Heterologous desensitization of T cell functions by CCR5 and CXCR4 ligands: inhibition of cellular signaling, adhesion and chemotaxis
Iris Hecht1,
Liora Cahalon1,
Rami Hershkoviz2,
Adi Lahat2,
Suzanne Franitza1 and
Ofer Lider1
1 Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel 2 Department of Internal Medicine, Assaf-HaRofe Hospital, Zrifin, Israel
Correspondence to: O. Lider; E-mail: ofer.lider{at}weizmann.ac.il
Transmitting editor: L. Steinman
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Abstract
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T cells migrate into inflamed sites through the extracellular matrix (ECM) in response to chemotactic areas and are then simultaneously or sequentially exposed to multiple chemotactic ligands. We examined the responses of human peripheral blood T cells, present in an ECM-like context, to combinatorial signaling transduced by SDF-1
(CXCL12), and two CCR5 ligands, RANTES (CCL5) and MIP-1ß (CCL4). Separately, these chemokines, at G protein-coupled receptor (GPCR)-stimulating concentrations, induced T cell adhesion to fibronectin (FN) and T cell chemotaxis. However, the pro-adhesive and pro-migratory capacities of SDF-1
and RANTES or MIP-1ß were mutually suppressed by the simultaneous or sequential exposure of the cells to these CCR5 or CXCR4 ligands. This cross-talk did not involve the internalization of the SDF-1
receptor, CXCR4, but rather, a decrease in phosphorylation of ERK and Pyk-2, as well as inhibition of Ca2+ mobilization. Strikingly, early CXCR4 signaling of phosphatidylinositol-3-kinase, detected by SDF-1
-induced AKT phosphorylation, was insensitive to RANTESCCR5 signals. Accordingly, early chemotaxis to SDF-1
was not susceptible to CCR5 occupancy, whereas late stages of T cell chemotaxis were markedly down-regulated. This is an example of a specialized functional desensitization of heterologous chemokine receptors that induces GPCR interference with T cell adhesion to ECM ligands and chemotaxis within chemokine-rich extravascular contexts.
Keywords: extracellular matrix, fibronectin, MIP-1ß, RANTES, SDF-1
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Introduction
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Migration of immune cells to extravascular sites of inflammation within the extracellular matrix (ECM) is affected by multiple pro-inflammatory signals, which are provided by various ECM-associated mediators, such as chemokines and cytokines (15). During migration in such signal-rich environments, T cells may encounter, either sequentially or simultaneously, a wide variety of immunomodulators (68). Furthermore, while T cells adhere to and migrate within the ECM, they express and secrete cytokines and chemokines into their environment (2). These inflammatory mediators appear to affect T cell behavior via specific cytokine and chemokine receptors, as well as by activating ß1 integrins.
Pro-migratory signals transmitted to migrating immune cells are complex; some chemoattractants can inhibit, by desensitization, the chemotactic migration induced by others (7,9). For example, pre-exposure of leukocytes to a dominant attractant, such as formyl-Met-Leu-Phe peptide, can suppress their later migration toward IL-8 (9). Thus, it was proposed that in tissues T lymphocytes navigate by responding to complex chemoattractant fields in a step-by-step manner (8) and that the final destinations of migrating T cells are determined by the sequence of chemoattractive information they encounter during migration, as well as by information received via TCR (6,8). However, how the interactions of cytokines and chemokines actually transmit adhesion and migration-altering signals to T cells present within the inflamed ECM is not yet fully understood.
SDF-1
and its sole receptor, CXCR4, are involved in normal myelopoeisis and lymphopoeisis both in embryonic and adult development (9). However, recent studies show that SDF-1
and CXCR4 are also involved in the recruitment of blood- borne leukocytes to sites of inflammation (10). Since other chemokines such as the CCR5 chemokines RANTES and MIP-1ß also reside within the inflamed site (3,4), we found it intriguing to study the combinatorial effect of these chemokines on T cell functions. These chemokines affect leukocyte functions via their interactions with cell- surface-expressed G protein-coupled chemokine receptors (GPCR). Signaling through chemokine receptors, such as CXCR4 and CCR5, also involves ligand-induced receptor homo- or hetero-dimerization, followed by STAT activation and JAK signaling (7).
