocyte/Macrophage Chemotaxis to CX3CL1 (Fractalkine)1

Jean-Claude Gevrey,2* Beth M. Isaac,* and Dianne Cox*†

CX3CL1 (fractalkine), the only member of the ␦ subclass of chemokines, is a known chemotactic factor for monocytes/macrophages as well as NK cells and T lymphocytes. In several pathologies, excessive production of CX3CL1 at speciﬁc sites leads primarily to monocyte/macrophage recruitment, which causes tissue and vascular damage. Despite their clinical relevance, the mechanisms underlying monocyte/macrophage chemotaxis to CX3CL1 remain poorly documented. The present report addresses this issue and identiﬁes cell signaling crucial for this process. Using the murine monocyte/macrophage RAW cell line, we show that CX3CL1 treatment elicits a rapid and transient increase in F-actin and the formation of F-actin-enriched cell protrusions. CX3CL1 also triggers tyrosine phosphorylation of proteins localized in those protrusions. The protein tyrosine kinase Syk is

activated upon CX3CL1 treatment, and reduction of Syk expression using RNA-mediated interference results in a speciﬁc and Downloaded from massive impairment of RAW cell migration to CX3CL1. Similar results are obtained using the Syk inhibitor, piceatannol. Cells with reduced Syk expression also exhibit a major defect in CX3CL1-induced cytoskeletal remodeling. These data suggest that in monocytes/macrophages, Syk is essential for proper reorganization of the actin cytoskeleton in response to CX3CL1 and is therefore required for cell chemotaxis to CX3CL1. The Journal of Immunology, 2005, 175: 3737–3745.

lso known as fractalkine or neurotactin, CX3CL1 is as of marked decrease in monocyte/macrophage accumulation com- http://www.jimmunol.org/ now the only member of the ␦/CX3C subclass of che- pared with CX3CR1ϩ/ϩ animals (8, 9). Also, soluble CX3CL1 is A mokines, established on the basis of the arrangement of up-regulated in the synovial fluid of rheumatoid arthritis patients N-terminal conserved cysteine residues (1, 2). In vivo, CX3CL1 and induces the migration of CX3CR1-expressing monocytes (4). appears to be expressed by a variety of tissues and cell types. In Despite the clinical relevance of the interaction between cells of particular, it is found as a transmembrane-anchored molecule at the the monocyte/macrophage lineage and CX3CL1, there is no infor- surface of endothelial cells activated with inflammatory cytokines, mation available regarding the mechanisms underlying their ability where it behaves as an adhesion molecule. When released from the to chemotax toward this molecule. Using the MonoMac6 cell line cell surface through proteolytic cleavage, the soluble domain of in response to soluble CX3CL1, Cambien et al. (11) have identi- CX3CL1 acts as a chemotactic factor for monocytes, NK cells, and fied PI3K and members of the MAPK family, ERK1/2, p38MAPK, by guest on September 26, 2021 T cells (1, 2). CX3CL1 binds exclusively the cell surface, seven- and JNK1, as signaling components required for cell adhesion to transmembrane, G protein-coupled receptor CX3CR1 and repre- fibronectin. Activation of Src and phosphorylation of Syk tyrosine sents its sole cognate ligand to date (3). kinases downstream of CX3CR1 have also been reported, although Originally considered a homeostatic chemokine involved in a putative functional role for these enzymes in any CX3CL1-in- basal trafficking/homing of leukocytes, CX3CL1 is now increas- duced cell response has not been investigated (11). Surprisingly, ingly recognized as a molecule whose expression is enhanced un- MonoMac6 cells fail to achieve chemotaxis or transendothelial der particular pathological situations, causing an excessive recruit- migration in response to CX3CL1 (12). The THP-1 monocytic cell ment of cytotoxic leukocytes and therefore extensive vascular and line, also used in studies addressing the mechanism of CX3CL1- tissue injury. Recently, accumulating evidence has indicated a role for CX3CL1 in the pathogenesis of a number of diseases, includ- dependent cell adhesion (13), has similarly been reported to have ing, but not limited to, rheumatoid arthritis (4–6) and atheroscle- a negligible chemotactic response to CX3CL1 (14). These obser- rosis (7–10). Interestingly, mononuclear phagocytes represent the vations indicate that MonoMac6 and THP-1 cell lines, both estab- key component of the cellular infiltrates in those two cases, and lished from an acute monocytic leukemia patient, do not mimic the their recruitment in response to CX3CL1 has been shown to sig- behavior of monocytes/macrophages in vivo or freshly isolated nificantly contribute to the extent of the disease. Indeed, monocytes as far as chemotaxis toward CX3CL1 is concerned (1, CX3CR1Ϫ/Ϫ mice placed into an atherosclerosis-prone back- 8, 9, 15–17). The reasons for this apparent discrepancy are ground have reduced atherosclerotic lesion formation as well as a unknown. Using cells derived from the murine monocyte/macrophage RAW 264.7 cell line, we aimed at identifying signaling compo- *Department of Anatomy and Structural Biology and †Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461 nents required for cell migration in response to soluble CX3CL1. Received for publication February 23, 2005. Accepted for publication July 12, 2005. In the present report we provide evidence that these cells represent a valuable model to study CX3CL1-induced chemotaxis and ex- 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 hibit morphological changes and remodeling of the actin cytoskel- with 18 U.S.C. Section 1734 solely to indicate this fact. eton upon CX3CL1 treatment. Because tyrosine phosphorylation 1 This work was supported by Grant KO1AR02158 from the National Institutes of has been reported to be important in the function of several che- Health (to D.C.). mokine receptors, we explored this possibility with respect to 2 Address correspondence and reprint requests to Dr. Jean-Claude Gevrey, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Gruss/MRRC Room 306, CX3CR1-mediated cell chemotaxis. We show that the cytoplasmic Bronx, NY 10461. E-mail address: [email protected] protein tyrosine kinase Syk is rapidly activated by CX3CL1, and

