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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

To examine the migratory properties of cytokine-activated T (Tck) cells.

Methods

Tck cells were generated by culture of peripheral blood T cells in the presence of interleukin-6 (IL-6), tumor necrosis factor α, and IL-2. Changes in cell surface phenotype were analyzed by flow cytometry. Chemotactic responsiveness was measured using in vitro chemotaxis assays and transendothelial migration through human umbilical vein endothelial cell monolayers. Levels of vascular cell adhesion molecule 1 (VCAM-1) were measured by sandwich enzyme-linked immunosorbent assay.

Results

Cytokine stimulation up-regulated the expression of chemokine receptors and integrins on Tck cells, including CXCR4, very late activation antigen 4 (VLA-4), and lymphocyte function–associated antigen 1. Increased expression of CXCR4 and VLA-4 integrin resulted in concentration-dependent chemotaxis to their ligands, stromal cell–derived factor 1 (SDF-1) and VCAM-1, which could be selectively blocked using a specific CXCR4 inhibitor and antibodies against VLA-4. Increased expression of VLA-4 also resulted in increased transendothelial migration of Tck cells, which could be abrogated using blocking antibodies against VLA-4. Tck cells also showed an increased chemotactic response to rheumatoid arthritis (RA) fibroblast-like synoviocytes cultured in vitro, which could be blocked using inhibitors against VLA-4 and CXCR4.

Conclusion

The activated phenotype of Tck cells results in increased migratory responsiveness to SDF-1 and soluble VCAM-1, which are among the chemokines and proteins found elevated in the RA synovial joint environment. Cytokine-dependent activation may contribute to RA pathogenicity by promoting T cell recruitment to and retention in the joint, perpetuating the inflammatory cascade in RA.

Rheumatoid arthritis (RA) has a prevalence of ∼1% of the population. It results in reduced mobility and significant morbidity and is characterized by chronic inflammation arising from the activities of cells within the synovial tissue (1). In healthy individuals, the synovial tissue is relatively acellular; however, in RA, there is an influx of immune cells, which causes and maintains inflammation. These immune cells consist mainly of macrophages and T lymphocytes (2, 3), which together with the resident cell types, such as synovial fibroblasts, contribute to the disease pathology. The importance of T cells in the disease is evidenced by the presence of the shared epitope in the HLA class II antigen, polymorphisms in T cell signaling molecules increasing RA susceptibility, and, more recently, T cell reactivity to citrullinated proteins (4–6).

It has been shown that large numbers of T cells are recruited to the synovium in clinical disease (1) including both typical and atypical T cell phenotypes. These consist mainly of CD4+CD45RO+CD45RBlow memory T cells; however, there is evidence of CD45RO−CD45RA+CD45RBhigh naive T cells and double-positive CD45RO+CD45RA+ T cells (7, 8). These cells also show varying expression of the costimulatory molecules CD27 and CD28 as well as the cell adhesion marker CD62L, which is expressed mainly by naive T cells (7–9). RA synovial T cells exhibit differential expression of adhesion molecules and chemokine receptors compared to peripheral blood T cells from RA patients and healthy controls, which may affect their homing and migration to the joints (refs.10 and11, and for review, see ref.12). These markers may also differentially affect their interaction with other cells present within the RA synovium, including M1-type macrophages, mature and immature plasmacytoid dendritic cells, endothelial cells, and the expanded resident fibroblast-like synoviocytes (FLS).

It has also been shown that T cells can be activated independently of antigen by cytokine stimulation. The first finding that T cells could be activated independently of antigen was described in 1994 by Unutmaz et al (13) and was subsequently confirmed by our group and others (14–16). Our group hypothesized that the activation state of T cells within the joints of RA patients was maintained by cytokines present within the disease tissue in a bystander manner and independently of antigen. We subsequently showed that peripheral blood T cells cultured for 8 days in the presence of tumor necrosis factor α (TNFα), interleukin-2 (IL-2), and IL-6 could stimulate production of proinflammatory cytokines from monocytes in a contact-dependent manner. Furthermore, these cytokine-activated T (Tck) cells demonstrated an effector function identical to that of T cells isolated from the RA synovium (16, 17).

