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The Chemokine Stromal Cell-Derived Factor-1 Modulates ₄₇ Integrin-Mediated Lymphocyte Adhesion to Mucosal Addressin Cell Adhesion Molecule-1 and Fibronectin¹

Natalia Wright^*, Andrés Hidalgo^2,*, José Miguel Rodríguez-Frade, Silvia F. Soriano, Mario Mellado, Marisa Parmo-Cabañas^*, Michael J. Briskin and Joaquin Teixidó^3,*

^* Department of Immunology, Centro de Investigaciones Biológicas, and Department of Immunology and Oncology, Centro Nacional de Biotecnología, Madrid, Spain; and Millenium Pharmaceuticals, Inc., Cambridge, MA 02139

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The interaction between the integrin ₄₇ and its ligand, mucosal addressin cell adhesion molecule-1, on high endothelial venules represents a key adhesion event during lymphocyte homing to secondary lymphoid tissue. Stromal cell-derived factor-1 (SDF-1) is a chemokine that attracts T and B lymphocytes and has been hypothesized to be involved in lymphocyte homing. In this work we show that ₄₇-mediated adhesion of CD4⁺ T lymphocytes and the RPMI 8866 cell line to mucosal addressin cell adhesion molecule-1 was up-regulated by SDF-1 in both static adhesion and cell detachment under shear stress assays. Both naive and memory phenotype CD4⁺ T cells were targets of SDF-1-triggered increased adhesion. In addition, SDF-1 augmented ₄₇-dependent adhesion of RPMI 8866 cells to connecting segment-1 of fibronectin. While pertussis toxin totally blocked chemotaxis of CD4⁺ and RPMI 8866 cells to SDF-1, enhanced ₄₇-dependent adhesion triggered by this chemokine was partially inhibited, indicating the participation of G_i-dependent as well as G_i-independent signaling. Accordingly, we show that SDF-1 induced a rapid and transient association between its receptor CXCR4 and G_i, whereas association of pertussis toxin-insensitive G₁₃ with CXCR4 was slower and of a lesser extent. SDF-1 also activated the small GTPases RhoA and Rac1, and inhibition of RhoA activation reduced the up-regulation of ₄₇-mediated lymphocyte adhesion in response to SDF-1, suggesting that activation of RhoA could play an important role in the enhanced adhesion. These data indicate that up-regulation by SDF-1 of lymphocyte adhesion mediated by ₄₇ could contribute to lymphocyte homing to secondary lymphoid tissues.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lymphocytes recirculate from blood to secondary lymphoid tissues and back again into the blood circulation, a process that is of fundamental importance for normal immune surveillance (1, 2, 3). Homing of lymphocytes to lymphoid tissues is mediated by selective interactions between adhesion receptors expressed on lymphocytes and their ligands displayed on high endothelial venules (HEV)⁴ located on postcapillary venules (4). The integrin ₄₇ is a cell adhesion receptor expressed on different T and B lymphocyte subsets that mediates their attachment to HEV in mucosa-associated lymphoid tissues by interacting with mucosal vascular addressin mucosal addressin cell adhesion molecule-1 (MAdCAM-1) (5, 6, 7, 8, 9). MAdCAM-1, a 60-kDa glycoprotein that belongs to the Ig superfamily, is expressed on HEV in Peyer’s patches, mesenteric lymph nodes, and lamina propria venules within the gut, and its expression represents sites of lymphocyte extravasation into these intestine-associated lymphoid tissues (4, 9, 10). Naive T lymphocytes (CD45RA⁺ among CD4⁺) show relatively homogeneous intermediate levels of ₄₇ expression, whereas memory B and T lymphocytes can be subdivided into ₄₇^high and ₄₇^- populations (6, 11, 12). Both naive and memory T cells home efficiently to Peyer’s patches, but ₄₇^- lymphocytes are excluded (12, 13), indicating that ₄₇ expression is associated with homing to intestinal lymphoid tissues.

Apart from mediating cell adhesion to MAdCAM-1, ₄₇ can also interact with the connecting segment-1 region of fibronectin (CS-1/FN) (14, 15). This interaction might play an important role in ₄₇^high lymphocyte homing within lymphoid tissues rather than in recruitment of lymphocytes from the blood (4). Several amino acids on both ₄ and ₇ subunits that are critical for ₄₇-dependent cell adhesion to MAdCAM-1 and CS-1/FN have recently been identified (16, 17, 18).

After tethering and rolling of lymphocytes on HEV, they rapidly stick and arrest, a process involving ₄₇/MAdCAM-1 interactions and favored by chemokine-triggered integrin activation (19). The stromal cell-derived factor-1 (SDF-1; CXCL12) is a CXC chemokine that potently attracts lymphocytes (20, 21, 22) and exerts chemoattractive and activating functions upon binding to its G protein-coupled receptor CXCR4, which is expressed on B and T lymphocytes, including CD4⁺ and CD8⁺ cells (23, 24, 25, 26). In addition, CXCR4 acts as a coreceptor for T-tropic HIV, and SDF-1 inhibits T tropic HIV infection (23, 25).