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 3738 Syk IN CX3CL1 CHEMOTAXIS using RNA-mediated interference to reduce endogenous Syk lev- fixation and permeabilization as described in Immunofluorescence micros- els, we show that Syk is required for proper formation of mem- copy above. Fixed cells were incubated with saturating concentrations of brane protrusions and cell chemotaxis in response to CX3CL1. Our rhodamine-phalloidin and YO-PRO-1 (both from Molecular Probes) to stain F-actin and nucleic acids, respectively. Fluorescence intensities of data demonstrate a new function of Syk downstream of a chemo- rhodamine (excitation wavelength, 545 nm; emission wavelength, 590 nm) kine receptor besides its well-documented roles in signal transduc- and YO-PRO-1 (excitation wavelength, 485 nm; emission wavelength, 520 tion from immunoreceptors and integrins in hemopoietic cells. nm) were measured using a plate reader (Polarstar Optima), and the nor- malized F-actin cellular content (calculated as the ratio of rhodamine to YO-PRO-1 fluorescence) was expressed as the percent increase in response Materials and Methods to CX3CL1 compared with the unstimulated condition. Cells, Abs, and reagents RAW/LR5 cells (RAW), derived from the murine monocyte/macrophage Immunoprecipitation and Western blotting RAW 264.7 cell line, have been described previously (18) and were cultured in RPMI 1640 medium (Mediatech) supplemented with 10% heat- Cells were lysed in ice-cold buffer A containing 25 mM Tris, 137 mM inactivated FBS (Sigma-Aldrich) and antibiotics (100 IU/ml penicillin/100 NaCl, 1% Triton X-100, 1% SDS, 2 mM EDTA, 1 mM orthovanadate, 1 ␮g/ml streptomycin). Murine bone marrow-derived macrophages were iso- mM benzamidine, 10 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin, pH 7.4. For lated and prepared as previously described (19) and were cultured in immunoprecipitation, the SDS concentration of the total cell lysate was ␣-MEM supplemented with 15% FBS, 360 ng/ml recombinant human lowered to Ͻ0.2% by dilution in buffer A without SDS before incubation CSF-1 (Chiron), and antibiotics. All cells were maintained at 37°C in a 5% with Abs prebound to Protein A/G Plus agarose beads (Santa Cruz Bio-

CO2 atmosphere. Recombinant mouse CX3CL1 (aa 25–105) and mouse technology) overnight at 4°C. Beads were then pelleted, washed three CSF-1 were purchased from R&D Systems. Rabbit anti-CX3CR1 Ab times, resuspended in Laemmli buffer, and boiled for 5 min. Total cell (TP501) was obtained from Torrey Pines Biolabs; mouse anti- lysates and/or immunoprecipitates were resolved by SDS-PAGE, and pro- Downloaded from phosphotyrosine (PY99), rabbit anti-Syk (LR and N19), rabbit anti- teins were transferred onto polyvinylidene diﬂuoride membranes (Immo- ERK1/2 (C14), and mouse anti-JNK1 (F3) Abs were purchased from Santa bilon-P; Millipore) that were subsequently blocked using 3% BSA/1% Cruz Biotechnology; rabbit anti-phospho-Syk (Y519/520 in mouse, Y525/ OVA in TBS containing 0.1% Tween 20 before incubation with primary 526 in human), mouse anti-phospho-ERK1/2 Abs (T202/Y204), rabbit Abs overnight at 4°C. Membranes were then washed and incubated with anti-phospho-p38MAPK (T180/Y182), rabbit anti-p38MAPK, and mouse anti- secondary Abs conjugated to HRP. Signals were visualized using the Su- phospho-JNK (T183/Y185) were obtained from Cell Signaling Technol- perSignal West Pico chemiluminescent substrate (Pierce), and images were ogy; anti-␤-actin (AC-15) Ab was purchased from Sigma-Aldrich. Alexa acquired using a Kodak Image Station 440.