More recently, we have shown that the most potent Tck effector cells reside within the CD4+CD45RO+ effector memory population (17). We reported the increased expression of a number of activation markers, chemokine receptors, and adhesion and integrin molecules on Tck cells, a phenotype that resembled that of T cells extracted from synovial tissue of RA patients. In that previous study we highlighted the significantly increased expression of the chemokine receptor CXCR4 and the adhesion molecules CD18, CD29, and CD49d on Tck cells, suggesting that Tck cells may show increased migration and trafficking to the synovial microenvironment.

Cell migration has been implicated in the pathogenesis of RA due to an influx of cells into the joint, and it is controlled by the chemokine receptor/ligand network. Receptor/ligand pairs show high levels of promiscuity, which leads to heterogenous cell types migrating to and being retained in the RA synovium; however, there is a degree of specificity. Stromal cell–derived factor 1 (SDF-1), the specific ligand for CXCR4, has been detected in the RA synovium and is of special interest since it predominantly attracts CD4+CD45RO+ memory T cells which express the receptor CXCR4 and are the main T cell subset implicated in RA pathogenesis (18, 19).

Cell migration also involves affinity up-regulation of integrins such as very late activation antigen 4 (VLA-4) and VLA-5, shown to be involved in the binding of inflammatory cells to extracellular matrix components such as fibronectin (11). VLA-4 is increased up to 10-fold in synovial cells compared to resting peripheral blood lymphocytes (PBLs) (10) and binds vascular cell adhesion molecule 1 (VCAM-1), which is expressed on both endothelium and macrophages in RA synovium. Furthermore, soluble VCAM-1 (sVCAM-1) is found circulating at high levels in RA and has been shown to induce migration of a number of cell types, including synovial fibroblasts, eosinophils, T cells, and monocytes (20–22).

In the present study, we confirm and extend our previous findings on the up-regulation of chemokine receptors and adhesion molecules on Tck cells and investigate the chemotactic and migratory properties of these cells. We demonstrate that Tck cells undergo increased migration in response to SDF-1 and sVCAM-1 when compared to matched resting T cells or T cells activated through the T cell receptor (TCR), and this is dependent on expression of CXCR4 and VLA-4, respectively. Furthermore, Tck demonstrate increased transendothelial migration through TNFα-stimulated human umbilical vein endothelial cell (HUVEC) monolayers, which is partially mediated through VLA-4/VCAM-1. We also show increased chemotaxis of Tck cells to RA FLS.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Patients.

Synovial membranes were obtained from patients undergoing elective joint replacement procedures. All procedures received local ethics committee approval (Riverside Research Ethics Committee no. 1752). Tissue was obtained from RA patients fulfilling the American College of Rheumatology 1987 revised classification criteria (23). Single-cell suspensions were prepared by mechanical and enzymatic disruption as previously described (24). RA FLS were generated by subculture of synovial cells and removal of the nonadherent population. RA FLS were used between the first and second passages. Human umbilical cords were collected from Chelsea and Westminster Hospital (London, UK) with informed consent after approval (no. 2948) by the Riverside Research Ethics Committee. HUVECs were isolated by enzymatic digestion as previously described (25). Endothelial cells were used between the second and fourth passages for all experiments.

Proteins and antibodies.

TNFα, IL-2, and IL-6 were kindly gifted by Boehringer Ingelheim, the National Institutes of Health, and Novartis, respectively. Flow cytometry antibodies to CXCR4, CXCR7, CCR6, CCR8, and CD49e were purchased from R&D Systems. Flow cytometry antibodies to CD49d, CD29, CD18, CD11a, β7 integrin, CXCR2, CXCR3, and CCR5 were from BD Biosciences. Blocking antibodies against CD29 (clone 4B4), CD49d (HP2/1), and VCAM-1 (P8B1) were from Beckman Coulter, Serotec, and Millipore, respectively. Isotype control antibodies IgG1 (107.3) and IgG2a (G155-178) were from BD Biosciences. CXCR4 antagonist AMD3100 was purchased from Sigma-Aldrich. Recombinant SDF-1 and recombinant sVCAM-1 were from PeproTech and R&D Systems, respectively. Anti-CD3 and anti-CD28 antibodies were from BD Biosciences. Carboxyfluorescein succinimidyl ester (CFSE) was from Invitrogen. Cells were cultured in RPMI 1640/L-glutamine (PAA Laboratories) supplemented with 10% normal AB human serum (Sigma-Aldrich) or 10% fetal calf serum (Biosera), as appropriate.

Lymphocyte purification and CD4+ cell enrichment.