Previous studies have shown that SDF-1 can modulate the adhesive activity of the VLA-4 integrin on CD34⁺ human bone marrow hemopoietic progenitors, myeloma cells, and T lymphocytes (27, 28, 29, 30). SDF-1 is constitutively expressed on many tissues, including secondary lymphoid tissues (20, 31), and is therefore a potential candidate to contribute to lymphocyte homing during recirculation. In the present work we have investigated whether ₄₇-dependent lymphocyte adhesion to MAdCAM-1 and CS-1/FN can be subjected to regulation by SDF-1. Modulation of this adhesion could contribute to lymphocyte homing to mucosa-associated lymphoid tissue.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and Abs

The human B lymphoblastoid cell line RPMI 8866 was cultured in RPMI 1640 medium (BioWhittaker, Verviers, Belgium) supplemented with 10% FBS (BioWhittaker) and antibiotics (complete medium). The Chinese hamster ovary (CHO)-MAdCAM-1 transfectants were maintained in -MEM (BioWhittaker) supplemented with 10% FBS and containing 0.8 mg/ml G418 (Calbiochem, San Diego, CA). Human PBMC were isolated from buffy coats using a Ficoll density gradient (Biochrom, Berlin, Germany). After attachment to plastic for 1.5 h at 37°C in complete medium, nonadhered cells were recovered, and CD4⁺ T lymphocytes were purified with the CD4 Positive Isolation kit (Dynal Biotech, Oslo, Norway). Purity was >99% for each sample, as analyzed by flow cytometry (EPICS XL; Coulter, Hialeah, FL). The mAbs used in this study included anti-₁ Lia 1/2.1, anti-CD4 T4, anti-CD45RA RP 2/2.1, P3X63 (all gifts from Dr. F. Sánchez-Madrid, Hospital de la Princesa, Madrid, Spain), anti-₄₇ Act-1 (32), anti-CD45RO (Caltag Laboratories, Burlingame, CA), anti-CXCR4 44.717.111 (R&D Systems, London, U.K.), and CXCR4-01 (33). Anti-G_i polyclonal Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-G₁₃ was a gift from Dr. P. C. Sternweis (University of Texas, Dallas, TX).

Static adhesion assays

The generation of a soluble human MAdCAM-1-Ig fusion protein (sMAdCAM-1-IgG) has been previously described (18). The FN-H89 fragment of fibronectin, which contains the CS-1 site and lacks the RGD central binding domain, was generated as previously reported (34). Cells were labeled for 20 min at 37°C with the fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) (Molecular Probes, Leiden, The Netherlands), washed, resuspended in adhesion medium (RPMI/0.5% BSA), and preincubated with or without inhibitors or Abs. In those experiments in which soluble recombinant human (rh)SDF-1 (R&D Systems) was used, cells were incubated with the chemokine and added (6 x 10⁴ RPMI 8866 or 10⁵ CD4⁺) in triplicate to 96-well dishes (High-binding; Costar, Cambridge, MA) coated with 5 µg/ml FN-H89 as previously described (35) or containing a CHO-MAdCAM-1 monolayer. For adhesions to FN-H89, plates were spun for 15 s to place cells in contact with the ligand and allowed to adhere for 5 min at 37°C. For adhesions to CHO-MAdCAM-1, cells were incubated for 10 min at 37°C with no previous centrifugation. Unbound cells were removed by three washes with RPMI 1640 medium. When rhSDF-1 was coimmobilized with ligands, the chemokine was prepared at 0.5 µg/ml in sodium bicarbonate buffer (0.1 M, pH 8.8) for FN-H89 (5 µg/ml) or in carbonate buffer (pH 9.5) for sMAdCAM-1-Ig (300 ng/well) adhesions. The mixtures (50 µl/well) were coated on 96-well dishes for 2 h at 37°C, and wells were finally blocked with 0.5% BSA in the same corresponding buffers. For sMAdCAM-1-Ig, cells were allowed to settle for 5 min at 4°C, and then adhesion was conducted for 30 min at 37°C, followed by three gentle washes with RPMI 1640. Bound cells were lysed with 1% SDS in PBS, and the extent of adhesion was quantified using a fluorescence analyzer (POLARstar Galaxy; BMG Labtechnologies, Offenburg, Germany). Inhibitors included cytochalasin D and pertussis toxin (PTX; Calbiochem). Recombinant C3 transferase was expressed and purified as previously described (36).

Flow chamber adhesion assays

Several 15-mm² areas on petri dishes were coated for 2 h at 37°C with 15 µl PBS containing sMAdCAM-1-IgG alone (4 µg/ml), sMAdCAM-1-IgG with SDF-1 (2 µg/ml), or SDF-1 or PBS alone. The coated spots were washed with PBS and blocked for 1 h at 37°C with FCS. Petri dishes were incorporated as the lower wall of a parallel flow chamber (IQUUM, Boston, MA) and mounted on an inverted microscope (IX-70, Olympus, Tokyo, Japan) connected to a CCD camera (Cohu, San Diego, CA). CD4⁺ or RPMI 8866 cells (10⁶/ml) preincubated in the absence or the presence of PTX and/or anti-₄₇ Act-1 Ab (10 µg/ml) were infused at 37°C for 2 min at a flow rate of 1 dyne/cm². Flow was then stopped, and cells were allowed to settle for different times. Total cells from different fields were counted before flow was restored (0.5–2 dyne/cm²), and cells remaining tightly bound for >3 min were counted. Data are presented as the percentage of cells remaining bound compared with total cells in each field before reestablishing the flow.