We have assumed that T cell behavior within the ECM during inflammation is influenced by the simultaneous or sequential presence of SDF-1
with RANTES and MIP-1ß in a milieu of ECM glycoproteins. This hypothesis was tested here by examining the adhesion and migration of human T cells upon exposure to SDF-1
together with a pro-inflammatory mediator (RANTES or MIP-1ß) in the presence of fibronectin (FN), a major cell-adhesive glycoprotein ligand of the ECM. Separately, these mediators elevate adhesiveness and chemotaxis of leukocytes (5,6,1113), although with different kinetic patterns. Based on the findings presented herein, we postulate that chemotactic stimuli, encountered simultaneously or sequentially by ECM-penetrating T cells, can inhibit the T cells adhesion, migration and specific intracellular tyrosine phosphorylation and Ca2+ mobilization, probably due to heterologous receptor desensitization.
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Methods
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Reagents
The reagents and antibodies were obtained as noted: recombinant human IL-2 (sp. act. 18 x 106 U/mg; Chiron, Amsterdam, The Netherlands); human RANTES, MIP-1ß and SDF-1
(PeproTech, Rocky Hill, NJ); FN (Chemicon, Temecula, CA); collagen type I (Cellagen; ICN, Costa Mesa, CA); laminin (Sigma, St Louis, MO); BSA (Sigma); HEPES buffer, antibiotics, FCS and RPMI 1640 (Kibbutz Beit-Haemek, Israel); Na51Cr (Amersham, Little Chalfont, UK). The following anti-human mAb were also used: anti-IFN-
IL-10 (PharMingen, San Diego, CA), tumor necrosis factor (TNF)-
(Endogen, Woburn, MA); anti-phosphorylated Pyk2 (p-Pyk2), clone py881 (Biosource, Camarillo, CA); anti-Pyk2 (clone N-19), anti-phosphorylated ERK (pERK; clone E-4; Santa Cruz Biotechnology, Santa Cruz, CA); anti-ERK (Sigma); mAb anti-phosphorylated AKT (pAKT) and anti-AKT (Cell Signaling Technology, Beverly MA); mAb anti-CXCR4 (R & D Systems, Abingdon, UK); anti-CD44 (clone MCA P89; Serotec, Oxford, UK); anti-CCR5 (clone 45529.111; R & D Systems, Minneapolis, MN).
Purification of human T cells
T cells from human peripheral blood (PBL) were isolated on Ficoll gradients, washed, resuspended in PBS containing 3% heat-inactivated FCS and incubated (45 min, 37°C, 7% CO2-humidified atmosphere) on nylon wool columns (NovaMed, Jerusalem, Israel), as previously described (12,13). Non-adherent cells were eluted and washed, and platelets were removed by centrifuging (700 r.p.m., 15 min, 18°C) the cells. Residual monocytes were removed by incubating (2 h, 37°C) the cells on tissue culture plates and collecting the non-adherent cells. The PBL thus obtained contained >95% CD3+ cells.
T cell adhesion
Adhesion of T cells to FN was analyzed as previously described (12). Briefly, flat-bottomed microtiter (96-well) plates were pre-coated with FN (0.5 µg/well). 51Cr-labeled PBL were either left untreated or were pre-incubated (usually 30 min, 37°C) with the indicated concentrations of IL-2, RANTES, SDF-1
or MIP-1ß before exposure (usually 30 min, 37°C) to a second chemokine. The cells (105 cells in 100 µl RPMI containing 0.1% BSA) were then added to the FN-coated plates, incubated (30 min, 37°C) and then washed. Adherent cells were lysed, and the resulting supernatants removed and counted in a
-counter. The results were expressed as the mean percentage (±SD) of bound T cells from quadruplicate wells for each experimental group.