Single-donor plateletpheresis residues were purchased from the North London Blood Transfusion service and then diluted 1:1 with sterile phosphate buffered saline and human peripheral blood mononuclear cells isolated by density-gradient centrifugation using Lympholyte (Cedarlane). Lymphocytes were then enriched by elutriation (Beckman Coulter). CD4+ T cells were isolated from PBLs using CD4 immunomagnetic bead isolation kits (Invitrogen) in accordance with the manufacturer's instructions.

CD4+ T cell culture and phenotypic analysis.

Tck cells were generated from CD4+ T cells by culture in RPMI 1640 supplemented with 10% human AB+ serum in the presence of IL-2 (25 ng/ml), TNF (25 ng/ml), and IL-6 (100 ng/ml) for 8 days at 37°C in 5% CO2. TCR-stimulated T (Ttcr) cells were prepared by stimulation of CD4+ T cells with plate-bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (4 μg/ml) for 2 days at 37°C in 5% CO2. Cell surface marker expression was analyzed on a Becton Dickinson FACSCanto II flow cytometer after staining with fluorochrome-conjugated antibodies.

In vitro chemotaxis assay.

Cell migration was investigated using 96-well chemotaxis plates with 3-μm–pore size polycarbonate filters (Neuroprobe). Plate wells were blocked with 1% bovine serum albumin (BSA) followed by placing a range of concentrations of SDF-1 and sVCAM-1 diluted in assay buffer (RPMI 1640 with 0.1% BSA) in the lower plate wells. Cells (2.4 × 105) in a 60-μl volume were placed in the upper wells. In some cases, Tck cells were pretreated with neutralizing antibodies (anti-CD29 and anti-CD49d) at 10 μg/ml, or with a small molecule inhibitor (AMD3100) at 50 ng/ml for 30 minutes prior to addition to the assay. After a 2-hour incubation, the migrated cells in the lower wells were collected and counted using flow cytometry. Checkerboard analysis was used to determine if cell migration was gradient-dependent by placing a range of concentrations of SDF-1 or sVCAM-1 in the upper wells as well as in the lower wells of the assay.

In vitro chemotaxis assay of Tck cell migration to cultured RA FLS.

Tck cell migration to RA FLS was investigated using 96-well chemotaxis plates with 3-μm–pore size polycarbonate filters. FLS were cultured in the lower wells at a concentration of 1 × 104/well for 18 hours prior to the assay. T cells were stained with 5 μM CFSE as previously described (17) and added to the upper wells. After a 2-hour incubation, CFSE+ migrated cells were collected and counted using flow cytometry.

In vitro transmigration assay.

HUVECs were stimulated for 18 hours in the presence of 10 ng/ml TNF prior to addition of 5 × 104 cells to fibronectin-coated Transwell inserts (Corning) with 3-μm–pore size filters. HUVECs were allowed to adhere overnight to form a monolayer. Resting T, Ttcr, or Tck cells (1 × 106 each) in a 500-μl volume were added to each monolayer, and the assay was incubated for 24 hours at 37°C in 5% CO2. Migrated cells in the lower chamber were sampled and counted using trypan blue exclusion at various time points as indicated. For some studies, Tck cells and HUVECs were pretreated with neutralizing antibodies at a final concentration of 10 μg/ml for 30 minutes prior to addition of T cells to the monolayers. Soluble VCAM-1 produced by HUVECs was measured in cell culture supernatants by sandwich enzyme-linked immunosorbent assay (ELISA).

Statistical analysis.

Results were analyzed using GraphPad Prism 5 software, and statistical differences between groups were analyzed using Wilcoxon's rank sum test, paired t-test, or one-way analysis of variance with Bonferroni adjustment or Dunnett's post-test, as appropriate. Associations between groups of data were analyzed using Pearson correlation analysis.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Tck cells up-regulate expression of integrins and chemokine receptors.

Stimulation of CD4+ T cells in the presence of IL-6, TNFα, and IL-2 up-regulated expression of a number of chemokine receptors and integrin molecules. In particular, cytokine stimulation resulted in a dramatic up-regulation of CXCR4 expression (9.5-fold; P < 0.0001), as measured by flow cytometry (Table 1). In addition, we observed increased expression of CXCR2 (1.5-fold; P = 0.0181), CXCR7 (1.9-fold; P = 0.0195), CCR5 (2-fold; P = 0.0106), CCR6 (2.5-fold; P = 0.0053), and CCR8 (2.5-fold; P = 0.0204), although to a lesser degree than that seen for CXCR4. Expression of the chemokine receptors CXCR3 and CCR7 was not significantly modulated by cytokine stimulation.