Actin polymerization and chemotactic assays

To determine the content of polymerized actin (F-actin), 10⁵ cells per condition were permeabilized, fixed, and stained in a single step by addition of a 2x solution containing 0.5 mg/ml L--lysophosphatidyl-choline (Sigma-Aldrich, St. Louis, MO), 8% formaldehyde, and 4 U/ml FITC-phalloidin (Molecular Probes, Eugene, OR). Cells were incubated at 22°C for 10 min, washed with PBS, and subjected to flow cytometry. For chemotactic assays, RPMI 8866 (2 x 10⁵) or CD4⁺ cells (3 x 10⁵) in 100 µl adhesion medium were placed in the upper chamber of a Transwell (5-µm pore size; Costar). Then 600 µl adhesion medium with or without rhSDF-1 (200 ng/ml) was added to the lower chamber, and cells were allowed to migrate for 3 h at 37°C. Viable migrated cells were counted in a flow cytometer, analyzing each sample in the same predetermined time and flow conditions. Where indicated, cells were treated with 500 ng/ml PTX for 2 h at 37°C.

GTPase activity assays

We followed essentially the method previously reported (37). The GST-C21 and GST-PAK-CD fusion proteins were generated as previously described (38). To determine the effect of SDF-1 on RhoA and Rac1 activation, cells were treated with or without 150 ng/ml rhSDF-1, washed in ice-cold PBS, and incubated for 15 min at 4°C in lysis buffer (37). For RhoA and Rac1 activation upon ₄₇/sMAdCAM-1-Ig interaction, we performed soluble binding assays of RPMI 8866 cells with sMAdCAM-1-Ig (30–40 µg/ml, 30 min at 23°C), following the method previously described (16). Cells were then collected, washed, and lysed as described above. Lysates were centrifuged, 15 µl was kept for total lysate samples, and the remaining 185 µl was mixed with fusion proteins precoupled to glutathione-agarose beads. The beads and proteins bound to the fusion protein were washed in an excess of lysis buffer, eluted in Laemmli sample buffer, and analyzed for bound Rac1 or RhoA by Western blotting using mAbs against human Rac1 (BD PharMingen/Transduction Laboratories, San Diego, CA) or RhoA (Santa Cruz Biotechnology).

Immunoprecipitation, SDS-PAGE, and Western blot

After SDF-1 stimulation, untreated or PTX-treated cells were solubilized (20 mM triethanolamine (pH 8), 300 mM NaCl, 2 mM EDTA, 20% glycerol, and 1% digitonin, with 10 µM sodium orthovanadate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). Following centrifugation, lysates were immunoprecipitated as previously described (39) using CXCR4-01 Ab. Precipitates or protein extracts were separated in SDS-PAGE and transferred to nitrocellulose membranes. Western blot analysis was performed as previously reported (39), using 5% nonfatty dry milk in TBS as the blocking agent. Protein loading was controlled using a protein detection kit (Pierce, Rockford, IL) as well as by reprobing the membrane with the immunoprecipitating Ab.

Statistical analysis

The results are expressed as the mean ± SD of data obtained from three or more experiments performed in triplicate unless otherwise stated. Statistical significance was determined using two-tailed Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
SDF-1 up-regulates ₄₇-mediated lymphocyte adhesion to MAdCAM-1

Expression of CXCR4 on human CD4⁺ T lymphocytes and that on the ₄₇⁺₄₁^- B lymphoblastoid cell line RPMI 8866 have been previously described (24, 26). CXCR4 expression conveyed a chemotactic response of CD4⁺ and RPMI 8866 cells to SDF-1 that was abolished by PTX, indicating the participation of heterotrimeric G proteins of the G_i family (Fig. 1A). In addition, SDF-1 triggered a rapid and transient increase in F-actin polymerization in both cell types (Fig. 1B). These results indicated that CXCR4 on CD4⁺ and RPMI 8866 cells mediated cellular responses leading to cell motility and changes in the organization of actin cytoskeleton.

View larger version (13K): [in this window] [in a new window]   FIGURE 1. SDF-1 triggers chemotaxis and F-actin polymerization on human CD4+ T lymphocytes and RPMI 8866 cells. A, Cells were preincubated with or without PTX and allowed to migrate in Transwell chemotaxis chambers to medium alone (control) or SDF-1-containing lower chambers. Data represent the mean ± SD of two experiments, each performed with duplicate samples. B, Cells were incubated for the indicated times with SDF-1, stained with FITC-phalloidin, and subjected to flow cytometry. No change in F-actin content was detected when cells were incubated with medium alone (data not shown).