Chemotaxis assays
Chemotaxis of T cells within three-dimensional ECM-like gels, containing FN, laminin, and collagen types I and IV, was performed as previously described (6,13,14). Briefly, three drops of medium containing ECM components were placed 1.5 mm from each other on a microscope slide. The first drop contained cells, the second ECM medium and the third a chemoattractant. Once the drops started to polymerize, they were connected to form a continuous matrix, placed inside CO2-filled chambers and incubated at 37°C to allow the T cells to reach a steady-state dispersion in the gel. T cell migration within these gels was monitored using an inverted phase-contrast microscope and a video camera connected to a time-lapse VCR. Cell images were recorded in real-time, and cell locomotion and distance traveled were analyzed manually (with a Dynamic Image Analysis System) from computerized movies (made from played-back video segments). Whether cells were randomly migrating (motile, but moving randomly in the gel or in a direction away from the chemoattractant) or directionally moving [moving towards the chemoattractant, defined as migrating in the direction of the chemoattractant for at least 1520 min during the period of the assay (60 min)] was determined. T cell movement was analyzed for each 10-min interval. The migration of 51Cr-labeled T cells was examined in a 48-well Transwell chemotaxis apparatus (6.5-mm diameter; Corning, Corning, NY), consisting of two compartments separated by polycarbonate filters (5 µm pore size) pre-treated (1 h, 37°C) with FN (25 µg/ml). 51Cr-labeled T cells (2 x 105 in 100 µl of RPMI containing 0.1% BSA, antibiotics) were added to the upper chambers. The bottom chambers contained 0.6 ml of the same media, with or without human SDF-1
(250 ng/ml). After incubation at 37°C for 3 h (7.5% CO2-humidified atmosphere), cells that had transmigrated into the lower wells were collected, centrifuged and lysed (in 100 µl of distilled water containing 1 M NaOH and 0.1% Triton X-100), and the radioactivity in the resulting supernatants was determined with a
-counter. The percentage (±SD) of cell migration was calculated as the radioactivity counts in the lysates of the lower chambers (representative of the migrating cells) divided by the total counts (representative of 2 x 105 cells).
Ca2+ mobilization
Chemokine-induced Ca2+ mobilization was assessed in Jurkat cells. Jurkat cells were washed with PBS at room temperature and resuspended (50 x 106/ml) in modified Gays buffer (MGB; 5 mM NaCl, 0.22 mM KH2PO4, 1.1 mM Na2HPO4, 5 mM glucose, 0.3 mM MgSO4, 1 mM MgCl2 and 10 mM HEPES, pH 7.4). Fura-2AM (final concentration 5 µM) (Molecular Probes, Eugene, OR) mixed with pluronic acid F-127 (0.02%, final concentration; Molecular Probes) was added to the suspended cells. After 1 h at 37°C, the cells were washed (700 r.p.m., 20 min) and resuspended (50 x 106/ml) in MGB. CaCl2 (1.3 mM, final concentration) was then added and Ca2+ mobilization was measured at 340/380 nm immediately after the addition of chemokines.
FACS analysis
PBL (5 x 105/sample) were treated (37°C, 30 min) with SDF-1
alone, or together with RANTES or MIP-1ß, or left untreated. The cells were then washed in FACS buffer (0.01% Azid and 1% BSA in PBS) and incubated (30 min on ice) with anti-CXCR4 mAb (1 µg in 50 µl). Unbound antibody was washed as above and the cells were incubated (30 min on ice) with secondary antibody (FITC-conjugated goat anti-mouse). The cells were then washed and resuspended in FACS buffer. CXCR4 expression was measured by FACS.
Phosphorylation of Pyk-2, ERK and AKT
Human T cells were maintained (37°C, 7% CO2, humidified atmosphere) for 48 h in RPMI containing 1% HEPES and antibiotics, and then resuspended (107 cells/ml) in RPMI containing 0.1% BSA. The cells (5 x 106/sample) were then activated with SDF-1
(250 ng/ml), MIP-1ß (125 ng/ml) or RANTES (125 ng/ml) and incubated for 7 min in FN-precoated wells (24 well plates, 8.5 µg/well). The reactions were terminated by keeping the plates at 70°C for at least 30 min. The cells were thawed at 4°C and lysed using a lysis buffer (0.5 mM EDTA, 150 mM NaCl2, 10 nM NaF, 25 mM Tris, pH 7.5, 1% Triton X-100, 200 mg/ml PMSF and 1% phosphatase-inhibiting cocktail). The lysates were centrifuged (15 min, 10,000 g, 4°C) and the protein content of the resulting supernatants was determined. Sample buffer was then added to the samples, which were boiled, and equal amounts of proteins were subjected to 7.5% SDSPAGE and then transferred to nitrocellulose membranes. The nitrocellulose membranes were blocked with TBST (20 mM Tris, 135 mM NaCl2 and 0.1 Tween 20, pH 7.5) containing 5% low-fat milk and probed with the following mAb in blocking buffer: anti-phosphorylated (p)*Pyk2 (1.5 µg/ml), anti-total (t) Pyk2 (0.2 µg/ml), anti-pERK (0.2 µg/ml) or anti-tERK (1:20,000 from stock), anti-pAKT (1:1000 from stock) or anti-tAKT (1:1000 from stock). Immunoreactive protein bands were visualized using a horseradish peroxidase-conjugated secondary antibody and the enhanced chemiluminescent system (14).