Table 1.  Expression of chemokine receptors and integrin chains on Tck cells*
 nMFI fold increase, mean ± SDP
  • *

    Lymphocytes were isolated from peripheral blood mononuclear cells by elutriation, followed by purification of the CD4+ T cell population by positive immunomagnetic bead selection. CD4+ T cells were cultured in the presence of tumor necrosis factor α (25 ng/ml), interleukin-6 (IL-6) (100 ng/ml), and IL-2 (25 ng/ml) for 8 days. Cells were washed, stained, and analyzed by flow cytometry on the day of isolation (resting T cells) and after cytokine stimulation (cytokine-activated T [Tck] cells). Shown is the fold increase in mean fluorescence intensity (MFI) for each molecule, calculated with reference to resting T cell values. n = number of donors tested or number of individual experiments; SDF-1 = stromal cell–derived factor 1; GROα = growth-related oncogene α; IP-10 = interferon-γ–inducible 10-kd protein; I-TAC = interferon-inducible T cell α chemoattractant; NS = not significant; MIP-3α = macrophage inflammatory protein 3α; ELC = Epstein-Barr virus–induced gene 1 ligand chemokine; SLC = secondary lymphoid-tissue chemokine.

  • By Wilcoxon's rank sum test.

Chemokine receptor/ligand(s)   
 CXCR4/CXCL12 (SDF-1)349.5 ± 30<0.0001
 CXCR7/CXCL12 (SDF-1)101.9 ± 1.70.0195
 CXCR2/CXCL1 (GROα)151.5 ± 1.70.0181
 CXCR3/CXCL10 (IP-10), CXCL11 (I-TAC)16NS
 CCR5/CCL5 (RANTES)172 ± 1.70.0106
 CCR6/CCL20 (MIP-3α)182.5 ± 50.0053
 CCR7/CCL19 (ELC), CCL21 (SLC)4NS
 CCR8/CCL1 (I-309)172.5 ± 5.40.0204
Integrin/alternative nomenclature   
 CD18/β2 integrin344.6 ± 12.2<0.0001
 CD11a/αL integrin112.5 ± 1.60.0029
 CD29/β1 integrin153.4 ± 4.90.0034
 β7 integrin132.3 ± 3.50.0024
 CD49a/α1 integrin152.7 ± 3.40.0002
 CD49d/α4 integrin402.2 ± 2.5<0.0001
 CD49e/α5 integrin141.5 ± 1.1NS

Integrins are heterodimeric cell adhesion proteins consisting of an α-chain and a β-chain that are important in cell–cell and cell–extracellular matrix interactions. Analysis of integrin expression on cytokine-stimulated CD4+ T cells using flow cytometry revealed a significant up-regulation of expression of lymphocyte function–associated antigen 1 (LFA-1), an integrin consisting of the αL-chain CD11a and the β2-chain CD18. While both CD11a and CD18 were highly expressed on unstimulated CD4+ T cells, cytokine stimulation increased the mean mean fluorescence intensity of both molecules (2.5-fold [P = 0.0029] and 4.6-fold [P < 0.0001], respectively). In addition, cytokine stimulation of CD4+ T cells increased expression of VLA-4, an integrin consisting of the α4-chain CD49d (2.2-fold; P < 0.0001) and the β1-chain CD29 (3.4-fold; P = 0.0034). CD29 can also associate with α-chains 1–9 to constitute other VLA molecules. Expression of the α-chain CD49a was also increased in response to cytokine stimulation (2.7-fold; P = 0.0002), while expression of CD49e was unaffected. In addition, expression of β7 integrin, also known to associate with α4 integrin chain CD49d, was up-regulated by cytokine stimulation (2.3-fold; P = 0.0024).

Increased expression of CXCR4 on Tck cells corresponds to increased chemotaxis in response to SDF-1.

We used in vitro chemotaxis assays to determine if the observed increased expression of chemokine receptors on CD4+ T cells after cytokine stimulation corresponded to an increased chemotactic response to ligands of their receptors. Interestingly, in the absence of any chemotactic gradient, Tck cells showed an increased ability to migrate in these assays (10% of total cells added) compared to donor-matched resting T cells or Ttcr cells (<2% of total cells added) (P < 0.0001) (Figure 1A).