View larger version (21K): [in this window] [in a new window]   FIGURE 2. SDF-1 increases 47-mediated cell adhesion to CHO-MAdCAM-1 transfectants. BCECF-AM-labeled RPMI 8866 or CD4+ T cells were subjected to adhesion for 10 min at 37°C to CHO-MAdCAM-1 monolayers. For 5-min samples, SDF-1 (200 ng/ml) was added to wells 5 min before the end of adhesion, whereas in the case of 10-min samples SDF-1 was added from the start of adhesion. For 20-min samples, cells were preincubated with the chemokine for 10 min at 37°C and then added to the adhesion assay. Control samples were subjected to adhesion in the absence of SDF-1. Also shown is the adhesion of cells preincubated with anti-47 Act-1 Ab (10 µg/ml) before the adhesion assay. Adhesions were quantified in a fluorescence analyzer, and data represent the mean ± SD of triplicate samples from one representative result of six (for RPMI 8866) or three (for CD4+) independent experiments. **, Adhesion was significantly stimulated (p < 0.05, by Student’s two-tailed t test).

View larger version (31K): [in this window] [in a new window]   FIGURE 3. SDF-1 coimmobilized with sMAdCAM-1-IgG up-regulates 47-dependent cell adhesion. A, Static cell adhesion. SDF-1, sMAdCAM-1-IgG, or both proteins together were immobilized in adhesion wells as described in Materials and Methods. BCECF-AM-labeled CD4+ T lymphocytes or RPMI 8866 cells were subjected to adhesion for 30 min at 37°C. Some samples were preincubated with anti-47 Act-1 (10 µg/ml) Ab before the adhesion assay. Adhesions were quantified in a fluorescence analyzer, and data shown are from two independent experiments. Basal adhesion to wells coated with BSA alone is also shown. B, Adhesion under flow conditions. CD4+ or RPMI 8866 cells incubated with or without Act-1 were infused under shear flow over immobilized SDF-1, sMAdCAM-1-IgG, or both proteins together. Flow was then stopped, and cells were allowed to settle for 5 min for CD4+ or for 3 min for RPMI 8866 cells. Nonbound cells were washed off with adhesion medium at flow rates of 0.5 or 2 dynes/cm2, as indicated by the numbers in parentheses. Resistance to detachment by shear flow is represented as the mean percentage ± SD (three experiments) of cells remaining bound after 3-min washing from at least four different counted fields compared with the total cells in each field before washing. The percentage of cells remaining bound to FCS is also shown. **, Adhesion was significantly stimulated (p < 0.05, by Student’s two-tailed t test).

Naive and memory CD4⁺ T cell subpopulations are both targets for SDF-1-triggered up-regulation of adhesion to MAdCAM-1

To determine whether SDF-1 was preferentially targeting naive CD45RA⁺ or memory CD45RO⁺ phenotype cells among human CD4⁺ T lymphocytes for increased adhesion to MAdCAM-1, we compared by FACS analysis cells recovered from adhesion to MAdCAM-1 alone, MAdCAM-1 coimmobilized with SDF-1, or the whole cell population before the adhesion assay. The results showed that the proportions of CD45RA⁺ and CD45RO⁺ cells were similar inside the CD4⁺ cell population attached to MAdCAM-1 immobilized with or without SDF-1. However, a minor, but consistent, decrease in naive CD45RA⁺ cells and a concomitant slight increase in memory cells in the population recovered from adhesions to coimmobilized SDF-1 and MAdCAM-1 were detected (Fig. 4). These data suggest that SDF-1 targets both memory and naive CD4⁺ T lymphocytes in the up-regulation of ₄₇-mediated adhesion to MAdCAM-1, with a slight preference for memory CD45RO⁺ cells.

View larger version (28K): [in this window] [in a new window]   FIGURE 4. Expression of CD45RA and CD45RO on CD4+ T cells bound to MAdCAM-1/SDF-1. CD4+ T lymphocytes recovered from adhesions to sMAdCAM-1-Ig alone or from sMAdCAM-1-Ig coimmobilized with SDF-1 or total CD4+ cells before adhesion were analyzed by FACS using anti-CD45RA or anti-CD45RO Abs. The dashed line in each panel represents control staining obtained with the P3X63 Ab. The percentages of cells stained with Abs are indicated. Shown are two results representative of five separate experiments.

SDF-1 increases ₄₇-mediated adhesion to CS-1/fibronectin

The CS-1 region of fibronectin represents an additional binding site for ₄₇ (14, 15). We used RPMI 8866 cells to test the effect of SDF-1 on adhesion to FN-H89, a CS-1-containing fragment of fibronectin. A short (2-min) incubation of RPMI 8866 cells with soluble SDF-1 augmented the adhesion to FN-H89, which was blocked by Act-1, but not by Lia 1/2.1 anti-₁ Ab (Fig. 5, left panel). Similar to sMAdCAM-1-IgG, a substantially higher up-regulation of adhesion of RPMI 8866 cells was obtained when both FN-H89 and SDF-1 were coimmobilized in the same well compared with the adhesion to FN-H89 alone (Fig. 5, right panel). The adhesion was totally inhibited by Act-1, indicating that ₄₇ was mediating this increased adhesion.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Effect of SDF-1 on 47-dependent cell adhesion to FN-H89. Left panel, BCECF-AM-labeled RPMI 8866 cells were incubated for 2 min at 37°C in the absence (control) or the presence of SDF-1 and subjected to adhesion for 5 min at 37°C to FN-H89 after a short spin. Some samples were preincubated with Act-1 anti-47 or Lia 1/2.1 anti-1 Abs. Right panel, Labeled cells were directly assayed in adhesion to FN-H89 alone or coimmobilized with SDF-1. Also shown is adhesion of cells preincubated with Act-1 before the adhesion assay. Adhesions were quantified in a fluorescence analyzer, and data represent the mean ± SD of triplicate samples from a representative result of six (left panel) or three (right panel) experiments. **, Adhesion was significantly stimulated (p < 0.05, by Student’s two-tailed t test).