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Results
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Bi-directional inhibition of T cell adhesion to FN by SDF-1
and RANTES
The effects of SDF-1
and RANTES, separately and together, on T cell adhesion to immobilized FN was examined. As previously shown (12,14), exposure of T cells to SDF-1
or RANTES alone caused appreciable and dose-dependent adhesion of T cells to FN, with maximal adhesion occurring with 20200 ng/ml (2.525 nM; see Fig. 1A and B). Next, the adhesion of T cells to FN after pre-treatment with one chemokine and activation with the other was examined. Pre-treatment (30 min) of T cells with adhesion-inducing concentrations of SDF-1
(i.e. 20 ng/ml) caused a marked inhibition in the RANTES-induced adhesive response of T cells (Fig. 1B; P < 0.05). Interestingly, pre-treatment of T cells with RANTES markedly inhibited the SDF-1
-induced T cell adhesion to FN, even when non-adhesion-inducing concentrations, i.e. 0.2 (data not shown) and 2 ng/ml, of RANTES were used (Fig. 1A; P < 0.05). Thus, RANTES and SDF-1
mutually inhibit the ability of the other to induce T cell adhesion, with RANTES being the more potent inhibitor.
The specificity of the chemokinechemokine inhibitory effect was probed by further examining the effects of RANTES, MIP-1ß (each at 20 ng/ml) and IL-2 (10 IU/ml) on SDF-1
-induced T cell adhesion to FN. Exposure of T cells to each of these mediators separately resulted in marked adhesion of the cells to FN (Fig. 2A). In contrast, when the cells were exposed to either RANTES or MIP-1ß and then to SDF-1
, their adhesion to FN was inhibited (P < 0.05). This phenomenon was chemokine specific; sequential exposure of T cells to IL-2 and SDF-1
did not affect (decrease or increase) their adhesion to FN (Fig. 2A). Similar results were obtained with rat basophilic leukemia (RBL-2H3) cells, a tumor analogue of mucosal mast cells that also adhere to ECM components (data not shown), implying that the anti-adhesive combinatorial effects of these chemokines are not restricted to T cells.

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Fig. 2. Inhibition of SDF-1 -induced adhesion by MIP-1ß and RANTES is chemokine-specific and mediated through CCR5. (A) T cells were exposed (30 min, 37°C) to MIP-1ß, RANTES (20 ng/ml each) or IL-2 (10 IU/ml). SDF-1 was then added to the cells and following an additional 30 min incubation the cells were plated on FN-coated microtiter plates. The percentage of cells that adhered was determined 30 min later. *P < 0.05 compared to the effect of SDF-1 alone. One experiment representative of five. (B) Specific abrogation, by anti-CCR5 mAb, of RANTES- or MIP-1ß-induced inhibition of SDF-1 -mediated adhesion. T cells were incubated (30 min, 37°C) with anti-CCR5 (125 ng/ml, solid bars) or anti-CD44 (125 ng/ml, striped) mAb, or were left untreated (open bars). The cells were then exposed to the indicated chemokines (20 ng/ml each), alone or together, for an additional 30 min and plated onto FN-coated microtiter plates. Cell adhesion was determined 30 min later. */**P < 0.05. One experiment representative of three.
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The adhesion-abrogating effect of RANTES on SDF-1
-induced T cell adhesion, when encountered simultaneously, was CCR5, specific; mAb specific for CCR5 (the RANTES and MIP-1ß receptor) that blocked RANTES- and MIP-1ß-induced T cell adhesion abrogated the suppressive effects of these chemokines on the SDF-1
-induced T cell adhesion, in contrast to the control anti-CD44 mAb, which did not have any inhibiting effect (Fig. 2B). Thus, abrogation of T cell adhesion induced by exposure of the cells to SDF-1
in combination with RANTES or MIP-1ß involves the chemokine-specific CCR5 receptor.