Fluor 568-phalloidin and all secondary Abs conjugated to Alexa Fluor 488 http://www.jimmunol.org/ or 568 used for immunostaining were obtained from Molecular Probes. All secondary Abs conjugated to HRP (used for Western blotting) were pur- Generation of cells with stable reduced Syk expression using chased from Jackson ImmunoResearch Laboratories. Pertussis toxin from RNA-mediated interference Bordetella pertussis was obtained from Sigma-Aldrich. The Syk inhibitor, piceatannol, and the MEK inhibitor, PD98059, were obtained from EMD Reduction of Syk expression was achieved through the retroviral infection Biosciences. of RAW cells with short hairpin RNAs directed against the Syk mRNA, using pSUPER.retro.puro (pSUP)3 plasmids (Oligoengine). Two different Immunofluorescence microscopy 19-nt target sequences were selected in the mouse Syk cDNA coding sequence as follows: sh#1, 5Ј-GGCACACCACTACACCATC-3Ј (177–195; RAW cells or bone marrow-derived macrophages were plated on 12-mm SH2 domain); and sh#2, 5Ј-GGGAAAGTATTGCACTACC-3Ј (627–645, glass coverslips and serum-starved for at least 3 h before stimulation. For kinase domain); each one was used to design a 60-nt oligo, which was by guest on September 26, 2021 stimulation, cells were treated with BWD buffer (20 mM HEPES, 125 mM subcloned between the BglII and HindIII restriction sites of the pSUP vec- NaCl, 5 mM KCl, 5 mM dextrose, 10 mM NaHCO3,1mMKH2PO4, 1 tor according to the manufacturer’s instructions. The resulting pSUP/ mM CaCl2, and 1 mM MgCl2, pH 7.4) in the presence or the absence of Syksh#1 and pSUP/Syksh#2 plasmids were transfected into a 293T-based CX3CL1 at 37°C for the indicated times. Cells were then fixed for 7 min packaging cell line. The corresponding culture supernatants were then used using 3.7% formaldehyde in BWD and were permeabilized for 5 min using to retrovirally infect RAW cells. Infected cells were selected on the basis 0.2% Triton X-100 in BWD before blocking and incubation with Abs of their resistance to puromycin (brought by the pSUP plasmids), and their and/or phalloidin. Coverslips were mounted on slides using a medium con- level of Syk protein expression was monitored by Western blotting. Puro- taining 100 mM n-propylgallate. For extracellular staining of CX3CR1, mycin-resistant heterogeneous cell populations as well as single cell-de- live cells were preincubated on ice in BWD containing 1% BSA and a rived populations were obtained with significant (Ͼ70%) and stable (for a 1/100 dilution of mouse FcBlock (2.4G2 Ab; BD Pharmingen) before add- period of at least 3 wk) reduction of Syk. As a control, the same set of ing the primary Ab, followed by the secondary Ab solution, both in cold experiments was performed using a mock short hairpin RNA (shRNA) 1% BSA/BWD. Cells were kept on ice for Ͻ1 h, then fixed and mounted sequence corresponding to sh#1 in a scrambled order (5Ј-TCCACACCAC as described above. Images shown in Figs. 1, 2, and 8 were taken using the TACACGAGC-3Ј), referred to as scrambled later in the text. ϫ60 oil/1.40 phase 3 objective of an Olympus IX71 microscope coupled to a Sensicam-cooled CCD camera. Images shown in Fig. 3 were taken using a ϫ60 immersion objective of a confocal laser-scanning microscope Phagocytosis assay (model radiance 2000; Bio-Rad). The ability of cells to perform Fc␥R-mediated phagocytosis was assessed Chemotaxis assay as described previously (18). Briefly, cells were incubated with a suspen- sion of sheep erythrocytes opsonized with rabbit IgG (IgG-RBC) for 30 Cell chemotaxis was measured using a transmigration chamber assay with min at 37°C in BWD. Noninternalized IgG-RBCs were then removed by 8-␮m pore size inserts (Falcon; BD Biosciences) according to the manu- washes, followed by hypotonic lysis. At least 100 cells were observed by facturer’s instructions. Briefly, the inserts were placed into 24-well plates phase contrast microscopy, and the number of erythrocytes in each cell was containing RPMI 1640 in the presence or the absence of CX3CL1. Serum- counted and expressed as the average number of particles ingested per cell starved cells (n ϭ 500,000) were then loaded onto the inserts and incubated (phagocytic index). at 37°C for 4 h. Cell migration was quantified by counting the number of cells that migrated through the insert (at least 10 different randomly selected fields in each well were counted under a phase contrast microscope) Data analysis and was expressed as the percentage of migrated cells in the absence of any Ϯ stimulant. All results were calculated as the mean SEM. Data were analyzed using Student’s t test, and differences between two means with a value of p Ͻ Cellular F-actin measurement 0.05 were regarded as significant. Measurement of total F-actin content was mainly performed as described previously (20). Briefly, 50,000 cells were plated into wells of a 96-well plate (in quadruplicate for each condition) and serum-starved for at least 3 Abbreviations used in this paper: pSUP, pSUPER.retro.puro; scr, scrambled-treated; 3 h. Cells were then incubated with BWD in the presence or the absence shRNA, short hairpin RNA; WASp, Wiskott-Aldrich syndrome protein; NI, of CX3CL1 for various times at room temperature, followed by prompt noninfected. The Journal of Immunology 3739