Effect of PTX on SDF-1-triggered increase in ₄₇-mediated cell adhesion

As shown above, chemotaxis of CD4⁺ and RPMI 8866 cells in response to SDF-1 was abolished by PTX, an agent that catalyzes the ADP ribosylation of specific G protein subunits of the G_i family, preventing receptor-G protein interactions (40). PTX did not reduce the enhancement of static adhesion of CD4⁺ T cells to MAdCAM-1 coimmobilized with SDF-1 when a 30-min adhesion time was used or when we performed shorter (7.5-min) adhesions after a spin to place cells in contact with ligands, an assay condition that resulted in similar basal adhesion levels and up-regulated CD4⁺ adhesion (Fig. 6A, left panel). Only a small fraction (<20%) of the increased adhesion of RPMI 8866 cells to MAdCAM-1 immobilized with SDF-1 was inhibited by PTX when adhesion was conducted for 30 min (Fig. 6A, right panel). When we shortened the time of adhesion to 2 min after cell centrifugation, a 40–50% inhibition by PTX was obtained. In addition, the SDF-1-triggered increase in RPMI 8866 cell adhesion to FN-H89 was only partially reduced by PTX both when soluble SDF-1 was used and when it was coimmobilized with FN-H89 (Fig. 6B).

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Effect of PTX on SDF-1-triggered up-regulation of 47-dependent cell adhesion under static conditions. BCECF-AM-labeled CD4+ or RPMI 8866 cells were exposed to PTX (500 ng/ml, 2 h) or adhesion medium alone. A, Cells were allowed to adhere for the indicated times to sMAdCAM-1-IgG coimmobilized with SDF-1. B, left panel, RPMI 8866 cells were incubated for 2 min in the absence (control) or the presence of SDF-1 and were subjected to adhesion for 5 min at 37°C to FN-H89 after a short spin. B, right panel. The results when cells were directly assayed for adhesion to FN-H89 coimmobilized with SDF-1. Adhesions were quantified in a fluorescence analyzer, and data represent the mean ± SD from at least two independent experiments, each performed with triplicate samples. **, Adhesion was significantly inhibited (p < 0.05 by Student’s two-tailed t test); *, inhibition was observed, but did not reach the p < 0.05 confidence level.

View larger version (16K): [in this window] [in a new window]   FIGURE 7. Resistance to detachment by shear flow of PTX-treated cells attached to MAdCAM-1/SDF-1. Cells incubated with or without PTX (500 ng/ml, 2 h) and/or Act-1 anti-47 Ab were infused through areas coated with the indicated ligands. Flow was stopped, cells were allowed to settle for 5 min for CD4+ or 3 min for RPMI 8866 cells, and flow (0.5 and 1 dynes/cm2, respectively) was restored. Bound cells from at least four different fields were counted after a 3-min washing. Data represent the mean percentage ± SD (three experiments) of cells remaining bound after washing from at least four different counted fields compared with total cells in each field before washing. **, Adhesion was significantly inhibited (p < 0.05, by Student’s two-tailed t test).

Role of Rho GTPases and actin cytoskeleton in up-regulation of ₄₇-mediated cell adhesion by SDF-1

Small GTPases of the Rho family, like RhoA and Rac1, are key regulators of the organization of actin cytoskeleton (41). Upstream events leading to the exchange of GDP for GTP bound to Rho GTPases activate them so they can then interact with downstream targets to produce different biological responses. We used RPMI 8866 cells to study whether SDF-1 influences the activation of RhoA and Rac1, by performing pull-down assays with GST fusion proteins containing domains derived from Rho GTPase targets. SDF-1 rapidly (1 min) and substantially increased the amount of active RhoA and Rac1 from basal levels, as detected with the GST-C21 and GST-PAK-CD fusion proteins, respectively, and this activation was still detected, although at lower levels, after 15-min incubations (Fig. 8A). To obtain some insight into a potential participation of RhoA activation in the SDF-1-triggered increase in RPMI 8866 adhesion, we used C3 transferase, an enzyme that specifically ADP-ribosylates and inhibits Rho activation (42). C3 reduced to approximately half the increase in RPMI 8866 cell adhesion to MAdCAM-1 immobilized with SDF-1, whereas it totally blocked their up-regulated adhesion to FN-H89 (Fig. 8B), suggesting that activation of RhoA could be involved in the increased adhesion. Cytochalasin D, an agent that disrupts actin filaments, blocked the up-regulation of F-actin polymerization triggered by SDF-1 on CD4⁺ and RPMI 8866 cells (data not shown). Preincubation with cytochalasin D at concentrations of up to 5 µg/ml substantially inhibited the enhanced adhesion of CD4⁺ T lymphocytes without affecting their basal adhesion to MAdCAM-1 alone (Fig. 8C). This same concentration partially inhibited (30%) the increase in adhesion of RPMI 8866 cells, and we did not use higher concentrations because basal adhesion was affected. These results suggest that the actin cytoskeleton plays a relevant role in SDF-1-induced enhancement of cell adhesion to MAdCAM-1 mediated by ₄₇.

