Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.1206726 on May 17, 2007

Published online before print May 17, 2007
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(Journal of Leukocyte Biology. 2007;82:380-391.)
© 2007 by Society for Leukocyte Biology

Intracellular signaling required for CCL25-stimulated T cell adhesion mediated by the integrin {alpha}4β1

Marisa Parmo-Cabañas*,1, David García-Bernal*,1, Rosa García-Verdugo*, Leonor Kremer{dagger}, Gabriel Márquez{ddagger},2 and Joaquin Teixidó*,3

* Department of Molecular and Cellular Physiopathology, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain; and
{dagger} Protein Tools Unit and
{ddagger} Department of Immunology and Oncology, Centro Nacional de Biotecnología (CSIC), Madrid, Spain

3 Correspondence: Centro de Investigaciones Biológicas, Department of Physiopathology, Ramiro de Maeztu 9, 28040 Madrid, Spain. E-mail: joaquint{at}cib.csic.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The {alpha}4β1 integrin is expressed on thymocytes and mediates cell attachment to its ligands CS-1/fibronectin (CS-1/FN) and VCAM-1 in the thymus. The chemokine CCL25 is highly expressed in the thymus, where it binds to its receptor CCR9 on thymocytes promoting migration and activation. We show here that {alpha}4β1 and CCR9 are coexpressed mainly on double- and single-positive thymocytes and that CCL25 strongly stimulates CD4+CD8+ and CD4+CD8– adhesion to CS-1/FN and VCAM-1. CCL25 rapidly activated the GTPases Rac and Rap1 on thymocytes, and this activation was required for stimulation of adhesion, as detected using the CCR9+/{alpha}4β1+ human T cell line Molt-4. To study the role on CCL25-stimulated adhesion of the Rac downstream effector Wiskott-Aldrich syndrome protein family verproline-homologous protein 2 (WAVE2) as well as of Rap1-GTP-interacting proteins, regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and Rap1-GTP-interacting adapter molecule (RIAM), we knocked down their expression and tested transfectant attachment to {alpha}4β1 ligands. We found that WAVE2 and RAPL but not RIAM were required for efficient triggering by CCL25 of T cell adhesion to CS-1/FN and VCAM-1. Although Rac and Rap1 activation was required during early steps of T cell adhesion stimulated by CCL25, WAVE2 was needed for the development of actin-dependent T cell spreading subsequent to adhesion strengthening but not during initial {alpha}4β1-ligand interactions. These results suggest that regulation by CCL25 of adhesion of thymocyte subpopulations mediated by {alpha}4β1 could contribute to control their trafficking in the thymus during maturation, and identify Rac-WAVE2 and Rap1-RAPL as pathways whose activation is required in inside-out signaling, leading to stimulated adhesion.

Key Words: chemokine • fibronectin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The {alpha}4β1 integrin is a heterodimer cell adhesion receptor expressed mainly on cells of hematopoietic origin, which mediates cell-cell and cell-extracellular matrix interactions [1 , 2 ]. VCAM-1 and the alternatively spliced CS-1 region of fibronectin (FN) are main ligands for {alpha}4β1, and these interactions play key roles in leukocyte trafficking during immune surveillance and inflammation [2 ]. Previous studies demonstrated that {alpha}4β1 expression was diminished gradually through thymocyte maturation [3 4 5 ] and that the expression pattern correlated with differences in {alpha}4β1-dependent thymocyte adhesion [3 , 5 6 7 ]. Expression of CS-1/FN has been found predominantly on the thymic medulla, whereas VCAM-1 is expressed by cortical thymic epithelial cells [3 , 8 , 9 ], suggesting that differential localization of these {alpha}4β1 ligands might influence the trafficking and maturation of thymocytes.

Chemokines are a family of low molecular weight cytokines, which elicit cell migration and activation upon binding to their seven-transmembrane, G-protein-coupled receptors [10 11 12 13 ]. Expression of the chemokine CCL25 is mainly restricted to thymus and small intestine [14 15 16 ], and this chemokine exerts its functions through interaction with the receptor CCR9 [17 18 19 ]. CCL25 is expressed by epithelial cells in the thymic cortex and by some epithelial cells in the medulla [15 ]. Furthermore, CD4+CD8+ thymocytes express high levels of CCR9 and migrate toward CCL25 [15 , 18 , 20 , 21 ], suggesting that the CCL25/CCR9 axis could play important roles in T lymphocyte trafficking in the thymus.

The control of cell adhesion represents one of the targets regulated by chemokine signaling. Thus, {alpha}4β1-mediated T lymphocyte adhesion is modulated by chemokines, a process controlling lymphocyte trafficking [22 , 23 ]. The expression of {alpha}4β1 and CCR9 on thymocytes and the specific localization of {alpha}4β1 ligands and CCL25 in the thymus raise the possibility that {alpha}4β1 function might constitute a target of regulation by CCL25, thus representing a mechanism controlling thymocyte migration.

Characterization of the signaling generated upon binding of chemokines to their receptors, which finally impinges on {alpha}4β1 and influences the avidity for its ligands, has started to elucidate the molecular components whose activation is critical for final stimulation of integrin function. These include the GTPases Rac and Rap1, which are activated rapidly and transiently by chemokines such as CXCL12 and CCL21 on T lymphocytes [24 25 26 ], which is required for efficient stimulation of {alpha}4β1-dependent adhesion.

Although upstream molecules involved in chemokine-promoted Rac activation, such as Vav1, have been shown to be needed for efficient stimulation of {alpha}4β1-dependent T cell adhesion [24 ], little is known about the role of Rac downstream effectors in this process. A potential candidate is Wiskott-Aldrich syndrome protein family verproline-homologous protein 2 (WAVE2), a Rac effector, which forms multimolecular complexes with abscisic acid insensitive 1 (Abi1), nucleosome assembly protein 1 (Nap1), and p53-inducible mRNA 121 (PIR121) and links Rho GTPase activation and actin assembly through stimulation of actin-related protein-2/3-nucleating activity, resulting in lamellipodia protrusions and dorsal ruffling [27 28 29 30 ].

Regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and Rap1-GTP-interacting adapter molecule (RIAM) are downstream effectors for Rap1, which mediate activation signals from this GTPase, leading to stimulation of β1 and β2 integrin-mediated adhesion [26 , 31 , 32 ]. RAPL is a Rap1-GTP-binding protein, highly expressed on lymphocytes, which forms a complex with LFA-1 upon Rap1 activation by chemokines and is required for CCL21-stimulated spleen T and B cell attachment to β1 and β2 integrin ligands [26 , 33 ]. RIAM was identified as a Rap1-GTP-interacting protein in a yeast two-hybrid screen [31 ], and it was found that interfering with its expression caused a reduction in adhesion of Jurkat T cells to FN. However, its role in chemokine-promoted T cell adhesion, dependent on {alpha}4β1, has not been addressed.

In the present work, we have investigated whether {alpha}4β1-mediated thymocyte adhesion can be controlled by the thymic chemokine CCL25 and studied the role of the Rac and Rap1 downstream effectors WAVE2, RAPL, and RIAM in CCL25-stimulated T cell adhesion, dependent on the integrin {alpha}4β1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells, antibodies, and reagents
Thymuses from 2- to 3-week-old BALB/c mice were gently disrupted and filtered through nylon mesh to remove aggregates, and primary thymocytes were resuspended in adhesion medium (RPMI 1640/BSA 0.5%). The human T cell line Molt-4 was cultured in RPMI-1640 medium (Invitrogen Gibco, Paisley, Scotland) supplemented with 10% FBS (BioWhittaker, Verviers, Belgium; growth medium). For sorting, 6 x 108 thymocytes were centrifuged and washed with PBS containing 2% BSA, 2% FBS, and 0.05% sodium azide (PBSst). Cells were double-stained with rat anti-mouse CD4-PE (Clone L3T4, BD Biosciences PharMingen, San Diego, CA, USA) and rat anti-mouse CD8{alpha} spectral Red (Clone 53-6.7, Southern Biotech, Birmingham, AL, USA) at 4°C for 25 min and washed twice with PBSst. Thymocyte subsets were selected as CD4+CD8–, CD4–CD8+, and CD4+CD8+ populations (>98% pure) in an Epics Altra sorter (Beckman Coulter, Hialeah, FL, USA). Antibodies to human (96-1.5) and mouse (K629) CCR9 have been described [18 , 20 ], whereas a new anti-human CCR9 mAb to be described (Laura Carramolino, Maria Lozano, Beatriz Salvador, Carlos Martinez-A., L. Kremer, manuscript in preparation) was used in confocal microscopy. Antibodies to murine {alpha}4 integrin (PS/2) and CD4 (GK1.5) were obtained from American Type Culture Collection (Manassas, VA, USA). The human anti-{alpha}4 ALC1.63, anti-WAVE2, anti-β1 mAb 15/7, and control P3X63 mAb were gifts from Drs. Angel L. Corbí (Centro de Investigaciones Biológicas, Madrid, Spain), Tadaomi Takenawa (Institute of Medical Science, University of Tokyo, Japan), Francisco Sánchez-Madrid (Hospital de la Princesa, Madrid, Spain), and Ronen Alon (The Weizmann Institute of Science, Rehovot, Israel), respectively. Dr. Theresia Stradal (German Research Centre for Biotechnology, Braunschweig, Germany) provided anti-PIR121 antibodies, anti-Abi1 and anti-Rap1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-Rac was from BD Biosciences PharMingen. Human and murine CCL25 were purchased from R&D Systems (Abingdon, UK). Inhibitors included pertussis toxin, piceatannol (Calbiochem-Novabiochem Co., Darmstadt, Germany), and cytochalasin D (Sigma-Aldrich, St. Louis, MO, USA).

RNA interference, transfections, and RT-PCR
A small interfering (si)RNA duplex for Rap1A was designed targeting bases 534–554, sense strand: AUCAUGUCUGCUGCUCUAGdTdT (Ambion Inc., Austin, TX, USA). We also used a predesigned siRNA for human Rap1A (Rap1A/B, targeted to bases 103–123) and a cocktail of four siRNA for human RAPL (Dharmacon Inc., Lafayette, CO, USA). Sequences for Rac1, Vav1, and control siRNA have been reported earlier [24 ]. Three siRNA duplexes were designed corresponding to human WAVE2 (WAVE2.A, targeted to bases 433–453, sense strand: GAGGCACUCAAAUUCUACAdTdT; WAVE2.B, bases 214–235, sense strand: GUCGACCGACUACAGGUUAdTdT; WAVE2.C, bases 719–739, sense strand: ACGUGGAUGCAAGUAGCUAdTdT; Ambion Inc.). RIAM short hairpin (sh)RNA [31 ] was a gift from Dr. Vassiliki A. Boussiotis (Dana Farber Cancer Institute and Brigham and Women’s Hospital, Boston, MA, USA), and CXCR4 shRNA has been generated recently and will be described (Rubén Bartolomé, M. Eugenia Miguelena, Raphael Delgado, Lorena Martinez-Prats, Sergio Ferreiro, Marisa Soto Manuel Desco, Paloma Sánchez-Mateos, Reuben Agami, J. Teixido; manuscript in preparation). shRNA and siRNA were nucleofected in Molt-4 cells resuspended in Nucleofector V solution (2–2.5x106 cells/100 µl) using an Amaxa Nucleofector device (Amaxa, Cologne, Germany). After transfection, cells were cultured for 16 h in growth medium and used for expression or functional analyses. siRNA transfections did not affect cell viability, as assessed in cell cycle analyses, by flow cytometry and by trypan blue exclusion (not shown). Amplification of RIAM and RAPL was performed using primers 5'-AAAGCGCCCACTGACTATTG-3' and 5'-CGTTGCCTCTCTTCTTTTGC-3' for RIAM and 5'-CTGGACGAGGAACTGGAAGACTGCTTC-3' and 5'-CATCTCACCCTACGGAAGAGGTAGGGA-3' for RAPL and Taq DNA polymerase (Invitrogen Gibco). The PCR profile consisted of 1-min initial denaturation at 95°C, followed by 35 cycles of 45 s denaturation at 95°C, 1-min second annealing at 66°C for RAPL and 60°C for RIAM, 1.5-min polymerization at 72°C, and finally, by 10-min extension at 72°C. Aliquots of each sample were amplified using the same conditions with human GAPDH primers [24 ].