Results RAW cell migration in response to CX3CL1. Preincubation of RAW cells as a model to study CX3CL1 chemotaxis RAW cells with either CX3CR1 neutralizing Ab (the same as used in Fig. 1A) or pertussis toxin both resulted in a massive inhibition To study monocyte/macrophage chemotaxis in response to of CX3CL1-induced cell migration, indicating a specific involve- CX3CL1 (fractalkine), we first selected a cell model that was ap- ment of CX3CR1 as well as one or more pertussis toxin-sensitive propriate for this purpose. RAW 264.7 cells have been extensively G proteins in this process (Fig. 1, D and E). These data indicated used as a bona fide replica for moderately differentiated macro- that RAW cells expressed a functional receptor for CX3CL1 and phages and have been shown to respond to physiological stimuli exhibited genuine chemotaxis toward this molecule, in accordance regulating proliferation (21), production of NO (22, 23) and cyto- with the behavior of monocytes/macrophages in vivo. kines (22, 24), phagocytosis (18, 25, 26), or chemotaxis (27). To determine whether RAW cells could also represent a valid model to study CX3CL1-induced chemotaxis, we first verified that they CX3CL1 induces reorganization of the actin cytoskeleton expressed CX3CR1 at their surface by performing immunostaining on live cells at 4°C, a temperature known to prevent internalization The ability of cells to move implies a dynamic remodeling of the (18). A bright staining was observed when cells were incubated actin cytoskeleton that governs the extension of cell protrusions with CX3CR1 Ab, but no significant staining was detected using and the retraction of the cell body (reviewed in Ref. 28). To doc- corresponding matching isotype IgG (Fig. 1A). The ability of ument the cell response to CX3CL1 in terms of morphology and RAW cells to chemotax to soluble CX3CL1 was then assessed actin dynamics, we stimulated RAW cells for various times with using a transmigration chamber assay as described in Materials 50 ng/ml CX3CL1 before fixation and F-actin staining using phal- Downloaded from and Methods. Increasing the dose of CX3CL1 in the lower com- loidin. We typically observed a robust formation of thin and un- partment of the chamber resulted in a significant increase in cell dulating F-actin-enriched membrane protrusions (also termed ruf- migration starting at the dose of 1 ng/ml (Fig. 1B). Both 50 and fles) on the dorsal and lateral sides of the cells, which appeared to 150 ng/ml CX3CL1 appeared to trigger a comparable maximal cell be maximal after 1-min stimulation (Fig. 2A). Quantitative mea- response. To determine whether CX3CL1 stimulated RAW cell surement of the total F-actin cellular content showed a rapid in- chemotaxis (i.e., directional cell movement dependent on a con- crease detectable within 10 s after addition of CX3CL1 and peak- centration gradient) and/or chemokinesis (i.e., random cell motility ing at 1 min to represent an ϳ30% increase of F-actin compared http://www.jimmunol.org/ stimulated in a gradient-independent manner), we added 50 ng/ml with nonstimulated cells (Fig. 2B). The amount of F-actin then CX3CL1 only in the top compartment of the chamber. In this case, decreased to reach levels close to the basal after 3 min. Therefore, cell migration remained similar to the basal level (Fig. 1C), clearly RAW cells massively reorganized their cytoskeleton upon indicating that a concentration gradient was required for efficient CX3CL1 treatment, resulting in a rapid and transient increase in by guest on September 26, 2021 FIGURE 1. RAW cells chemotax toward CX3CL1 (fractalkine). A, Live cells were incubated with rabbit anti-CX3CR1 Ab or control IgG at 4°C before fixation and incubation with anti-rabbit IgG Ab conjugated to Alexa Fluor 488. Immunofluores- cence images are shown as well as the corresponding phase contrast images (see insets). Scale bar ϭ 10 ␮m. B, Cell migration in response to increasing doses of CX3CL1 was measured as described in Materials and Methods. C, To distinguish between chemotaxis and chemokinesis, cell migration was assessed in response to 50 ng/ml CX3CL1 added in either the bottom chamber or the top chamber of the transmigration apparatus. D, The specificity of CX3CL1-induced chemotaxis was verified by preincu- bating the cells with 10 ␮g/ml CX3CR1 neutralizing Ab or control IgG for 1 h before subjecting the cells to the transmigration assay. E, Cells were preincubated for 5 h with or without 250 ng/ml pertussis toxin (PTX) before measuring cell migra- p Ͻ ,ء .tion as described above. n ϭ 3 0.05 compared with the corresponding controls. 3740 Syk IN CX3CL1 CHEMOTAXIS Downloaded from http://www.jimmunol.org/

FIGURE 2. CX3CL1 treatment induces a rearrangement of the actin cytoskeleton. A, RAW cells were either untreated (Untr) or treated with 50 ng/ml CX3CL1 for 1 min before fixation and staining of F-actin using phalloidin conjugated to Alexa Fluor 568. Images are representatives of at least five independent experiments. Scale bar ϭ 10 ␮m. B, Cells were treated for various times with CX3CL1, then fixed, and total F-actin, nor-