View larger version (27K): [in this window] [in a new window]   FIGURE 8. Role of Rho GTPases and actin cytoskeleton in up-regulation of 47-mediated cell adhesion by SDF-1. A, SDF-1 activates RhoA and Rac1 on RPMI 8866 cells. Cells were treated with SDF-1 for the stated times and solubilized, and lysates were incubated with glutathione agarose-bound fusion proteins to detect active RhoA and Rac1. Beads and proteins bound to fusion proteins were eluted in Laemmli sample buffer and analyzed for bound RhoA and Rac1 by Western blot using anti-RhoA and anti-Rac1 Abs. Aliquots of the respective lysates served as controls for analyzing the total amounts of RhoA and Rac1. B, Effect of C3 on the up-regulation of 47-dependent cell adhesion by SDF-1. RPMI 8866 cells were exposed to C3 (50 µg/ml, 16 h) or adhesion medium alone (control). After labeling with BCECF-AM, cells were allowed to adhere for 30 min at 37°C to sMAdCAM-1-IgG alone or coimmobilized with SDF-1 (left panel) or were incubated for 2 min with or without SDF-1 and assayed for 5 min at 37°C in adhesion to FN-H89 (right panel). C, Role of actin cytoskeleton in SDF-1-triggered increase in 47-mediated adhesion to MAdCAM-1. BCECF-AM-labeled CD4+ or RPMI 8866 cells were incubated for 30 min at 37°C in adhesion medium alone (control) or with the indicated amounts of cytochalasin D (Cyt D) and allowed to adhere to sMAdCAM-1-IgG alone or coimmobilized with SDF-1. Adhesions were quantified in a fluorescence analyzer, and data represent the mean ± SD from at least three independent experiments, each performed with triplicate samples. **, Adhesion was significantly inhibited (p < 0.05, by Student’s two-tailed t test); *, inhibition was observed but did not reach the p < 0.05 confidence level.

View larger version (19K): [in this window] [in a new window]   FIGURE 9. Activation of RhoA and Rac1 upon 47/MAdCAM-1 interaction. RPMI 8866 cells were incubated with medium alone (Control) or with soluble sMAdCAM-1-Ig in the presence or the absence of the Act-1 anti-47 Ab. Cells were solubilized, lysates were incubated with glutathione agarose-bound fusion proteins to detect active RhoA and Rac1, and samples were processed and analyzed as described in Fig. 8.

SDF-1 induces association of CXCR4 with heterotrimeric G_i and G₁₃ proteins

Western blot analysis of RPMI 8866 lysates using Abs to several heterotrimeric G proteins revealed significant expression of G_i as well as PTX-resistant G₁₃ and G_q/11 (data not shown). To characterize G protein association with CXCR4 upon SDF-1 stimulation, we incubated RPMI 8866 cells with SDF-1, and cell lysates were immunoprecipitated with anti-CXCR4 Abs, followed by Western blotting using anti-G_i and G₁₃ Abs. The results showed that G_i rapidly and transiently interacted with CXCR4 upon SDF-1 activation, with a maximum association detected between 1 and 3 min (Fig. 10). G₁₃ also associated with CXCR4 after SDF-1 stimulation, but with slower kinetics (maximum at 5 min) and to a lesser extent compared with CXCR4/G_i association, as longer gel exposures were needed to sufficiently detect G₁₃. G_i was barely detected associated with CXCR4 when these experiments were conducted with PTX-treated cells. Interestingly, higher amounts of G₁₃ were found associated with CXCR4, and time kinetics revealed a faster CXCR4/G₁₃ interaction compared with nontreated RPMI 8866 cells (Fig. 10). These data indicate that G_i and G₁₃ are involved in SDF-1-induced RPMI 8866 cell activation.

View larger version (34K): [in this window] [in a new window]   FIGURE 10. SDF-1 induces association of CXCR4 with Gi and G13. Untreated (Control) or PTX-treated (500 ng/ml, 2 h) RPMI 8866 cells were stimulated with SDF-1 for the indicated times. Cells were solubilized, and lysates were immunoprecipitated with anti-CXCR4–01 Ab and tested in a Western blot with anti-Gi or G13 Ab as indicated. Equivalent receptor loading was controlled by reprobing each membrane with CXCR4–01 Ab. As a positive control, unprecipitated RPMI 8866 cell lysates were tested in a Western blot with Gi or G13 Ab (lysate). A representative experiment of three performed is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Enhancement of cell adhesion to sMAdCAM-1-Ig was detected by coimmobilizing SDF-1 with this ₄₇ ligand and was achieved in both static adhesion assays as well as under shear stress by measuring the resistance to cell detachment after applying shear flow. The data suggested that the effect of SDF-1 on ₄₇-mediated adhesion to sMAdCAM-1 probably involved strengthened cell adhesion to this ligand, leading to firm attachment. The rational for using immobilized SDF-1 was based on the observation that it can be found attached to bone marrow endothelium (27) and can be displayed on the surface of HUVEC (30). Although we do not have experimental evidence, it is possible that up-regulation of ₄₇-mediated lymphocyte adhesion to CHO-MAdCAM-1 induced by soluble SDF-1 might be due to its attachment to the surface of CHO-MAdCAM-1 monolayers, which could stabilize its binding to CXCR4 on lymphocytes.