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Figure 1. Stromal cell–derived factor 1 (SDF-1) (CXCL12) is chemotactic for cytokine-activated T (Tck) cells and acts through CXCR4. Increasing concentrations of SDF-1 were added to the lower wells of a 96-well chemotaxis plate with 3-μm pores. T cells (2.4 × 105) were added to the top wells, and following a 2-hour incubation, migrated cells were removed from the lower wells and counted by flow cytometry. A, Shown is migration of T cell receptor–stimulated T (Ttcr) cells, Tck cells, and resting T cells in response to media in the absence of chemokine (n = 30 experiments). B, Shown is migration of Tck and resting T cells in response to increasing concentrations of SDF-1 (n = 8 experiments). C, Shown is migration of Tck cells in response to increasing concentrations of SDF-1 (n = 8 experiments). D, Tck cells were pretreated with 50 ng/ml of the CXCR4 antagonist AMD3100 for 30 minutes prior to addition to the top wells (n = 5 experiments). Values in A and B are the mean ± SD percent migration of total cells added. Values in C and D are the mean ± SD percent chemotaxis. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001. NS = not significant.

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In response to increasing concentrations of SDF-1, Tck cells migrated to a higher level compared to donor-matched resting T cells in accordance with their increased expression of the receptor CXCR4 (Figure 1B). While resting T cells displayed a varied migratory response to SDF-1, Tck cells displayed a consistently increased chemotactic response. Tck cell chemotaxis to SDF-1 at concentrations of 3 and 30 ng/ml was significantly increased compared to background migration (P = 0.0051 and P = 0.0006, respectively) (Figure 1C). Less chemotaxis was observed at higher concentrations of SDF-1, consistent with the characteristic bell-shaped chemotactic response. Pretreatment of Tck cells with a small-molecule inhibitor of CXCR4 abrogated migration to 20 ng/ml SDF-1 (P = 0.0185), confirming that this effect was mediated through CXCR4 receptor expression on Tck cells (Figure 1D). However, blockade of CXCR4 in the absence of SDF-1 had no inhibitory effect on the background migration observed for Tck cells (Figure 1D).

In light of the observed migration of Tck cells in the absence of a chemokine gradient (Figure 1A), we sought to ensure that the increased response to SDF-1 was truly chemotactic (directed movement) and not chemokinetic (random movement) in nature. Results of a checkerboard assay investigating chemotaxis at different SDF-1 concentrations in the presence and absence of a chemokine gradient confirmed that Tck cells migrated to a positive gradient of SDF-1, but no migration above control levels was observed when this gradient was absent (Figure 2A). These results demonstrate that SDF-1 is indeed chemotactic and not chemokinetic for Tck. Despite the up-regulation of CCR5, CCR6, and, in some donors, CXCR3, no chemotaxis of Tck cells to their ligands RANTES, macrophage inflammatory protein 3α, and interferon-γ–inducible 10-kd protein was observed (data not shown).

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Figure 2. Tck cell migratory response is chemotactic in response to SDF-1 and chemokinetic in response to soluble vascular cell adhesion molecule 1 (sVCAM-1). A and B, Checkerboard analysis of Tck cell migration in response to increasing concentrations of SDF-1 (A) or sVCAM-1 (B) in both the upper and lower wells in in vitro chemotaxis assays. Numbers in boxes are the percent migration in response to the various concentrations of SDF-1 or sVCAM-1. Shaded boxes are indicative of the same concentration in both wells and hence the absence of a chemokine gradient (data are representative of 3 independent experiments). See Figure 1 for other definitions.

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Increased expression of VLA-4 (CD49d/CD29) on Tck corresponds to increased migration in response to sVCAM-1.