malized to the cell number, was quantitatively measured as described in by guest on September 26, 2021 Materials and Methods. n ϭ 3–7 independent determinations for each time point. the total F-actin amount and the formation of dorso-lateral membrane ruffles, suggesting the acquisition of a motile phenotype. FIGURE 3. CX3CL1 stimulates tyrosine phosphorylation. A, RAW cells were treated for various times with 50 ng/ml CX3CL1, and total cell CX3CL1 stimulates discrete tyrosine phosphorylation lysates were subjected to Western blotting using a phosphotyrosine-spe- After binding to G protein-coupled receptors, several chemokines cific Ab (PY) or using anti-␤-actin Ab as a verification of equal protein have been shown to stimulate tyrosine kinase activity, which ap- loading. A Western blot representative of three independent experiments is shown. B, RAW cells were either untreated or treated with CX3CL1 for 1 pears to be required for accurate signaling and cell chemotaxis min before fixation and costaining of F-actin and phosphotyrosine-contain- (29). We therefore asked whether CX3CL1 could trigger tyrosine ing proteins. C, Murine bone marrow-derived macrophages, prepared as phosphorylation in monocytes/macrophages. Total lysates were described in Materials and Methods, were stimulated with CX3CL1 for 1 prepared from RAW cells treated with 50 ng/ml CX3CL1 for var- min before costaining as shown for RAW cells. Unless otherwise noted, all ious times and subjected to Western blotting using an Ab recog- images shown are z-sections collected 2.5 ␮m above the cell-substratum nizing phosphotyrosine (PY) residues (Fig. 3A). Multiple bands interface (cell midsections) using a confocal microscope. Images noted top corresponding to phosphotyrosine-containing proteins were clearly represent the same cells as those shown below, but were taken 5 ␮m above induced after 30 s and 1 min of CX3CL1 treatment before return- the coverslip level to focus on dorsal ruffles. All images are representative ing to levels comparable to unstimulated cells after 5 min (Fig. of at least three independent experiments. Scale bars ϭ 10 ␮m. 3A). To determine the intracellular localization of these tyrosine- phosphorylated proteins, we performed immunofluorescence ex- The CX3CL1-induced F-actin rearrangement was temporally periments using the phosphotyrosine-specific Ab in costaining ex- and spatially correlated to tyrosine phosphorylation. This sug- periments together with F-actin (Fig. 3, B and C). Confocal gested a possible role of phosphotyrosine-containing proteins in microscopy indicated that tyrosine-phosphorylated proteins were the control of the actin cytoskeleton in monocytes/macrophages significantly colocalized with the typical F-actin-rich protrusions and led us to search for a specific candidate capable of fulfilling induced in RAW cells upon CX3CL1 treatment, especially at the this function. dorsal surface (see Fig. 3B). CX3CL1 stimulation of primary macrophages derived from progenitors isolated from mouse bone mar- Involvement of Syk downstream of CX3CR1 row led to a similar cell response with the formation of membrane Two lines of evidence prompted us to investigate the putative in- ruffles enriched in both F-actin and tyrosine-phosphorylated pro- volvement of the Syk tyrosine kinase in the CX3CL1-induced sig- teins (Fig. 3C). naling pathways. Firstly, Syk has been shown to be phosphorylated The Journal of Immunology 3741 in the MonoMac6 monocyte cell line even though the functional CX3CL1, we used the Syk-selective inhibitor piceatannol. RAW relevance of this modification in the CX3CL1 signal transduction cells were pretreated with 50 ␮M piceatannol or with DMSO as a had not been explored (11), and secondly, CX3CL1 treatment of control (vehicle) for 1 h before being subjected to a transmigration RAW cells resulted in the prominent increase in the phosphoty- assay in response to CX3CL1 in the presence or the absence of the rosine content of immunoreactive species with an electrophoresis inhibitor. Piceatannol treatment of cells led to the abolishment of mobility slightly below 75 kDa, consistent with the molecular their ability to chemotax to CX3CL1 (Fig. 4B). No difference in mass of Syk (see Fig. 3A). We first sought to establish whether Syk cell viability was observed between DMSO- and piceatannol-pre- was activated downstream of CX3CR1 in RAW cells by perform- treated cells, as assessed by the trypan blue exclusion method. ing immunoprecipitation experiments. Cells were incubated with CX3CL1 treatment of RAW cells increased the level of tyrosine or without 50 ng/ml CX3CL1 for 1 min before lysis under dena- phosphorylation of Syk, in particular on key residues required for turing conditions and immunoprecipitation (IP) of Syk. Immuno- its activity and pharmacological inhibition of Syk activity dramat- precipitants were then subjected to anti-phosphotyrosine Western ically inhibited cell chemotaxis in response to CX3CL1. blotting (WB PY). The amount of Syk immunoprecipitated in each condition was determined by probing the same samples with the Reduction of Syk expression in RAW cells results in a severely Syk Ab used for the immunoprecipitation. CX3CL1 treatment of impaired ability of cells to chemotax to CX3CL1 RAW cells resulted in an increase in the tyrosine phosphorylation To demonstrate the specific involvement of Syk in CX3CL1-in- level of Syk (Fig. 4A, upper panel). We also performed the reverse duced cell chemotaxis, we chose to inhibit Syk expression using an experiment where phosphotyrosine-containing proteins were im- RNA-mediated interference approach. RAW cells were retrovi- munoprecipitated (IP PY) from lysates of cells treated or not with rally infected with pSUP plasmids containing either two different Downloaded from CX3CL1. Immunoprecipitates were then probed for the presence sequences coding for shRNA targeting the Syk transcript or a con- of Syk specifically phosphorylated on tyrosine residues 519/520 trol scrambled sequence coding for a mock shRNA, as described in (WB P-Syk). These residues lie within the Syk activation loop, and Materials and Methods. After puromycin selection, infected cells their phosphorylation has been shown to be required for Syk ac- were screened for Syk expression by Western blotting. As shown tivity (30). CX3CL1 treatment resulted in an increased amount of in Fig. 5A, 70–80% of Syk protein levels were reduced in Syk activated Syk in RAW cells (Fig. 4A, lower panel). To determine shRNA-treated cells compared with scrambled-treated (scr) or http://www.jimmunol.org/ whether Syk activity could play a role in cell chemotaxis toward noninfected (NI) cells, without affecting the expression of by guest on September 26, 2021

FIGURE 4. Syk is activated upon CX3CL1 treatment, and piceatannol inhibits CX3CL1-induced cell migration. A, RAW cells were either untreated (Ϫ) or treated (ϩ) with 50 ng/ml CX3CL1 for 1 min before lysis and immunoprecipitation of Syk (IP Syk) or phosphotyrosine-containing FIGURE 5. Characterization of Syk shRNA-treated cells. A, Western proteins (IP PY) using specific Abs. Samples were then subjected to West- blot analysis of Syk, CX3CR1, and ␤-actin expression in RAW cells either ern blotting (WB) using the indicated Abs. The phosphospecific Ab used NI or retrovirally infected with pSUP plasmids containing a scrambled (P-Syk) specifically recognizes activated Syk (phosphorylated on residues control sequence (scr) or two different shRNA sequences targeting Syk Y519/520). Input represents the amount of Syk present in total cell lysates (sh#1 and sh#2). Samples used in this study are lysates from puromycin- for each condition. Western blots representative of three independent ex- resistant heterogeneous cell populations 2 wk after infection; similar results periments are shown. B, Cells were pretreated with 50 ␮m piceatannol were obtained with single cell-derived clones. Quantification of Syk/␤- (Syk inhibitor) or DMSO (vehicle) and then subjected to a transmigration actin signals on three different Western blots is shown for each condition. assay in response to CX3CL1 as described in Materials and Methods. n ϭ B, The ability of cells to undergo Fc␥R-mediated phagocytosis was deter- p Ͻ 0.05 compared ,ء .p Ͻ 0.05 compared with CX3CL1-induced stimulation in vehicle- mined as described in Materials and Methods. n ϭ 3 ,ء .3 treated cells. with NI control cells. 3742 Syk IN CX3CL1 CHEMOTAXIS

FIGURE 6. Cells with reduced Syk expression show impaired migration toward CX3CL1. A, Syk shRNA- treated cell migration in response to CX3CL1 was determined using a Transwell assay as described previ- p Ͻ ,ء .ously and compared with NI and scr cells. n Ն 4 0.05 compared with CX3CL1-induced migration in NI cells. B, The ability of Syk shRNA and scr cells to migrate in response to 20 ng/ml CSF-1 was also evaluated. n ϭ 3.