Cell adhesion and soluble binding assays
Recombinant human VCAM-1 {soluble VCAM-1 (sVCAM-1) [34 ]} and the FN-H89 fragment of FN, which contains the CS-1 site (CS-1/FN) [35 ], were coated on 96-well plates (Costar, Cambridge, MA, USA). Cells were labeled with 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Molecular Probes, Leiden, The Netherlands) in adhesion medium, followed by preincubation with inhibitors or antibodies before addition to wells (6x104 or 5x105 in 100 µl for Molt-4 cells or murine thymocytes, respectively) containing sVCAM-1 or FN-H89 (1–4 µg/ml), alone or immobilized with CCL25. After a 15-s spin, plates were incubated for 5 min (thymocytes) or 2 min (Molt-4) at 37°C, and subsequently, nonbound cells were removed. Following three washes, adhered cells were lysed, and the extent of adhesion was quantified using a fluorescence analyzer (BMG Labtechnologies, Offenburg, Germany). For experiments of soluble binding using sVCAM-1-Fc, cells were exposed for 45 s to CCL25 (150 nM) or MnCl2 (1 mM) before adding sVCAM-1-Fc at saturating concentrations (20 µg/ml) as described [24 ]. Bound sVCAM-1-Fc was detected by flow cytometry using PE-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG, Fc{gamma} fragment-specific (Jackson ImmunoResearch Lab., West Grove, PA, USA). For soluble-binding assays using VCAM-1{kappa}, we followed the method already described [24 ]. Briefly, cells were incubated for 45 s at 37°C with CCL25 or binding buffer alone, and then VCAM-1{kappa} (from Dr. Gabriele Weitz-Schmidt, Novartis Institutes for BioMedical Research, Basel, Switzerland) was added and incubated with cells for 75 s at 37°C. When Mn2+ was used, cells were incubated directly with VCAM-1{kappa} in binding buffer with 1 mM MnCl2. Bound VCAM-1{kappa} was detected by flow cytometry using FITC-conjugated goat anti-mouse {kappa} (Caltag Laboratories, Burlingame, CA, USA).

Immunoprecipitation, Western blotting, and GTPase assays
Cell lysates [24 ] were precleared with protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden), and supernatants were incubated with antibodies, followed by specific coupling to protein A-Sepharose beads. Proteins were eluted in Laemmli buffer, resolved by SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Pharmacia Biotech), which were incubated with antibodies, followed by washing and incubation with HRP-conjugated secondary antibodies. Proteins were visualized using SuperSignal chemiluminiscent substrate (Pierce, Rockford, IL, USA). For GTPase assays, we followed the method described [36 ]. Briefly, cells exposed to CCL25 (150 nM) were lysed, and aliquots of extracts were kept for total lysate controls or mixed with GST-p21-activated kinase-CD or GST-Ral-guanine-nucleotide dissociation stimulation-Ras-binding domain fusion proteins (gifts from Drs. John G. Collard and Johannes L. Bos, from The Netherlands Cancer Institute, Amsterdam, and University Medical Center, Utrecht, respectively), in the presence of glutathione-agarose beads. Bound proteins were eluted and separated by SDS-PAGE and transferred to PVDF membranes, which were incubated with antibodies against Rac or Rap1. Protein detection was performed as above.

Confocal microscopy, flow cytometry, and actin polymerization
To analyze cellular spreading, transfectants were allowed to adhere to FN-H89 and subsequently, were exposed to CCL25 for different times. Unbound cells were removed carefully, and cells remaining attached were fixed and mounted with mowiol, and images were captured by confocal microscopy (Leica TCS-SP2-AOBS-UV, Leica, Mannheim, Germany) with 100x oil immersion objective. For one-color flow cytometry, 2–3 x 105 cells were incubated at 4°C with primary antibodies, followed by incubation with FITC-conjugated secondary antibodies (Dako A/S, Denmark) and analysis in a Coulter Epics XL flow cytometer. For three-color staining, the following antibodies were used: rat anti-mouse CD4-FITC or anti-mouse CD4-PE (BD Biosciences PharMingen), rat anti-mouse CD8{alpha} spectral Red (Southern Biotech), rat anti-mouse CD49d-biotin (BD Biosciences PharMingen), rabbit anti-mouse CCR9 K629 and donkey anti-rabbit Ig-FITC (Amersham Biosciences, Little Chalfont, UK), and streptavidin-PE (Southern Biotech). Staining was performed with 1.5 x 106 thymocytes per sample, following the method described [20 ]. We used flow cytometry to determine the content of F-actin in cells stimulated with CCL25, as reported previously [37 ].

Statistical analyses
The results are expressed as the mean ± SD of triplicate samples obtained from two or more experiments. Statistical significance was determined using the two-tailed Student’s t-test. For three or more conditions, data were analyzed by one-way ANOVA, followed by Tukey-Kramer multiple comparisons. In both analyses, the minimun acceptable level of significance was P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CCL25 stimulates {alpha}4β1-dependent thymocyte adhesion
To identify thymocyte subpopulations which coexpress {alpha}4β1 and CCR9, we performed three-color flow cytometry on thymocytes from BALB/c mice. Double-negative CD4–CD8– thymocytes expressed high levels of {alpha}4/CD49d and low amounts of CCR9, whereas {alpha}4/CD49d diminished with thymocyte maturation, and CCR9 expression was highest at double-positive CD4+CD8+, decreasing on CD4+ and CD8+ single-positive thymocytes (Fig. 1A ), in agreement with previous reports [3 , 6 , 15 , 18 , 20 , 21 ]. More than 80% of thymocytes coexpressed {alpha}4 and CCR9, indicating that CD4+CD8+, CD4+, and CD8+ but not CD4–CD8– thymocytes represented subpopulations potentially responsive to CCL25 and {alpha}4β1 ligands.