Time-kinetic analysis of the combined effects of SDF-1
and RANTES on T cellFN interactions
Next, we examined whether the duration of the exposure of T cells to SDF-1
and RANTES (20 ng/ml each) influences their adhesive effect on T cells. T cells were exposed for 30, 90 and 120 min to either one or both of the chemokines simultaneously and then incubated on FN for an additional 30 min. The amount of SDF-1
-induced T cell adhesion to FN depended on the duration of exposure to the chemokine and exhibited a bell-shaped pattern; maximal adhesion occurred upon exposure for 3090 min and adhesion following longer exposures (i.e. 120 min) declined (Fig. 3), probably due to homologous desensitization of CXCR4 receptors. In contrast, RANTES-induced T cell adhesion to FN was not time dependent, with similar adhesion occurring upon exposure for 30120 min. Thus, T cell adhesion induced by SDF-1
and RANTES separately exhibited two distinct time-dependent patterns.
Next, the kinetic patterns of T cell adhesion to FN following simultaneous exposure to both SDF-1
and RANTES were examined. The co-inhibitory effect of both chemokines persisted for co-incubations of 3090 min. However, at 120 min, T cells adhered to FN as if only RANTES was present (Fig. 3; P < 0.05). This suggests that the mutual inhibitory effect of the two chemokines is due to the presence of both chemokines in a functionally active state. The time-dependent loss of activity of one of the chemokines, in this case the pro-adhesive properties of SDF-1
at 120 min, probably enables the other chemokine (RANTES) to exert its pro-adhesive activity.
Inhibition of SDF-1
-induced chemotaxis through ECM glycoprotein ligands by RANTES or MIP-1ß
The ability of T cells to chemotactically navigate through the ECM depends on combined signals provided by chemotactic cytokines present within the ECM (1,15). Therefore, we first analyzed human T cell migration in the Transwell apparatus, through FN-coated membranes, in which net leukocyte migration toward a chemokine over time (3 h) can be measured. Using this system, we measured T cell responses to SDF-1
present in the lower wells, in the absence or presence of RANTES or MIP-1ß in the upper and lower compartments. When added, the CCR5 chemokines were applied in equal concentrations in both compartments in order to create a uniform (non-gradient) chemokine field (8). Using this set-up, we found that a substantial 18% migration of T cells occurred when only SDF-1
was present in the lower chambers, i.e. no additional chemokine was present (data not shown). However, SDF-1
-induced migration of T cells was significantly (P < 0.05) reduced if either RANTES or MIP-1ß (20200 ng/ml) was present in both the upper and lower chambers, although with distinct dose-dependency patterns. T cell chemotaxis toward SDF-1
was not affected by the presence of IL-2 (data not shown), which is in agreement with IL-2 not affecting T cell adhesion to FN (Fig. 2A).
Next, we used a three-dimensional ECM-like gel system, containing laminin, collagen types I and IV, and FN, with which we planned to monitor, in real time, the migration of individual T cells along chemotactic gradients in vitro. Previously, we showed that IL-2, RANTES and SDF-1
gradients can activate human CD4+ T cell chemokinesis (i.e. random movements) and chemotaxis (i.e. directional migration) along the ECM (6,13). As shown here, directional migration of T cells within a RANTES gradient in an ECM-like gel peaks (7%) at 40 min (Fig. 4C) and is lower than that obtained in a SDF-1
gradient, in which 20% migration occurs in 4050 min (Fig. 4B). Interestingly, SDF-1
pre-treatment of human T cells completely abolished their chemotaxis toward RANTES (Fig. 4C).
Next, T cells were pre-treated with RANTES and their chemotaxis toward SDF-1
present in the chemotactic zone was examined. RANTES appeared to affect SDF-1
-induced T cell chemotaxis in ECM-like gels in two distinct and opposite fashions. During the first 1030 min of the T cell chemotaxis assay, when the level of chemotaxis induced by SDF-1
alone was relatively low (512%), RANTES elevated synergistically the SDF-1
-induced T cell chemotaxis (Fig. 4B). However, after 3050 min, when exposure to SDF-1
alone induced higher T cell chemotaxis (1720%), chemotaxis of T cells pre-treated with RANTES appeared to decrease to the level of migration induced by SDF-1
alone (7%). Thus, the adhesive and chemotactic responses of T cells to dual chemokine signaling appears to depend on the concentrations of the chemokines used (Fig. 1), and on the duration of T cell exposure to these chemokines (Fig. 3).