In light of previous studies documenting the ability of soluble integrin ligands to induce cell migration (20–22), coupled with our findings of increased VLA-4 expression on Tck cells, we investigated the migration of Tck in response to sVCAM-1 (Figure 3A). Results of in vitro chemotaxis assays revealed a dose-dependent increase in Tck migration in response to sVCAM-1, while in contrast migration of resting T cells or Ttcr cells was minimal. Approximately 30% of Tck cells migrated in response to a concentration of 10 μg/ml of sVCAM-1, significantly higher than that of Ttcr cells (P = 0.0256) and Tck cells background migration (P = 0.0096). Pretreatment of Tck cells with neutralizing antibodies against CD29 and CD49d abrogated this increased migration (P < 0.0001 and P = 0.0012, respectively), confirming that this effect was mediated through VLA-4 expression on Tck cells (Figure 3B). However, VLA-4 blockade did not further inhibit the background levels of Tck cells migration in the absence of a chemoattractant gradient. The ability of sVCAM-1 to stimulate Tck cells migration was not shared by other integrin ligands such as soluble intercellular adhesion molecule 1 (sICAM-1) or sICAM-2, despite up-regulation of the counterreceptor LFA-1 on T cells in response to cytokine stimulation (Figure 3C).

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Figure 3. Soluble vascular cell adhesion molecule 1 (sVCAM-1) is a chemoattractant for Tck cells and acts through very late activation antigen 4. Increasing concentrations of sVCAM-1 were added to the lower wells of a 96-well chemotaxis plate with 3-μm pores. T cells (2.4 × 105) were added to the top wells, and following a 2-hour incubation, migrated cells were removed from the lower wells and counted by flow cytometry. A, Migration of Ttcr, Tck, and resting T cells in response to increasing concentrations of sVCAM-1 (n = 5 experiments). B, Migration of Tck cells in response to 0 μg/ml or 10 μg/ml of sVCAM-1, following pretreatment of Tck cells with neutralizing antibodies against CD29 (10 μg/ml) and CD49d (10 μg/ml) or with IgG1 control (10 μg/ml) for 30 minutes prior to addition to the upper wells (n = 6 experiments). C, Migration of Tck cells in response to 10 μg/ml of soluble intercellular adhesion molecule 1 (sICAM-1) or sICAM-2. Values are the mean ± SD percent chemotaxis of total cells added. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by paired t-test. See Figure 1 for other definitions.

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The nature of the Tck response to sVCAM-1 was investigated using checkerboard analysis (Figure 2B). Tck cell migration was observed in both the presence and the absence of a concentration gradient, indicating that the Tck cell response to sVCAM-1 is chemokinetic (random movement) in nature.

Blockade of VLA-4 integrins (CD29/CD49d) selectively inhibits Tck cell transendothelial migration.

The effect of increased integrin chain expression on Tck cells was further investigated using an in vitro transendothelial migration assay, involving the measurement of cell migration through a TNFα-stimulated HUVEC monolayer at various time points. Tck cells demonstrated a significantly increased capacity to transmigrate through an endothelial monolayer compared to resting T cells and Ttcr (P < 0.01). Approximately 30% of Tck cells had migrated at the 24-hour time point compared to 7% of Ttcr cells and 2% of resting T cells (Figure 4A). We investigated the contribution of VLA-4 to Tck cell transmigration in this system using neutralizing antibodies against CD29 and CD49d. Pretreatment of Tck cells with neutralizing antibodies against CD29 inhibited migration by ∼70% (P = 0.0006) (Figure 4B), while neutralizing antibodies against CD49d inhibited migration by ∼50% (P = 0.0068) (Figure 4C). Coblockade of both molecules simultaneously did not result in further inhibition compared to blockade of each molecule alone (data not shown).

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Figure 4. Tck cells demonstrate increased transmigration, which is mediated through very late activation antigen 4. Human umbilical vein endothelial cells were stimulated for 18 hours with 10 ng/ml tumor necrosis factor prior to culture on Transwell inserts containing 3-μm pores. T cells (1 × 106) were added to the upper well and incubated for 24 hours. Migrated cells were removed from the bottom well and counted at the indicated time points. A, Shown is the percent transmigration of resting T cells, Ttcr cells, and Tck cells at the indicated time points. Values are the mean ± SD (n = 4 experiments). ∗∗ = P < 0.01 versus resting T cells and Ttcr cells, by analysis of variance with Bonferroni adjustment. B and C, Tck cells were pretreated with neutralizing antibodies against CD29 (10 μg/ml) (B) and CD49d (10 μg/ml) (C) or with IgG1 control (10 μg/ml) for 30 minutes prior to addition to the upper wells. Values are the percent of cells migrated at 24 hours (n = 8 experiments). ∗∗∗ = P < 0.001. See Figure 1 for definitions.

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RA FLS play a role in inducing migration of Tck cells.