Figure 1
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  		Figure 1. CCL25 stimulates {alpha}4β1-dependent murine thymocyte adhesion. (A) Thymocytes were stained in three-color assays using fluorochrome-conjugated CD4 or CD8 mAb, with biotin-labeled anti-CD49d or nonlabeled rabbit anti-CCR9 (K629) antibodies, followed by streptavidin-PE or donkey anti-rabbit Ig-FITC. Control staining with streptavidin-PE and rabbit-FITC serum is also shown. (B, left) BCECF-AM-labeled thymocytes were subjected to adhesion assays to CS-1/FN or VCAM-1 alone (Control) or immobilized with mouse CCL25 (150 nM). Where indicated, cells were preincubated with control anti-CD4 GK1.5 or anti-{alpha}4 PS/2 mAb (10 µg/ml) or with pertussis toxin (PTx; 200 ng/ml). (Right) Thymocytes were preincubated with control or anti-{alpha}4 mAb and subsequently incubated in the absence (Medium) or presence of CCL25 or Mn2+, followed by binding to VCAM-1-Fc. Cell-bound ligand was detected as indicated in Materials and Methods. Basal binding in the absence of VCAM-1-Fc is also shown. Displayed is a representative result of three independent experiments. (C) Sorted thymocyte populations were labeled with BCECF-AM and subjected to adhesion assays to VCAM-1 alone or immobilized with mouse CCL25. Adhesions were quantified in a fluorescence analyzer, and data represent the mean ± SD of triplicate samples from a representative result of three independent experiments (B and C). ***, Adhesions were up-regulated significantly, P < 0.001, or **, P < 0.01, according to the one-way ANOVA test. (D, upper panels) Murine thymocytes were incubated for the indicated times, with or without CCL25 (300 nM), and subjected to GTPase assays to detect active Rac1 or Rap1. Bound Rac1- or Rap1-GTP was determined by Western blotting using anti-Rac1 or anti-Rap1 mAb. Aliquots from cell lysates were kept aside for detecting total protein. Shown is a representative result out of four independent experiments, whereas the lower panel displays densitometer analyses in arbitrary units, showing fold induction of Rap1 and Rac1 activation (mean±SD) from the four GTPase experiments after correcting for total protein. ***, Activation was stimulated significantly, P < 0.001, or **, P < 0.01, according to the one-way ANOVA test.

 
To investigate if CCL25 was capable of stimulating the adhesion of thymocytes mediated by {alpha}4β1, we tested them in adhesion assays to CS-1/FN and VCAM-1, which were immobilized alone or together with CCL25. This chemokine promoted a strong cell attachment to {alpha}4β1 ligands, which was blocked by anti-{alpha}4 mAb, as well as by pertussis toxin (Fig. 1B , left), demonstrating that enhanced attachment was mediated by {alpha}4β1 and indicating the participation of G{alpha}i-dependent signaling. In addition, CCL25 induced thymocyte adhesion to both ligands when it was used as a soluble form (not shown). Moreover, CCL25 stimulated {alpha}4β1-dependent binding to VCAM-1-Fc in solution on a cell population representing 40–50% of total thymocytes (Fig. 1B , right), indicating that a substantial proportion of thymocytes is capable to switch {alpha}4β1 into high-avidity conformations upon chemokine stimulation. In these experiments, Mn2+ was used as a positive control for triggering potent {alpha}4β1/VCAM-1 interactions.

To compare different CCR9+-thymocyte populations in CCL25-promoted attachment to {alpha}4β1 ligands, we sorted CD4+CD8+, CD4+CD8–, and CD4–CD8+ cells, which were subjected to adhesion assays to VCAM-1. CCL25 stimulated the attachment of CD4+CD8+ and CD4+CD8– to VCAM-1, whereas CD4–CD8+ thymocytes displayed no or minor induction of adhesion (Fig. 1C) , suggesting that double-positive and CD4+ single-positive cells represent the main subpopulations targeted by CCL25 for up-regulation of cell adhesion to {alpha}4β1 ligands.

Expression of constitutively active forms of GTPases Rac and Rap1 in thymocytes leads to increased adhesion to {alpha}4β1 ligands [38 , 39 ]. CCL25 activated Rac1 and Rap1 on murine thymocytes (Fig. 1D) , coincident with times used in adhesion assays for these cells. The data raise the possibility that signaling associated to activation of these GTPases by the thymic chemokine CCL25 could mediate triggering of thymocyte attachment to CS-1/FN and VCAM-1. To explore this possibility, we used the human T cell line Molt-4 as a model, as these cells express high levels of CCR9 and {alpha}4β1. CCL25 coimmobilized with {alpha}4β1 ligands stimulated a robust cell attachment, which was inhibited by anti-{alpha}4 mAb and by pertussis toxin (Fig. 2A and 2B , left panels). Furthermore, soluble CCL25 triggered a rapid and transient increase in cell attachment to CS-1/FN and VCAM-1, which was blocked by anti-{alpha}4 mAb and partially inhibited by anti-CCR9 mAb (Fig. 2A and 2B , right panels).


Figure 2
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  		Figure 2. Rac1 and Rap1 are required for CCL25-stimulated T cell adhesion mediated by {alpha}4β1 integrin. (A, B, left panels) BCECF-AM-labeled Molt-4 T cells were incubated in the absence or presence of pertussis toxin or anti-{alpha}4 mAb and subjected to adhesion to CS-1/FN or VCAM-1, immobilized without (Control) or with human CCL25. (Right panels) Cells were treated with the indicated mAb, incubated for the stated times with or without soluble human CCL25, and subjected to adhesion assays to the same ligands. (C, upper gels) Molt-4 cells were incubated for the indicated times with or without CCL25, and subjected to GTPase assays to detect active Rac1 or Rap1, as in Figure 1 . (Lower gels) Molt-4 cells were transfected with Rac1, Rap1A/B, Rap1A, or control siRNA, and after solubilization, cell lysates were analyzed by Western blotting using anti-Rac1 or anti-Rap1 mAb, followed by membrane reprobing with anti-β-actin antibodies. (D) Nontransfected Molt-4 cells or siRNA transfectants were subjected to adhesion assays to CS-1/FN or VCAM-1 alone (Medium) or immobilized with CCL25. Extent of adhesion was determined as in Figure 1 , and data represent the mean ± SD of triplicate samples from representative results from three independent experiments for each panel. ***, Adhesions were up-regulated significantly, P < 0.001; **, P < 0.01; or *, P < 0.05, according to the one-way ANOVA test.