Reduction by MIP-1ß or RANTES of SDF-1
-induced Ca2+ mobilization, but not CXCR4 expression in T cells
We explored the mechanisms underlying the observed anti-adhesive and anti-migratory effects, in the presence of SDF-1
, of the CC chemokines RANTES and MIP-1ß, by studying their effect on the surface expression (and internalization) of the SDF-1
receptor, CXCR4, and on SDF-1
-induced intracellular Ca2+ mobilization. As expected, exposure (30 min, 37°C) of human T cells to 250 ng/ml (31 nM) SDF-1
substantially decreased the surface expression of CXCR4. Interestingly, internalization of CXCR4 was not affected by sequential or simultaneous activation of the cells with either MIP-1ß or RANTES (100 ng/ml) plus SDF-1
(Fig. 5). Furthermore, exposure to either RANTES or MIP-1ß alone did not alter T cell expression of CXCR4 (data not shown). These findings, along with those of others (13,16,17), demonstrating that chemokine receptors are not internalized upon cross-desensitization, indicate that the regulatory effects of MIP-1ß and RANTES on SDF-1
-induced T cellFN interactions are not due to their interference with CXCR4 internalization.
Since mobilization of intracellular Ca2+ can link chemokine receptors to down-stream signaling events that consequently lead to cell adhesion and migration, we examined whether the two CC chemokines affect SDF-1
-induced Ca2+ mobilization. Due to the small size of most mammalian T cells and consequently the amount of cytoplasm, equivocable mobilization trends of intracellular Ca2+ have been hard to demonstrate; therefore, we used Jurkat cells, a human T cell line, for these experiments. Importantly, these line cells responded similarly to human PBL upon exposure to SDF-1
and RANTES (data not shown). In Jurkat cells, SDF-1
(125 ng/ml) induced substantial Ca2+ mobilization. However, this effect of SDF-1
was blocked by pre-exposure (10 min) of the cells to RANTES (125 ng/ml; Fig. 6) As expected, pre-treatment of the cells with SDF-1
(125 ng/ml) blocked their ability to mobilize intracellular Ca2+ upon a second exposure to SDF-1
(125 ng/ml). Exposure of the cells to IL-2 (100 U/ml) did not affect their SDF-1
-induced Ca2+ mobilization. Similar results were obtained with RBL-2H3 cells, in which RANTES and MIP-1ß, but not IL-2, down-regulated SDF-1
-induced Ca2+ mobilization (data not shown). Thus, RANTES-mediated inhibition of SDF-1
- induced T cell activation, as manifested by the regulation of adhesion and migration, might be due, at least partially, to the effects of the CC chemokines on SDF-1
-induced mobilization of Ca2+.
Inhibition, by RANTES and MIP-1ß, of SDF-1
-induced Pyk2 and ERK, but not AKT phosphorylation
We studied how MIP-1ß and RANTES affect the intracellular signaling associated with SDF-1
-induced adhesion and migration by determining the effects of MIP-1ß and RANTES on the phosphorylation of Pyk2 and ERK in SDF-1
-stimulated human T cells. The phosphorylation of Pyk2 and ERK in resting T cells was compared to that of SDF-1
-stimulated (7 min) cells, in the absence and presence of RANTES or MIP-1ß. Although Pyk2 was activated (phosphorylated) in resting T cells, even in the absence of SDF-1
, this activation increased (P < 0.05) when SDF-1
was present (Fig. 7A). In resting T cells, there was no basal phosphorylation of ERK (Fig. 7B). However, when FN-interacting T cells were exposed to SDF-1
in the presence of either MIP-1ß or RANTES, phosphorylation of both Pyk2 and ERK decreased significantly (Fig. 7A and B respectively, P < 0.05). Under similar conditions, IL-2 did not affect SDF-1
-induced phosphorylation of either Pyk2 or ERK (data not shown). Thus, co-exposure of FN-interacting T cells to SDF-1
and either RANTES or MIP-1ß is associated with a decrease in Ca2+ mobilization, and phosphorylation of downstream effector molecules.