RA pathology has previously been shown to be perpetuated by the continuous immigration of T cells and other immune cells into the joint. Synovial fibroblasts have been reported to be involved in the chemokine production that attracts these cells (26–29). To investigate if Tck cells are attracted to RA FLS in vitro, we used modified chemotaxis assays of Tck migration to cultured RA FLS.

The presence of RA FLS resulted in a modest yet significant increase of background Tck cell migration in the majority of donors (P = 0.0006) (Figure 5A). ELISA analysis of FLS supernatants revealed sVCAM-1 production, with a mean concentration of 620 pg/ml (range 19–1,400 pg/ml) (Figure 5B). To investigate if this sVCAM-1 was responsible for the increased Tck cell migration, Tck cells and FLS were pretreated with neutralizing antibodies against VLA-4 (CD29/CD49d) and VCAM-1, respectively. Migration was inhibited by up to 62% compared to the isotype control; however, this varied between donors (Figure 5C). Since RA FLS have previously been reported to produce SDF-1, we investigated whether CXCR4 inhibition could abrogate Tck cell migration. Pretreatment of Tck cells with AMD3100 could indeed inhibit Tck cell migration to FLS by up to 67% (P = 0.0423); again, however, this differed between donors (Figure 5D).

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Figure 5. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) play a role in inducing migration of cytokine-activated T (Tck) cells. RA FLS were cultured overnight in the lower wells of 96-well chemotaxis plates with 3-μm pores. Tck cells were stained with carboxyfluorescein succinimidyl ester and added to the upper wells. After a 2-hour incubation, migrated cells were collected and counted using flow cytometry. A, Shown is migration of Tck cells in response to media and RA FLS (n = 12 experiments). ∗∗∗ = P < 0.001 by Wilcoxon's rank sum test. B, Supernatants from cultured FLS were analyzed for levels of soluble vascular cell adhesion molecule 1 (sVCAM-1) by enzyme-linked immunosorbent assay (n = 14 experiments). Values are the mean ± SD. C, Tck cells and FLS were pretreated with neutralizing antibodies against very late activation antigen 4 (CD29) (10 μg/ml) and VCAM-1 (10 μg/ml), respectively, or with IgG control (10 μg/ml) for 30 minutes prior to addition to the upper wells (n = 4 experiments). D, Tck cells were pretreated with 50 ng/ml of the CXCR4 antagonist AMD3100 for 30 minutes prior to addition to the upper wells (n = 4 experiments). ∗ = P < 0.05 by paired t-test. Values in A, C, and D are the percent migration.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

The dysregulated production of inflammatory cytokines, particularly TNFα, is acknowledged to play a key role in the pathology of RA. Evidence from our group and others has shown that synovial T cells play a crucial role in the induction of macrophage-derived TNF production through a cell contact–dependent mechanism (15, 16, 30). We previously demonstrated that cytokine activation of peripheral blood T cells induced a phenotype that mimicked T cells isolated from synovial joint tissue in both effector function and cell surface expression (16, 17). In a previous study, we noted the up-regulation of a number of adhesion molecules and chemokine receptors on Tck cells (17), and in the present study, we extend these findings and demonstrate that Tck cells exhibit increased migration in response to SDF-1 and sVCAM-1 due to increased expression of CXCR4 and VLA-4. This may have implications for the trafficking and retention of T cells to the inflamed synovium in RA.

We analyzed the expression of chemokine receptors on Tck cells, which revealed a significant up-regulation of CXCR4. Tck cells also showed a dose-dependent migratory response to SDF-1, the ligand for CXCR4. Using a small-molecule inhibitor of CXCR4, we confirmed that the response to SDF-1 was CXCR4 dependent, as SDF-1 has recently been described as a ligand for a second chemokine receptor, CXCR7. Increased CXCR4 expression has previously been identified on RA synovial T cells (19). In addition, SDF-1 is expressed in the inflamed synovium and may play a role in the trafficking of T cells to this site in RA (19, 27).

Integrins and adhesion molecules are important in cell–cell and cell–extracellular matrix interactions and play a key role in cell trafficking. Analysis of adhesion molecule expression on Tck cells revealed up-regulation of VLA-4 (CD29/CD49d) and LFA-1 (CD11a/CD18) in response to cytokine stimulation. RA synovial T cells also express high levels of both CD29 and CD49d and show enhanced ability to bind the VLA-4 ligand fibronectin (31). In addition, VCAM-1, the principal ligand for VLA-4, is expressed selectively on RA endothelium and macrophages in the lining layer of the synovium, and its expression is decreased following anti-TNFα therapy (32).