 
CCL25 activated Rac1 and Rap1 on Molt-4 cells in a time window (1–2.5 min) inside the incubation times used in adhesion assays (Fig. 2C , upper gels). To analyze their role during stimulation by CCL25 of {alpha}4β1-dependent T cell adhesion, we transfected siRNA for these GTPases and tested transfectant attachment to {alpha}4β1 ligands. To this purpose, we used a siRNA for Rac1 previously reported [24 ] and two siRNA for Rap1, one specific only for Rap1A and the other showing predominant specificity for Rap1A but also some cross-reactivity with Rap1B (Rap1A/B; Fig. 2C , lower gels). Knocking down Rac1 or Rap1 expression resulted in a large impairment in stimulation by CCL25 of transfectant attachment to CS-1/FN and VCAM-1, compared with adhesion shown by control siRNA counterparts (Fig. 2D) . Impairment in up-regulated adhesion was not a result of alterations in {alpha}4β1 or CCR9 cell surface expression on transfectants, as assessed by flow cytometry (not shown).

Role of WAVE2, RAPL, and RIAM on CCL25-stimulated T cell adhesion mediated by {alpha}4β1
WAVE2 is a Rac1 downstream effector, which links Rho GTPase activation to de novo actin polymerization and is required for efficient lamellipodia and dorsal ruffling formation [30 , 40 ]. WAVE2 forms part of a multimolecular complex, which contains PIR121, responsible for binding to Rac1-GTP and recruitment of WAVE2 [41 ], Abi1, and Nap1 proteins [29 , 41 ]. Using antibodies against WAVE2, PIR121, and Abi1, which react with the human and mouse proteins, we found that WAVE2, on thymocytes and Molt-4 cells, existed in a complex with PIR121 and Abi1, as detected by immunoprecipitation with anti-WAVE2 antibodies, followed by Western blotting using antibodies to the above proteins (Fig. 3A ). Furthermore, the WAVE2 complex appeared intact upon 2–5 min stimulation with CCL25.


Figure 3
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  		Figure 3. Role of WAVE2 on CCL25-promoted T cell adhesion mediated by {alpha}4β1 integrin. (A) Thymocytes or Molt-4 cells were incubated for the indicated times, with or without CCL25 (mouse or human, respectively; 200 nM), and following solubilization lysates were immunoprecipitated (IP) with anti-WAVE2 or control (Ctr) antibodies. After SDS-PAGE and transfer, membranes were subjected to Western blotting (WB) using the indicated antibodies. (B, upper) Lysates from Molt-4 cells transfected with WAVE2 or control siRNA were analyzed by Western blotting using anti-WAVE2 antibodies. Membranes were reprobed with anti-β-actin mAb for protein loading control. (Lower) Nontransfected Molt-4 cells or WAVE2 or control siRNA transfectants were subjected to adhesion assays to CS-1/FN or VCAM-1 alone (Medium) or immobilized with CCL25. (C) WAVE2 or control siRNA transfectants in adhesion medium, with or without CCL25 (150 nM), were placed on coverslips coated with CS-1/FN and incubated at 37°C for the indicated times. After gently washing, bound cells were fixed and analyzed by confocal microscopy. Nomarski images displaying round and spread cells in representative fields are shown. Extent of adhesion was quantified as in Figure 1 , and data represent the mean ± SD of triplicate samples from a representative result from three independent experiments for each panel. ***, Adhesions were up-regulated significantly, P < 0.001, or **, P < 0.01, according to the two-tailed Student’s t-test.

 
Although it has been demonstrated recently that WAVE2 is a key component of the actin regulatory machinery in T cells [42 ], its role during chemokine-stimulated T cell adhesion has not been studied. To determine the role of WAVE2 in the activation by CCL25 of {alpha}4β1-mediated T cell adhesion, we knocked down its expression and tested transfectant attachment to CS-1/FN and VCAM-1. Transfection of WAVE2.A siRNA caused a large decrease in WAVE2 expression (90% inhibition), whereas transfection with WAVE2.B and WAVE2.C siRNA resulted in partial inhibitions (Fig. 3B , upper). Although basal, nonstimulated, WAVE2.A siRNA transfectant attachment was not affected compared with control siRNA transfectants, up-regulation of adhesion triggered by CCL25 was blocked in these WAVE2 knockdown cells (Fig. 3B , lower). Instead, no significant alterations in enhancement of adhesion were observed with WAVE2.C siRNA transfectants. Importantly, a clear defect in cell spreading on CS-1/FN in response to CCL25 was detected in WAVE2.A siRNA transfectants as compared with spreading of control siRNA counterparts (Fig. 3C) . When adhesion was prolonged, the spreading defects shown by WAVE2 knockdown cells were still evident, but more cells resisted the mild postadhesion washing step, suggesting that a certain amount of adhesion strengthening and/or undetectable low levels of spreading as a result of basal WAVE2 expression in these transfectants might account for the observed cell adhesion. These results indicate that CCL25-promoted T cell adhesion mediated by {alpha}4β1 requires the Rac1 effector WAVE2 and suggest that WAVE2 is needed for the development of cell spreading during T cell attachment.