CX3CR1 and ␤-actin. No significant difference in the level of Syk disrupted signaling downstream of the Fc␥R, as previously re- Ϫ/Ϫ reduction was observed using the two different target sequences, ported in Syk macrophages (31). Downloaded from sh#1 and sh#2. Significant Syk protein reduction was detectable in We then assessed the ability of cells with reduced Syk expres- puromycin-resistant heterogeneous cell populations (see Fig. 5A) sion to chemotax toward CX3CL1. Using the transmigration assay as well as in single cell-derived populations generated by limit with 50 ng/ml CX3CL1 in the lower chamber, we compared the dilution cloning (not shown). The cell viability of Syk or mock migration of cells with reduced Syk expression obtained from in- shRNA-treated cells was assessed with the trypan blue exclusion fection with two different shRNA (sh#1 and sh#2) to NI cells and method, and Ͼ95% of all cells were found to be viable. Unless scr cells (Fig. 6A). Cells with reduced Syk expression showed an http://www.jimmunol.org/ otherwise noted, all subsequent experiments were conducted using almost total inhibition of chemotaxis toward CX3CL1 compared at least two independent batches of shRNA-treated cells showing with control cells. Interestingly, the ability of Syk shRNA-treated at least 70% Syk protein reduction. As a control, we also tested the cells to migrate toward CSF-1, a well-known monocyte/macro- functional repercussion of Syk reduction in RAW cells on Fc␥R- phage chemoattractant that binds the receptor tyrosine kinase mediated phagocytosis, a process known to be Syk dependent (31). CSF-1R (32), was not significantly altered compared with that of As expected, the ability to internalize IgG-coated erythrocytes was mock shRNA-treated cells (Fig. 6B). markedly reduced in Syk shRNA-treated cells compared with scr We also compared RAW cells with normal and reduced Syk or NI cells (Fig. 5B). This observation is in agreement with a expression in terms of MAPK activation, because others have by guest on September 26, 2021

FIGURE 7. CX3CL1 activation of ERK1/2 is not required for RAW cell chemotaxis. A, RAW cells were stimulated with CX3CL1 for the indicated times before Western blotting of the corresponding Triton-soluble whole cell lysates. The activation statuses of ERK1/2 (p42/44MAPK), p38MAPK, and JNK1 were assessed using phosphospecific Abs, and the corresponding total (phosphorylation-independent) levels of MAPK expression are shown below as proof of equal protein loading. B, CX3CL1-induced ERK1/2 activation levels were compared between scrambled and Syk shRNA-treated cells. Results shown were obtained with a clone exhibiting 80% Syk protein reduction. C, RAW cells were preincubated for 1 h with 30 ␮M PD98059 (MEK inhibitor) or with DMSO (vehicle) before being subjected to a transmigration assay. PD98059 efficacy was confirmed through its ability to abolish ERK1/2 phosphorylation (see inset). For all experiments, n ϭ 3. The Journal of Immunology 3743 reported a piceatannol-sensitive activation of members of the MAPK family in chemokine-stimulated monocytes (11, 33). As shown in Fig. 7A, CX3CL1 treatment of RAW cells activated ERK1/2 (p42/44MAPK) within 3 min, and both proteins remained activated for at least 15 min. However, the phosphorylation levels of p38MAPK and JNK1 were not significantly increased in response to CX3CL1, even after a 3-h treatment (not shown). Consistent with the results reported by Cambien et al. (11) using piceatannol, CX3CL1-induced ERK1/2 activation was abolished in Syk shRNA-treated cells, suggesting that Syk might be upstream of ERK (Fig. 7B). Nevertheless, inhibition of the MEK/ERK pathway using PD98059 did not result in any reduction of the ability of RAW cells to chemotax toward CX3CL1 (Fig. 7C). These observations sug- gested that ERK is not a key mediator of the Syk function required for RAW cell migration in response to CX3CL1. Overall, these data sug- gested a MAPK-independent requirement for Syk in RAW cell chemotaxis in response to CX3CL1, but not to CSF-1.

Syk is required for the CX3CL1-induced reorganization of the Downloaded from actin cytoskeleton Finally, we analyzed the phenotype of RAW cells with reduced Syk expression with respect to F-actin rearrangement in response to CX3CL1. Strikingly, the increase in F-actin cellular content induced upon CX3CL1 treatment (as shown previously in Fig. 2B) was dramatically inhibited in Syk shRNA-treated cells compared http://www.jimmunol.org/ with scr cells or NI cells (Fig. 8A). Accordingly, the ability of cells with reduced Syk expression to extend membrane protrusions in response to CX3CL1 was also severely compromised (Fig. 8B). Quantitative analysis of the extent of protrusions in response to CX3CL1 was performed as described previously (18) and is shown below the representative images. Instead of exhibiting typical F- actin-rich rufﬂes upon CX3CL1 treatment, Syk shRNA-treated