A number of previous studies have shown that sVCAM-1, a ligand for VLA-4, can stimulate migratory responses in different cell types, including eosinophils, monocytes, and RA synovial T cells (20–22). We showed that VCAM-1 dose-dependently induced Tck cell migration. This migratory response was chemokinetic as opposed to chemotactic in nature and was mediated through VLA-4 expression on Tck cells, highlighted by the ability of neutralizing antibodies to CD29 and CD49d to inhibit the effect. Induction of VLA-4 expression on Tck cells also resulted in an increased ability to migrate through an endothelial cell monolayer, an in vitro model of extravasation.

Taken together, these results suggest that SDF-1 and sVCAM-1 may play a role in the recruitment and retention of T cells to the joint in RA. In our in vitro system, T cells acquire an increased chemotactic response to SDF-1 and VLA-4 after an 8-day activation in the presence of TNF, IL-6, and IL-2—cytokines whose levels are increased in the rheumatoid joint. This would initially suggest that T cells acquire this phenotype after they have entered the synovium; therefore, what is the relevance of the increased ability of the cells to respond to SDF-1 and VLA-4 if they are already outside the vasculature within the synovium, which seemingly obviates their need to respond to a chemotactic signal to enter the joint? We believe that the increased responsiveness of these cells to SDF-1 and VLA-4 will play a major role in retention of these cells within the synovium, where they subsequently contribute to cell contact–dependent induction of proinflammatory cytokine production from monocyte/macrophages and fibroblasts. The question remains, however, of whether SDF-1 and VLA-4 also play a role in the initial recruitment of these cells to the synovium in RA. It has previously been demonstrated that increased levels of both circulating SDF-1 and VLA-4 are present in the serum of RA patients, and levels are also increased in the synovium. This suggests the possibility that these molecules may also play a role in the initial recruitment of these cells to the synovium as well as in their retention at the site.

To address this question, we previously attempted to quantify Tck cell numbers in the peripheral blood of RA patients. However, as Tck cells represent a general cell phenotype rather than a distinct lineage, such studies are difficult due to the lack of specific markers for these cells; nevertheless, we did find an increased expression of CXCR4 on CD4+ T cells from the peripheral blood of RA patients as compared to healthy controls (data not shown). Increased SDF-1 production by the synovium in RA patients may therefore recruit these cells to the synovium in increased numbers, where they play a role in the induction of proinflammatory cytokine production contributing to the pathogenesis of RA.

It is acknowledged that synovial fibroblasts are among the principal cell types producing chemokines in RA synovial tissue. Of note, RA fibroblasts produce both SDF-1 and VCAM-1. We show that Tck cells are attracted to RA fibroblasts in vitro, and this is dependent on CXCR4 and VLA-4 expression. A previous study has documented similar findings for RA synovial T cells and B cells. In that study, RA synovial T cells showed an increased ability to migrate beneath fibroblasts isolated from RA tissue, and this effect was due to SDF-1 and VCAM-1 production (33).

Compared to resting T cells or Ttcr cells, Tck cells demonstrated an increased level of “background,” or basal, migration in the absence of chemoattractant in in vitro chemotaxis assays (Figure 1A). Increased basal migration has previously been reported for RA synovial T cells compared to RA peripheral blood T cells (20). This migration was not abrogated by blocking CXCR4 or VLA-4 and suggests that T cells activated by cytokines develop an increased level of basal migration, another phenotypic trait they share with T cells from RA synovial tissue.

In conclusion, we have expanded our previous studies on Tck cells. We have previously shown that cytokine activation induced a phenotype and effector function that mimicked RA T cells (17). In the present study, we demonstrate that cytokine activation up-regulates expression of CXCR4 and VLA-4 on T cells, resulting in increased migratory responses to their ligands SDF-1 and VCAM-1. This increased responsiveness to SDF-1 and sVCAM-1 may play a role in trafficking and retention of T cells to the inflamed synovium in RA.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Ahern had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Ahern, Brennan.

Acquisition of data. Bryant, Brennan.

Analysis and interpretation of data. Bryant, Ahern, Brennan.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
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