RAPL and RIAM are Rap1-GTP-binding proteins, which mediate Rap1 downstream signaling, leading to activation of β1 integrin-dependent cell adhesion [26 , 31 ]. Therefore, we compared RAPL and RIAM potential involvement in CCL25-stimulated adhesion by knocking down their expression and analyzed transfectant attachment to CS-1/FN. Expression of RAPL and RIAM in Molt-4 cells was reduced notably (60–70%) by RNA interference (Fig. 4A ), and triggering of adhesion by CCL25 was affected significantly (60%) in RAPL siRNA transfectants (Fig. 4B) . Instead, RIAM shRNA knockdown cells displayed up-regulation of adhesion to similar levels as those from control CXCR4 transfectants. We observed a consistent reduction (40–50%) in basal adhesion in RIAM shRNA transfectants compared with control counterparts, whereas no differences in basal adhesion were detected with RAPL siRNA knockdown cells. These data indicate that Rap1-RAPL but not Rap1-RIAM signaling is required for efficient stimulation of T cell adhesion mediated by {alpha}4β1 in response to CCL25.


Figure 4
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  		Figure 4. Role of RAPL and RIAM on CCL25-promoted T cell adhesion mediated by {alpha}4β1 integrin. (A) Molt-4 cells transfected with RAPL or control siRNA or with shRNA for RIAM or CXCR4 were analyzed by RT-PCR for RAPL or RIAM expression. Also shown are results from nontransfected (–) Molt-4 cells. Results were compared with GAPDH-loading controls. (B) Transfectants were tested in adhesion assays to CS-1/FN alone (Medium) or immobilized with CCL25. Extent of adhesion was quantified as in Figure 1 , and data represent the mean ± SD of triplicate samples from representative results from three independent experiments. **, Adhesions were up-regulated significantly, P < 0.01, according to the two-tailed Student’s t-test.

 
To compare the potential regulation by Rac1 and Rap1 of initial steps during CCL25-stimulated, {alpha}4β1-dependent adhesion, we subjected Molt-4 Rac1 and Rap1 siRNA transfectants to binding assays to VCAM-1-Fc in solution. This assay allows characterization of {alpha}4β1/VCAM-1 interactions in the absence of cell spreading and mimics the early steps in the cascade of adhesion events. CCL25 promoted VCAM-1-Fc binding to control siRNA transfectants, which was abolished by preincubation with anti-{alpha}4 mAb (Fig. 5 , left). A clear and consistent reduction in VCAM-1-Fc binding was detected on Rac1 and Rap1 siRNA transfectants (70–75% and 60–65%, respectively; n=4, P<0.01; Fig. 5A , left and right). In addition, Molt-4 cells transfected with siRNA for Vav1, a guanine-nucleotide exchange factor for Rac1 [43 ], similarly displayed a notable decrease in VCAM-1-Fc binding. Instead, WAVE2 knockdown cells showed only minor reductions in binding, which were not statistically significant. Control experiments revealed that all siRNA transfectants retained similar levels of VCAM-1-Fc binding upon exposure to Mn2+, and no binding was observed without VCAM-1-Fc or when Mn2+ was present, but no soluble ligand was added (not shown). These results indicate that Rac1 and Rap1, but not WAVE2, are required for efficient triggering by CCL25 of {alpha}4β1/VCAM-1 interactions at early steps of the adhesion process.


Figure 5
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  		Figure 5. CCL25-promoted, soluble binding of VCAM-1-Fc to Rac1, Rap1, and WAVE2 siRNA transfectants. (A) Molt-4 cells transfected with the indicated siRNA were preincubated with control or anti-{alpha}4 mAb and subsequently incubated in the absence (Medium) or presence of CCL25 or Mn2+, followed by binding to VCAM-1-Fc. Cell-bound ligand was detected as indicated in Materials and Methods. Basal binding in the absence of VCAM-1-Fc is also shown. Displayed is a representative result (left) and data corresponding to the mean ± SD (n=4) of VCAM-1-Fc binding (percent from mean fluorescence intensity values obtained with control siRNA transfectants; right). **, Binding was inhibited significantly, P < 0.01, according to the two-tailed Student’s t-test. (B) Cells were preincubated with cytochalasin D (Cyt D; 2.5 µg/ml), piceatannol (40 µM), or medium alone (Control), and CCL25-promoted binding of VCAM-1-Fc was measured as above (upper). Cells preincubated with the indicated concentrations of cytochalasin D were subjected to adhesion assays to VCAM-1 alone (Control) or immobilized with CCL25 (lower left). Following the washing step, pictures were taken of attached cells from adhesion assays performed in the presence or absence of cytochalasin D (lower right). (C) Molt-4 cells transfected with the indicated siRNA were exposed to CCL25 (300 nM) for different times, and following staining with FITC-phalloidin, they were subjected to flow cytometry to determine F-actin content. (D) Molt-4 cells were incubated in the absence (Medium) or presence of CCL25 or Mn2+, followed by binding to VCAM-1-{kappa}. Cell-bound ligand was detected by FITC-conjugated goat anti-mouse {kappa}. Inset numbers represent mean fluorescence intensity units.

 
As WAVE2 knockdown T cells display a large impairment in CCL25-stimulated adhesion to {alpha}4β1 ligands, which is associated with inhibition of spreading (Fig. 3B and 3C) but still maintaining binding to VCAM-1-Fc (Fig. 5A) , the data suggested that WAVE2 function might be needed mainly for actin-dependent spreading, after ligand binding and strengthening of adhesion have already occurred. Binding of VCAM-1-Fc to Molt-4 cells stimulated by CCL25 did not require F-actin polymerization, as an inhibitor of this process, such as cytochalasin D, did not affect VCAM-1-Fc binding, whereas piceatannol, a blocker of chemokine-stimulated Vav1 phosphorylation [44 ], inhibited VCAM-1-Fc binding by 70–75% (n=3; P<0.01; Fig. 5B , upper). Instead, cytochalasin D did impair CCL25-promoted T cell adhesion to VCAM-1 as a result of a defect in cell spreading (Fig. 5B , lower). Stimulation of F-actin polymerization in response to CCL25 required WAVE2 as well as Rac1 and Rap1 function, as knocking down these proteins in Molt-4 cells impaired actin polymerization in comparison with cells transfected with control siRNA (Fig. 5C) . Together, these results indicate that WAVE2 plays a key role during the actin-dependent spreading step in CCL25-promoted T cell adhesion mediated by {alpha}4β1 but not during the earlier steps of {alpha}4β1/VCAM-1 interaction.