Next, we measured the effect of RANTES on SDF-1
-induced phosphorylation of AKT in FN-associated T cells following 2, 7 and 15 min of T cell activation. The AKT protein kinase, which is activated in a pathway involving phosphatidylinositol-3-kinase (PI3-K), is associated with late chemokine-induced leukocyte functions (18). Interestingly, in contrast to the mutual down-regulatory effect of RANTES (or MIP-1ß) and SDF-1
on Pyk-2 and ERK signaling (Fig. 7A and B), which were involved in earlier chemokine-induced functions, AKT phosphorylation in T cells exposed to both chemokines appeared to remain intact (Fig. 7C).
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Discussion
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While migrating into and through tissues, T cells encounter a variety of molecules within the ECM that probably provide intrinsic signals that affect their behavior and function, including adhesion and migration, and inflammatory mediator production and secretion. Some of these molecules are structural components of the ECM, which act as a scaffold to support cell adhesion and tissue integrity; others are cytokines and chemokines secreted by cells within the inflammatory site. Here, the influence of a signal-rich environment on T cell function was examined in vitro by studying the effects (individual and combined) of chemokines (SDF-1
, RANTES and MIP-1ß) in the presence of immobilized FN and in ECM-like gels. We evaluated cellular activities, such as adhesion and migration, in response to multiple signals, and biochemical mechanisms (kinase activation and Ca2+ mobilization) underlying the changes in cell behavior.
T cell activation by, and responses to, chemokines are regulated by diverse factors, including other chemokines (19,20), TCR signaling (21), opiates (17) and gp120CD4 interactions (22). However, in contrast to the activatory capacity of a single mediator, here we have demonstrated a mutual inhibitory effect on T cell adhesion to FN and migration within ECM-like gels upon T cell co-exposure to SDF-1
together with either RANTES or MIP-1ß. The regulatory effect of one such chemokine on the activity of the other depends on the cells responsiveness to each of these chemokines when used alone, as well as on the chemokines time of exposure and its dosage. The anti-adhesive and anti-migratory activities of each of these chemokines, like their activation potentials, exhibited unique kinetic and dose-dependent patterns (Figs 3 and 4). SDF-1
exhibited a low adhesion inhibitory potential, since it only inhibited RANTES-induced T cell adhesion when used in high concentrations. However, when inhibiting concentrations of SDF-1
where used, the inhibition of T cell migration within ECM-like gels was independent of time. In contrast, RANTES efficiently inhibited SDF-1
-mediated adhesion, even at RANTES concentrations that did not induce adhesion when used alone. The inhibition of SDF-1
-induced T cell adhesion by RANTES was CCR5 specific, since anti-CCR5 mAb specifically blocked the inhibitory activity of RANTES and MIP1ß, and IL-2 did not inhibit SDF-1
-induced T cell adhesion to and migration through FN (Fig. 2). In the three-dimensional ECM-like gels, RANTES actually enhanced the early (1030 min) SDF-1
-induced T cell chemotaxis, but inhibited later (4060 min) T cell chemotaxis induced by SDF-1
. Thus, we suggest that the inhibitory effect, which is introduced to the migrating T cells by co-activation with the two chemokines, depends on the cells responsiveness to both chemokines. At time points in which responses of the cells to the chemokines were low and partial, the co-exposure resulted in no inhibition and even in additive activation. This is in agreement with the time-dependent inhibition of T cell adhesion to FN upon co-exposure to both RANTES and SDF-1
, which occurred at 3090 min (in which both RANTES and SDF-1
were shown to induce maximal adhesion), but not at 120 min (in which the cells were no longer sensitive to SDF-1
, probably due to homologous desensitization of CXCR4). This notion raises the possibility that within inflamed loci, T cell activation by both SDF-1
and RANTES will result in cell recruitment and consequent stoppage of the cells within the inflamed site.