The increase in ₄₇-mediated CD4⁺ cell adhesion to MAdCAM-1 by SDF-1 targeted both naive CD45RA⁺ and memory CD45RO⁺ subset cells. Memory cells express lower levels of CXCR4 than naive cells (24, 26, 43), but they respond to SDF-1 with equal or even slightly higher efficacy in up-regulation of adhesion to MAdCAM-1. This could be due to the fact that ₄₇⁺ memory cells express higher levels of ₄₇ than naive cells (11, 12, 44), which might allow them to attach with more strength to MAdCAM-1, enhancing the likelihood of SDF-1/CXCR4 binding. These results suggest that the pattern of ₄₇ expression among memory and naive subset cells and the subsequent ₄₇/MAdCAM-1 interaction might be dominant over the signaling originated upon SDF-1/CXCR4 binding, which triggers the increase in cell adhesion.

Chemotaxis of CD4⁺ and RPMI 8866 cells in response to SDF-1 was abolished by PTX, indicating an involvement of heterotrimeric G proteins of the G_i family. However, their up-regulated adhesion to sMAdCAM-1-Ig triggered by SDF-1 was only partially affected by PTX when tested on resistance to cell detachment under shear flow or in static adhesion assays. These data indicate that G_i-dependent signaling is involved in the subsequent increase in ₄₇ adhesive activity and suggest that additional, G_i-independent signaling plays a role in SDF-1-triggered enhanced adhesion. Reinforcing the involvement of G_i in the modulation of ₄₇-mediated cell adhesion, we detected a rapid and transient association with CXCR4 in RPMI 8866 cells incubated with SDF-1.

PTX-insensitive heterotrimeric G proteins include members of the G₁₂ and G_q families (45). We show in this work that in RPMI 8866 cells SDF-1 stimulated the interaction of CXCR4 with G₁₃, a G₁₂ family member, although to a lesser degree and with slower kinetics than CXCR4/G_i association. Interestingly, the amount of CXCR4-associated G₁₃ upon SDF-1 stimulation was notably enhanced in cells treated with PTX, and, as expected, there was a large reduction in G_i/CXCR4 interaction, raising the possibility that both G_i and G₁₃ interact with the same or closely located binding sites on CXCR4 and that G₁₃/CXCR4 association increases after G_i blockade. Alternatively, G_i might influence G₁₃ binding at a different site on CXCR4, which again could be increased upon inhibition of G_i binding. Therefore, the PTX-resistant increase in cell adhesion to MAdCAM-1 in response to SDF-1 could be the result of enhanced G₁₃ activation and its involvement in this adhesion. A known downstream effector target of G₁₃ is p115 RhoGEF (46), a Rho-activating guanine nucleotide exchange factor, and it has been reported that activation of G₁₃ mimicked the effects of activated forms of Rho on stress fiber formation that was inhibited by C3 (47, 48). In the present work we show that SDF-1 activated RhoA and Rac1 on RPMI 8866 cells, and inhibition of RhoA activation by C3 considerably reduced the up-regulation of ₄₇-dependent adhesion, suggesting that activation of RhoA represents an important point in the signals leading to increased adhesion. Previous work reported that expression of activated forms of RhoA caused an increase in ₄₇-dependent T lymphoma adhesion to MAdCAM-1 (49), an observation in the same line as the present findings on the proposed role of RhoA activation by SDF-1 in the up-regulation of ₄₇-mediated cell adhesion.

Additionally, we found that blockade of SDF-1-triggered F-actin polymerization in CD4⁺ and RPMI 8866 cells by cytochalasin D interfered with their response by increased adhesion to MAdCAM-1. As RhoA and Rac1 are key regulators of actin cytoskeleton organization, and their activity influences cell motility (41), it is possible that a functional link exists between SDF-1-induced activation of RhoA and Rac1 and reorganization of actin cytoskeleton, which could be associated with the modulation of ₄₇-dependent cell adhesion.