To get insights into potential involvement of Rac1 and Rap1 in the acquisition of high-affinity {alpha}4β1 conformations during stimulation with CCL25, we used monomeric VCAM-1{kappa} and the anti-β1 15/7 mAb. Monovalent ({alpha}4β1/VCAM-1-Fc) and bivalent [({alpha}4β1)2/VCAM-1-Fc] binding takes place during interaction between {alpha}4β1 and bivalent VCAM-1-Fc [45 ]. At high ligand concentrations, bivalent binding diminishes as a result of excess competing, low-affinity, monovalent binding, resulting in decreased, detectable binding. As monomeric VCAM-1{kappa} supports {alpha}4β1-mediated adhesion of Molt-4 cells [24 ], we used it in soluble-binding assays to the different siRNA transfectants. Mn2+ triggered binding of VCAM-1{kappa} to Molt-4 cells but to substantial lower levels compared with VCAM-1-Fc binding (Fig. 5D) , in agreement with previous data about the low affinity of {alpha}4β1/monovalent VCAM-1 interaction [45 ]. Furthermore, untransfected Molt-4 cells (Fig. 5D) or cells transfected with control, Rac1, or Rap1 siRNA (not shown) incubated with CCL25 were unable to bind VCAM-1{kappa}, while binding was preserved in transfectants exposed to Mn2+ (not shown). The 15/7 antibody [46 ] recognizes an activation epitope on β1 integrins and constitutes a useful tool for detection of {alpha}4β1 integrin high-affinity conformations. Although CCL25 did induce a consistent increase in 15/7 mAb binding compared with untreated control cells (not shown), enhanced 15/7 reactivity was modest and precluded us from testing the role of Rac1 and Rap1 in the generation of {alpha}4β1 high-affinity conformations. Therefore, we were unable to test directly with these two assays the role of Rac1 and Rap1 on potential changes in {alpha}4β1 affinity in response to CCL25.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cell migration promoted by chemokines is fundamental in processes such as hematopoiesis, immune surveillance, and angiogenesis [47 ]. A functional property of chemokines is their rapid and tight control of leukocyte adhesion [22 , 23 ], which regulates cell migration lately. Chemoattractants and cell adhesion molecules control thymocyte migration inside and out of the thymus [48 49 50 ]. In the present work, we have investigated whether CCL25, a chemokine expressed predominantly in the thymus, which promotes CCR9+ thymocyte migration [14 15 16 ], is capable of regulating thymocyte adhesion mediated by the {alpha}4β1 integrin. We show here that CCR9 and {alpha}4β1 are coexpressed mainly on immature, double-positive CD4+CD8+ thymocytes, whereas CD4+ and CD8+ single-positive cells coexpress lower levels of both proteins. Furthermore, we demonstrate that CCL25 stimulates CD4+CD8+ and CD4+CD8– adhesion to VCAM-1 and CS-1/FN, involving G{alpha}i-dependent signaling. Instead, up-regulation of CD4–CD8+ attachment by CCL25 could not be detected under our assay conditions. The basis for the lack of response in {alpha}4β1-mediated adhesion in this thymocyte population is currently unknown.

CCL25 is expressed by epithelial cells in the thymic cortex, as well as by some epithelial cells in the medulla [15 ], whereas VCAM-1 and CS-1/FN are found in the cortex and medulla, respectively [8 , 9 ]. Accordingly, CCL25 could promote CD4+CD8+ thymocyte trafficking through the cortex by triggering {alpha}4β1-dependent adhesion to VCAM-1 and later stimulate CD4+CD8– cell attachment to FN in the medulla, thus contributing to the correct localization of thymocyte subpopulations in the thymus during maturation. Importance of CCL25/CCR9 axis is highlighted by the fact that mutations on CCR9 lead to mislocalization of CD25+ thymocytes throughout the cortex [51 ] and that this interaction is required for homing of bone marrow lymphoid progenitors to the thymus [52 ]. Yet, CCR9 is dispensable for proper T cell development in the thymus [53 , 54 ], suggesting that other main thymic chemokines such as CXCL12 or CCL21 might trigger compensatory, adhesive events, which might obscure important roles of CCL25 in the correct trafficking of thymocytes during thymopoiesis.

Characterization of the intracellular signaling pathways required for stimulation of cell adhesion in response to chemokines is of key importance to identify the molecular mechanisms that control cell migration during differentiation and immune response. We found that the GTPases Rac1 and Rap1 were activated rapidly by CCL25 in thymocytes, as well as in the CCR9+/{alpha}4β1+ T cell line Molt-4, and knocking down their expression in Molt-4 cells resulted in a large impairment in CCL25-stimulated adhesion to CS-1/FN and VCAM-1. As {alpha}4β1 is responsible for enhanced adhesion to FN displayed by thymocytes expressing constitutively active forms of Rac or Rap1 [38 , 39 ], our data identify the activation of these GTPases by CCL25 in thymocytes as a potential mechanism controlling the stimulation of thymocyte attachment to {alpha}4β1 ligands.

WAVE2 is a main Rac downstream effector, which controls actin-based lamellipodia and dorsal ruffling protrusions and forms part of a multimolecular complex, including PIR121, Abi1, and Nap1 [30 , 40 ]. We show here that WAVE2 is expressed on thymocytes and Molt-4 T cells in a complex with PIR121 and Abi1. Recent data described that Jurkat T cells did not express Nap1 but instead, expressed a hematopoietic homologue, hematopoietic protein 1 (HEM-1) [42 ]. We have not analyzed here whether HEM-1 exists also in the WAVE2 complex in Molt-4 cells. Stimulation with CCL25 did not alter the integrity of the complex, and although early reports showed that Rac binding to PIR121 disrupted the multimolecular complex, producing an active WAVE/HSPC300 subunit [29 ], recent data indicated that the intact WAVE complex retains actin-nucleating activity [41 , 55 ]. Therefore, our results suggest that active WAVE2 complexes are still present in thymocytes and Molt-4 cells stimulated with CCL25.