cells appeared to be rounder, devoid of specific cytoskeletal struc- by guest on September 26, 2021 tures, and occasionally displayed small F-actin aggregates that remained identical in the presence or the absence of CX3CL1. Over- all, we concluded that reduction of Syk expression in RAW cells resulted in a major defect in their cytoskeletal reorganization in response to CX3CL1, which itself is presumably the cause of the FIGURE 8. CX3CL1-induced cytoskeletal reorganization is disrupted inability of cells to properly chemotax to CX3CL1. in cells with reduced Syk expression. A, The ability of cells to increase their F-actin content after a 1-min CX3CL1 stimulation was compared among NI, scr, and Syk shRNA-treated (sh) cells, as previously described. ,ء .Discussion n ϭ 4 independent experiments using heterogeneous cell populations The CX3CL1 (fractalkine)/CX3CR1 ligand/receptor pair has fos- p Ͻ 0.05 compared with NI cells. B, Scrambled and Syk shRNA-treated tered increasing interest in the past few years due to its emerging cells were either untreated or treated with CX3CL1 for 1 min, and their involvement in various clinical diseases, such as rheumatoid ar- ability to exhibit F-actin-rich cell protrusions (ruffles) was compared using thritis (4–6), cardiovascular diseases (7–10), cardiac allograft re- F-actin staining. Representative images of three independent experiments jection (34, 35), glomerulonephritis (36), or HIV infection (37, are shown. Scale bar ϭ 10 ␮m. The extent of CX3CL1-induced ruffles in 38). Indeed, overexpression or ectopic expression of CX3CL1 in individual cells was scored as described previously (18); protrusion in- various pathological tissues has been shown to be associated with dexes were calculated as the average of at least 50 cells in three different p Ͻ 0.05 ,ء .experiments and expressed as a percentage of scr cells. n ϭ 3 significant CX3CR1-expressing leukocyte infiltration, contributing compared with scrambled. to the progression of the disease (reviewed in Ref. 39). Additional pathologically relevant cell types susceptible to interact with CX3CL1 include human CMV, whose US28 broad-spectrum re- documenting intracellular signaling involved in monocyte/macro- ceptor has been shown to bind CX3CL1 with a high affinity (40), phage chemotaxis toward CX3CL1. and human cancer cells, which express functional CX3CR1 (41). In this study we have characterized the monocyte/macrophage Together, these observations support the idea of a broad function response to CX3CL1 in terms of actin dynamics with high tem- of the CX3CL1/CX3CR1 system in pathogenesis through the re- poral resolution. Very few studies have examined this aspect, and cruitment of various cell types to various sources of CX3CL1. quantitative measurement of F-actin cellular content in response to Cells of the monocyte/macrophage lineage represent the main leu- CX3CL1 has been performed only on dendritic cells (42) and T kocyte subset to accumulate at sites such as the thoracic aorta in lymphocyte subsets (43). These cell types were found to increase atherosclerosis or the synovium in rheumatoid arthritis and have their amount of F-actin within 30 s after addition of CX3CL1 even been shown to actively contribute to the progression of these dis- though, in both cases, the time scale used did not have sufficient eases. Their ability to chemotax to CX3CL1 is likely to account for resolution to determine whether the maximal response occurred at a major part of their recruitment. This report provides the first data this time point. In addition, a significant cytoskeletal reorganization 3744 Syk IN CX3CL1 CHEMOTAXIS has been observed in microglial cells, which started to round up and Syk shRNA-treated cells and control cells was significant after a display a peripheral band of actin bundles after 10 min of CX3CL1 4-h stimulation (Fig. 6) and was still comparable after a 16-h stim- treatment (44). This suggests that the morphological and temporal ulation (not shown), indicating that cells with reduced Syk expres- nature of the actin response to CX3CL1 depends on the cell type. sion were not simply delayed in their ability to respond to Our results demonstrate that the protein tyrosine kinase Syk is CX3CL1. In addition, cell migration to CSF-1, a chemotactic fac- activated in response to CX3CL1 and is required for the reorga- tor acting through a tyrosine kinase cell surface receptor, appeared nization of the actin cytoskeleton (i.e., formation of membrane to be normal in cells with reduced Syk expression. These data ruffles) and proper migration of RAW cells. Syk is primarily, but together with the observation that Syk does not appear to be es- not only, expressed in hemopoietic cells and is known to be essential for neutrophil migration in response to the bacterial peptide sential for the function of BCR, TCR, and FcR (reviewed in Ref. fMLP or MIP-2 (58) suggest that the role of Syk in cell signaling 45). Interestingly, signaling through ZAP-70, the second member is receptor selective and/or cell specific. Our results support the of the Syk/ZAP-70 family of protein tyrosine kinases, has been idea that Syk is required for the actin-driven cell motility in re- shown to be essential to mediate stromal cell-derived factor-1␣ sponse to CX3CL1, but we cannot rule out the possibility that (CXCL12) chemotaxis in T cells (46, 47). A potential role for Syk reduced migration in cells with reduced Syk expression could also itself downstream of chemokine receptors is more elusive even if be due to a defect in other steps involved in chemotaxis, i.e., sens- its activation has been reported in response to MIP-1␤/CCL4 in the ing of the chemokine gradient or establishment and maintenance of pre-B lymphoma cell line L1.2 transfected with CCR5 and in pri- cell polarity (28). Additional experiments are required to address mary T lymphocytes (48) as well as in hemopoietic progenitor these issues. Also, very little is known about CX3CL1 signal trans- cells after activation of CXCR3 (49). Phosphorylation of Syk has duction in monocytes/macrophages, and it is likely that other sig- Downloaded from also been reported in MonoMac6 cells in response to CX3CL1 naling components participate in the regulation of CX3CL1-in- (11) and CCL2 (MCP-1) (33). However, the functional signifi- duced chemotaxis. In particular, the possible contributions of a cance of these events in terms of cell chemotaxis was either ap- PI3K pathway, reported to be activated downstream of CX3CR1 parently negligible (49) or had not been investigated (11, 33, 48). (11), remain to be investigated as well as its potential interaction It should also be noted that the activation of Syk in hemopoietic with Syk, as described in neutrophils upon LPS stimulation (59).