We postulate that RANTES and MIP-1ß-mediated inhibition of SDF-1
-induced T cell activities is due to cross-regulation of the CXCR4 receptor, probably via CXCR4 desensitization. Homologous desensitization of chemokine receptors is often accompanied by receptor internalization (19). However, MIP-1ß and RANTES did not induce the internalization of CXCR4 present on the surface of the T cells (Fig. 5). Heterologous desensitization, which does not involve receptor internalization, occurs in opiate (met-enkephalin)-mediated desensitization of CXCR1 (16,17), and in TNF-
-mediated desensitization of CXCR4 and CCR5 (13), and can be explained by inhibition of down-stream signaling events. Indeed, pre-incubation of T cells with RANTES, but not IL-2, inhibited SDF-1
-induced Ca2+ mobilization (Fig. 6), which supported our hypothesis of CXCR4 being heterologously desensitized by the CC chemokines. Furthermore, in T cells exposed to SDF-1
together with either MIP-1ß or RANTES, phosphorylation of Pyk2 and ERK was inhibited (Fig. 7). Pyk2 is a member of a family of focal adhesion kinases that are highly expressed in T cells (23). Tyrosine phosphorylation of Pyk-2 (and related molecules) increases the enzymatic activity of this molecule and links ß1 integrins to signaling pathways that promote cell adhesion and migration (22,24,25). Phosphorylation of Pyk-2 also regulates signals to downstream molecules associated with cell adhesion and migration (2426), such as the mitogen-activated protein kinases ERK and Jun-NH2 kinase (27). ERK regulates cell adhesion and motility by enhancing the activity of the myosin light chain kinase, myosin phosphorylation and the consequent polymerization of actin fibers (2730). In fact, phosphorylation of ERK may be directly linked to that of Pyk2, since ERK can be activated upon phosphorylation of Tyr881 of Pyk2 (29,31). Therefore, the down-regulation of ERK phosphorylation we observed in T cells exposed to SDF-1
together with MIP-1ß or RANTES may be due to the inhibition of phosphorylation of Pyk-2. Down-stream signaling of ERK involves the regulation of those transcriptional events necessary for the proliferation of immune cells (32) and the regulation of the transcription of genes for several cytokines, such as IFN-
, IL-3, IL-6, IL-10 and TNF-
(33, 34). Further more, chemokine synthesis by T cells was recently shown to depend on Ca2+ mobilization and receptor phosphorylation (35). Interestingly, in contrast to the up-regulating effect of SDF-1
, when used alone, we found that the secretion of cytokines, such as TNF
, IFN-
and IL-10, is also impaired when SDF-1
and RANTES are encountered simultaneously by FN-interacting T cells, which are co-activated by mAb anti-CD3 (Hecht et al., in preparation).
Although there is controversial evidence as to the role of PI3-K in chemokine-induced chemotaxis of different cell types (36,37), we chose to analyze the effects of RANTES on SDF-1
-induced activation of this molecule. Interestingly, we found that in contrast to the down-regulatory effects caused by RANTES (or MIP-1ß) and SDF-1
on Pyk-2 and ERK phosphorylation, AKT phosphorylation, which was measured 2, 7 and 15 min post-activation in T cells maintained on FN, was not affected. It has been suggested that PI3-K activation and leukocyte chemotaxis depend on both the investigated cells lineage and the nature of the chemokine (36). The dichotomy in the interference with Pyk-2, ERK and AKT can be explained by the different sensitivities of the T cells to each of the tested chemokines and their resulting signals. We suggest that inhibition of PI3-K (and thus AKT) signaling requires a higher threshold of inhibitory signals.
Elucidating the effects of additive (simultaneous) and cumulative (sequential) exposure to molecules present on T lymphocytes during inflammation will provide insights into the events responsible for, and the mechanisms underlying induction and termination of immune reactions at sites of inflammation. The phenomena described here, which may be due to chemokine receptor desensitization via phosphorylation of CXCR4 on residues different from those mediating receptor internalization, or inhibiting heterodimerization of CXCR4 and CCR5 (7), may be relevant under physiological conditions. In vivo, inhibition of SDF-1
-induced T cell responses by MIP-1ß or RANTES may provide a mechanism whereby ECM-interacting cells can adapt and fine-tune their activation states and functions according to the diverse array of mediators present in inflammatory sites and within the lymph node micro-environment (18,22), and thus serve as potential targets for the treatment of chronic disease.
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Acknowledgements
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This study was supported by a research grant from the Israel Science Foundation, founded by the Israel Academy of Sciences and Humanities, and by The Robert Koch-Minnerva Center for Research in Autoimmune Diseases (Weizmann Institute) and the Center for the Study of Emerging Diseases (Jerusalem, Israel). O. L. is the incumbent of the Weizmann League Career Development Chair in Childrens Diseases.
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Abbreviations
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ECMextracellular matrix
FNfibronectin
GPCRG protein-coupled receptor
MGBmodified Gays buffer
PBLperipheral blood lymphocytes
PI3-Kphosphatidylinositol-3-kinase
TNFtumor necrosis factor
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