Importantly, we demonstrate that WAVE2 is required for CCL25-stimulated T cell adhesion mediated by {alpha}4β1 and provide evidence indicating that WAVE2 function is needed to generate an actin-dependent, cell-spread phenotype on {alpha}4β1 ligands, but it is not required during the initial steps of the development of {alpha}4β1/VCAM-1 interactions. Thus, WAVE2 was not or was minimally needed for actin-independent binding of VCAM-1-Fc promoted by CCL25, which is in contrast with the important role of Rac1 and its main upstream guanine nucleotide exchange factor Vav1, whose inhibition by RNA interference caused an important impairment in VCAM-1-Fc binding. Instead, WAVE2 and Rac1 functions were required for CCL25-triggered actin polymerization. The efficiency of soluble binding of ligands to integrins is considered to reflect integrin high-affinity conformations [56 , 57 ]. The experimental conditions required to obtain sufficient levels of VCAM-1-Fc binding (45 s preincubation with CCL25, followed by 75 s incubation with VCAM-1-Fc) allow detection of bound VCAM-1 in the absence of spreading but mainly reflect strengthening of {alpha}4β1/VCAM-1 interaction rather than earlier, rapid events involving changes in {alpha}4β1 conformations, leading to increased affinity for its ligands. Altogether, these data indicate that strengthening of {alpha}4β1/VCAM-1 interactions by CCL25 during the early steps of adhesion requires Vav1 and Rac1, whereas WAVE2 plays more prominent roles during post-binding events, requiring actin-based cell spreading. Reinforcement of this adhesion might reflect an increase in lateral mobility and clustering of {alpha}4β1 molecules, leading to higher numbers of adhesive bonds with VCAM-1, but we cannot rule out that Vav1-Rac1 signaling could also affect {alpha}4β1 affinity. Finally, these data also suggest the involvement of Rac1-dependent, WAVE2-independent signaling during stimulation of {alpha}4β1/VCAM-1 interaction in response to CCL25, which is currently under investigation.

RAPL and RIAM are Rap1-GTP-binding proteins, which convey stimulatory signals from Rap1 to activation of β1 integrins [26 , 31 ]. To compare their role during CCL25-stimulated T cell adhesion mediated by {alpha}4β1, we interfered with RAPL and RIAM expression in Molt-4 cells and tested transfectant attachment to CS-1/FN. We found that RAPL, but not RIAM, was needed for efficient triggering by CCL25 of T cell adhesion, revealing that Rap1-RAPL represents a signaling pathway activated by CCL25, which is required for proper T cell adhesion mediated by {alpha}4β1. The important role of RAPL during CCL25-promoted human T cell adhesion shown here using RNA interference for this protein, is consistent with earlier data demonstrating that T lymphocytes from RAPL–/– mice display deficient stimulation by CCL21 of {alpha}4β1-mediated attachment [26 ]. Moreover, a key role for RAPL was demonstrated for efficient emigration of thymocytes, suggesting that activation of Rap1-RAPL by chemokines controls thymocyte trafficking. Absence of inhibitory effects on T cell adhesion to {alpha}4β1 ligands upon stimulation by CCL25 seen on RIAM shRNA transfectants could reflect a compensatory role for RAPL, but it might also suggest that RIAM mediates Rap1-induced activation of β1 integrin-dependent cell attachment, independently of chemokine actions. Further work is necessary to characterize the mechanisms involved in RIAM-promoted activation of T cell attachment mediated by β1 integrins.

The following model can be proposed from the present results. Stimulation by CCL25 of Rac activation, possibly via Vav1, as well as of the Rap1-RAPL pathway, mediates the acquisition of high-avidity {alpha}4β1 conformations, which are responsible for the increase in T cell adhesion to its ligands. The Rac1 efector WAVE2 participates in late steps of the adhesion cascade involving actin-dependent cell spreading, an important event during cell migration (Fig. 6 ). It is possible that chemokines activate Rac-WAVE2 and Rap1-RAPL pathways in parallel, as previous work indicated that Rap1 does not activate Rac directly on T cells [25 ]. However, Rho might represent a convergence point between Rap1 and Rac cross-talks in thymocytes during regulation of integrin function, as reported [58 ]. More studies are required to characterize potential cross-talks between these two routes during chemokine-stimulated T cell adhesion to delineate how they influence the different steps in the adhesion cascade.


Figure 6
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  		Figure 6. Model for signaling from CCR9 to activation of {alpha}4β1 integrin. Rac1-WAVE2 and Rap1-RAPL pathways become stimulated upon CCL25 binding to CCR9 on T cells (a). Vav1 likely mediates the activation of Rac1 in response to CCL25, similarly to what has been observed with CXCL12 [24 ]. Activation of these pathways leads to an increase in VCAM-1 binding, suggesting that low- and intermediate-affinity {alpha}4β1 conformations (b) shift to high-affinity ones (c) and to clustering of active integrin heterodimers (d), providing the basis for increase in adhesion strengthening. Functional cross-talks between Rac1 and Rap1 may exist, as proposed earlier [58 , 59 ]. The possibility exists that RAPL could bind directly to β1, as demonstrated for the β2 integrin subunit [33 ]. WAVE2 does not play a role in initial {alpha}4β1/VCAM-1 interaction stimulated by CCL25 but rather, at later steps in the adhesion cascade, by activating actin-dependent cell spreading (e).

 


   ACKNOWLEDGEMENTS
 
This work was supported by grant SAF2005-02119 (from Ministerio de Educación y Ciencia) to J. T. We thank Pedro Lastres, Rafael Samaniego, Mª Teresa Seisdedos, and Mª del Carmen Moreno-Ortiz for their help in flow cytometry and confocal microscopy analyses and María Eugenia Miquilena for siRNA designing. Drs. José Luis Rodríguez-Fernández, Dulce Soler, and Michael J. Briskin are acknowledged for reagents and helpful discussions.


   FOOTNOTES
 
1 These authors contributed equally to this work. Back

2 Current address: R&D Department, Genetrix, Tres Cantos, Madrid, Spain. Back

Received December 12, 2006; revised April 2, 2007; accepted April 26, 2007.


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