Together these data indicate that G_i-dependent signaling plays an important role in the up-regulation of ₄₇-dependent cell adhesion to MAdCAM-1 in response to SDF-1 and raises the possibility that activation of G₁₃ could also contribute to the up-regulated adhesion. The kinetics of CXCR4 association with G_i and G₁₃ suggest that the faster signaling wave leading to increased adhesion originates from G_i activation. Activation of G_i results in inhibition of adenylyl cyclases (45), and it has been suggested that changes in cAMP levels due to modulation of adenylyl cyclase activity by chemoattractants could represent an important point in the regulation of integrin-dependent leukocyte adhesion (50). Therefore, alterations in cAMP levels by G_i-activated downstream signaling could also be involved in the SDF-1-triggered increase in ₄₇ adhesive activity. Several studies focused on ₄ integrin-mediated adhesion have also determined that G_i-dependent signaling is needed to activate this adhesion, based on PTX inhibition (19, 30, 51, 52). A second signaling wave, G_I independent and potentially arising from G₁₃, could activate downstream effectors, such as RhoA and perhaps also Rac1, which could result in an additional increase in and/or maintenance of ₄₇-mediated adhesion, favoring lymphocyte adhesion strengthening and resistance to detachment under shear stress. Studies addressing G_i and G₁₃ involvement in modulation of ₄₇-dependent lymphocyte adhesion using mutant forms of these G proteins will be of key importance to characterize their participation in this process. In addition, it has been recently reported that SDF-1 activates G_q, which mediates LFA-1 activation during in vivo migration of T cell hybridoma cells (53). A possible activation of G_q by SDF-1 in lymphocytes could represent an additional candidate mechanism contributing to the increase in ₄₇ adhesive activity.

Reorganization of actin cytoskeleton induced by SDF-1 might cluster ₄₇ on the cell membrane, which might influence the avidity for its ligands. In this regard, it has been proposed that integrin clustering plays important roles in changes in the avidity of ₄₁-mediated adhesion (30, 54). Additionally, affinity modulation of ₄₇-dependent adhesion by SDF-1 is not excluded, as this chemokine can influence ₄₁ affinity for VCAM-1 (55). We also show here that ₄₇/MAdCAM-1 interaction activates Rac1, which could contribute to the maintenance of enhanced lymphocyte adhesion and resistance to detachment.

The interaction of ₄₇ with MAdCAM-1 on Peyer’s patches and lamina propria HEV represents a key adhesion pathway in lymphocyte homing during recirculation (4). SDF-1 is expressed on most tissues, including secondary lymphoid tissues such as Peyer’s patches (31), and it has been proposed to be involved in lymphocyte recirculation (20, 31). SDF-1 displayed by HEV could bind to CXCR4 on lymphocytes, and our present data indicate that a functional consequence of this interaction is the up-regulation of ₄₇ adhesive activity, which might lead to lymphocyte adhesion strengthening to MAdCAM-1, representing a mechanism contributing to lymphocyte homing to secondary lymphoid tissues.

The CC chemokine secondary lymphoid tissue chemokine (SLC) (CXCL21) has also been reported to up-regulate ₄₇-dependent adhesion of human PBL and T cell lines to MAdCAM-1 in both static and flow chamber adhesion assays (51). It was found that SLC had a greater effect on naive than on memory CD4⁺ T cells, consistent with a preferential role of this chemokine for the homing of naive T cells, which has been further demonstrated with the use of plt mice that lack SLC expression (56, 57, 58). Both SDF-1 and SLC are probably involved in lymphocyte homing to secondary lymphoid tissues from the blood, but the present results together with SLC modulation of ₄₇-dependent lymphocyte adhesion suggest that SDF-1 might have a lesser role than SLC in defining the specificity of lymphocyte subset targeting.

₄₇ binding to CS-1/FN might be important in gut-homing lymphocyte-matrix interactions within lymphoid tissues (4). In addition, ₄₇/FN interaction has been suggested to play important roles in lymphoma dissemination (59). Consequently, modulation of this adhesion by SDF-1 could contribute to lymphocyte trafficking in normal immune surveillance as well as in lymphoma invasion.

Further research directed at characterizing the mechanisms underlying chemokine-triggered modulation of ₄₇-dependent lymphocyte adhesion, including the study of G_i and G₁₃ involvement, is required for a better characterization of the molecular events implicated in lymphocyte homing during recirculation.

	Acknowledgments

 
We thank Drs. Francisco Sánchez-Madrid, Angel Corbí, M. Luisa Toribio, Alan Hall, Paul C. Sternweis, and John Collard for reagents. We also thank Drs. Francisco Sanz-Rodríguez and Jens Stein for their contribution to parts of this work, and Pedro Lastres for help with flow cytometry.

	Footnotes

 
¹ This work was supported by Grant SAF99-0057 from Ministerio de Ciencia y Tecnología. N.W. is the recipient of a predoctoral fellowship from the Comunidad de Madrid.

² Current address: Department of Hematology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029.

³ Address correspondence and reprint requests to Dr. Joaquin Teixidó, Department of Immunology, Centro de Investigaciones Biológicas, Velázquez 144, 28006 Madrid, Spain. E-mail address: joaquint{at}cib.csic.es

⁴ Abbreviations used in this paper: HEV, high endothelial venule; CS-1/FN, connecting segment-1 of fibronectin; F-actin, polymerized actin; PTX, pertussis toxin; SDF-1, stromal cell-derived factor-1; rh, recombinant human; MAdCAM-1, mucosal addressin cell adhesion molecule-1; sMAdCAM-1-IgG, soluble human MAdCAM-1-Ig fusion protein; CHO, Chinese hamster ovary; BCECF-AM, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester; SLC, secondary lymphoid tissue chemokine.

Received for publication July 12, 2001. Accepted for publication March 5, 2002.

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