progenitor cells only occurred after 30 min of stimulation, at which In conclusion, we have identified the protein tyrosine kinase Syk http://www.jimmunol.org/ time the early events governing cell chemotaxis and motility have as a key signaling component in CX3CL1-induced chemotaxis of already taken place (49). Overall, the heterogeneity of cell models monocytes/macrophages, indicating for the first time a clear pos- and chemokines used in these different studies render comparisons itive requirement of Syk in a migratory process and shedding new difficult, because the kinetics and functional role of Syk activation light upon the as yet understudied cell signaling downstream of might be significantly different under these conditions. CX3CR1. In monocytes/macrophages, Syk has previously been shown to ␥ play a role in actin-dependent processes, such as Fc R-mediated Acknowledgments phagocytosis (31) and integrin-mediated adhesion (50). In this

We thank Drs. Fiona J. Pixley, Uma Siddhanta, and Yee-Guide Yeung for by guest on September 26, 2021 study we show that CX3CL1 induces a rise in F-actin in RAW providing cells and reagents; Dr. Hideki Yamaguchi for his help with con- cells that is dramatically inhibited in cells with reduced Syk ex- focal microscopy; as well as members of the laboratories of Drs. John pression, suggesting a function for Syk in the control of actin dy- Condeelis, Jeffrey Segall, and E. Richard Stanley for helpful discussions namics. How Syk could control such a process is unknown, but, and comments on this work. importantly, several studies indicate a link between Syk and the actin cytoskeleton. Syk has been reported to directly phosphorylate Disclosures at least two actin-binding proteins: the cortactin-related hemopoi- The authors have no financial conflict of interest. etic lineage cell-specific protein 1, which binds to the Arp2/3 complex and promotes actin assembly and branching (51, 52), and SH3P7, a protein of unknown function, isolated from the phos- References photyrosine immunoreactive fraction induced after lymphocyte ac- 1. Bazan, J. F., K. B. Bacon, G. Hardiman, W. Wang, K. Soo, D. Rossi, D. R. Greaves, A. Zlotnik, and T. J. Schall. 1997. A new class of membrane- tivation (53). Notably, Syk-mediated phosphorylation of hemopoi- bound chemokine with a CX3C motif. Nature 385: 640–644. etic lineage cell-specific protein 1 was required for its translocation 2. Pan, Y., C. Lloyd, H. Zhou, S. Dolich, J. Deeds, J. A. Gonzalo, J. Vath, to lipid raft microdomain together with Arp2/3 and the actin reg- M. Gosselin, J. Ma, B. Dussault, et al. 1997. Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation. Nature 387: 611–617. ulator Wiskott-Aldrich syndrome protein (WASp) after BCR 3. Imai, T., K. Hieshima, C. Haskell, M. Baba, M. Nagira, M. Nishimura, cross-linking (54). In addition, Syk has been shown to bind WASp M. Kakizaki, S. Takagi, H. Nomiyama, T. J. Schall, et al. 1997. Identification and through the adapter CrkL in platelets (55), and an interaction be- molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell 91: 521–530. tween Syk and the WASp-interacting protein has recently been 4. Ruth, J. H., M. V. Volin, G. K. Haines III, D. C. Woodruff, K. J. Katschke, Jr., reported upon Fc⑀RI activation in mast cells (56). A similar protein J. M. Woods, C. C. Park, J. C. Morel, and A. E. Koch. 2001. Fractalkine, a novel association has also been described in T cells, where the Syk- chemokine in rheumatoid arthritis and in rat adjuvant-induced arthritis. Arthritis Rheum. 44: 1568–1581. related protein ZAP-70 was shown to be part of a complex includ- 5. Blaschke, S., M. Koziolek, A. Schwarz, P. Benohr, P. Middel, G. Schwarz, ing WASp, WASp-interacting protein, and CrkL (57). Together, K. M. Hummel, and G. A. Muller. 2003. Proinflammatory role of fractalkine these data suggest that in hemopoietic cells, one or more protein (CX3CL1) in rheumatoid arthritis. J. Rheumatol. 30: 1918–1927. 6. Nanki, T., Y. Urasaki, T. Imai, M. Nishimura, K. Muramoto, T. Kubota, and complexes involving Syk might be relevant for the control of actin N. Miyasaka. 2004. Inhibition of fractalkine ameliorates murine collagen-induced assembly in response to different stimuli. Whether a similar sig- arthritis. J. Immunol. 173: 7010–7016. naling complex is present and/or can be induced after CX3CR1 7. Moatti, D., S. Faure, F. Fumeron, W. Amara Mel, P. Seknadji, D. H. McDermott, P. Debre, M. C. Aumont, P. M. Murphy, D. de Prost, et al. 2001. Polymorphism activation in monocytes/macrophages remains to be determined. in the fractalkine receptor CX3CR1 as a genetic risk factor for coronary artery Importantly, the cytoskeletal remodeling defect observed in disease. Blood 97: 1925–1928. cells with reduced Syk expression is clearly associated with their 8. Lesnik, P., C. A. Haskell, and I. F. Charo. 2003. Decreased atherosclerosis in CX3CR1Ϫ/Ϫ mice reveals a role for fractalkine in atherogenesis. J. Clin. Invest. inability to efficiently chemotax toward CX3CL1. Using a Trans- 111: 333–340. well assay, the difference in the number of migrated cells between 9. Combadiere, C., S. Potteaux, J. L. Gao, B. Esposito, S. Casanova, E. J. Lee, The Journal of Immunology 3745

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