In rheumatoid arthritis (RA), the synovium is infiltrated by mononuclear cells that influence the proliferation and activation of fibroblast-like synoviocytes (FLS) through soluble mediators as well as cell-to-cell contact. To identify receptor-ligand pairs involved in this cross-talk, we cocultured T cells with FLS lines isolated from synovial tissues from RA patients. Coculture with T cells induced phosphorylation of Akt (Ser473) and its downstream mediators, GSK-3α/GSK-β, FoxO1/3a, and mouse double minute-2, and enhanced FLS proliferation. T cell-mediated phospho-Akt up-regulation was unique for FLS as no such effect was observed upon interaction of T cells with dendritic cells and B cells. Akt activation was induced by all functional T cell subsets independent of MHC/Ag recognition and was also found with other leukocyte populations, suggesting the involvement of a common leukocyte cell surface molecule. Akt phosphorylation, enhanced in vitro FLS proliferation, and enhanced FLS IL-6 production was inhibited by blocking Abs to CD11a and ICAM-2 whereas Abs to ICAM-1 had a lesser effect. Selective involvement of the LFA-1–ICAM-2 pathway was confirmed by the finding of increased ezrin phosphorylation at Tyr353 that is known to be downstream of ICAM-2 and supports cell survival through Akt activation. CD28 T cells, which are overrepresented in RA patients, have high CD11a cell surface expression and induce Akt phosphorylation in FLS more potently than their CD28+ counterparts. These findings identify ICAM-2 as a potential therapeutic target to inhibit FLS activation in RA, allowing for a more selective intervention than broad LFA-1 inhibition.

Rheumatoid arthritis (RA)3 is a chronic inflammatory disease characterized by leukocyte infiltration into the synovium and synovial hyperplasia. Pannus, the aggressive front of the synovial tissue that destroys bone and cartilage, mainly consists of tissue-invading fibroblast-like synoviocytes (FLS) and macrophage-like synoviocytes. How synovial hyperplasia is regulated is pivotal for understanding and treating RA. FLS-intrinsic factors have been implicated that may favor FLS hyperproliferation or lack of apoptosis (1, 2, 3). The p53 tumor suppressor is abnormal in the synovial lining of RA, giving rise to the hypothesis that the p53-dependent apoptosis pathway is defective in RA. Additional genes that influence apoptotic behavior and that have altered expression in RA FLS include phosphatase and tensin homolog (PTEN), synoviolin, and sentrin (4, 5, 6).

In addition to these FLS-intrinsic factors, cell-to-cell interactions as well as various inflammatory mediators in the synovial milieu are involved (7, 8, 9, 10). Cadherin-11 is critical for the formation of the synovial lining and regulates the contact between FLS themselves (11, 12). Equally important are the interactions between FLS and mononuclear cells and, in particular, T cells that infiltrate the synovial sublining layer. In vitro, FLS and T cells interact in Ag-dependent and -independent manners. FLS express MHC class II molecules and can present Ag to CD4 T cells. Bidirectional signaling affects the behavior of both cell types (13, 14). FLS are able to provide costimulatory signals to T cells mostly through atypical pathways (such as killer Ig-like receptors, NKG2D, CD47, and fractalkine receptors) and not through the classical CD28–CD80/86 interaction, with CD80 and CD86 usually not being expressed on FLS (15, 16, 17, 18, 19). RA FLS, in contrast, respond to these interactions and mediators by activating signaling pathways such as PI3K/Akt, NF-κB, MAPKs, and STAT-3 that contribute to the expression of a variety of proinflammatory, proproliferation, and antiapoptosis molecules (20, 21, 22, 23, 24). Activation of these signaling pathways shifts the net balance between prosurvival and prodeath signals and contributes to the synovial hyperplasia. Of particular interest in apoptosis regulation is the PI3K/Akt pathway with activated Akt phosphorylating downstream targets GSK-3α/GSK-β, forkhead, mouse double minute-2 (MDM-2), Bad, and caspase-9 and mediating proliferative, prosurvival, and antiapoptotic effects (25).

Activation of the Akt pathway in FLS can be accomplished by soluble factors as well as cell-to-cell contact with other cells in the synovium. The current study was aimed at identifying receptor-ligand pairs in the T cell-FLS interaction that provide prosurvival signals. In this study, we report that T cell-induced activation of Akt in FLS is predominantly mediated by ICAM-2 that leads to PI3K/Akt activation through an ezrin-dependent mechanism. Stimulation of this pathway enhances proliferation of and cytokine production by FLS and is influenced by the cell surface density of LFA-1 (CD11a/CD18) that is highly expressed on CD28 T cells expanded in RA patients.

PBMC from healthy controls and RA patients meeting the 1987 American College of Rheumatology Criteria for RA were isolated using lymphocyte separation medium (Mediatech) by density centrifugation. This protocol was approved by the Emory University Institutional Review Board, and all subjects gave written, informed consent. PBMC were further fractionated using Ab-conjugated magnetic beads and an auto-MACS cell separator (Miltenyi Biotec).

T cell clones isolated from RA patients have been described earlier (26). Clones were maintained in RPMI 1640 medium supplemented with 10% FBS, 50 U/ml IL-2, penicillin, and streptomycin and were periodically stimulated with an anti-CD3 Ab (OKT3) and irradiated feeder cells.

FLS lines were established from synovial tissue samples of RA patients undergoing arthroplasty. Freshly obtained synovial tissues were washed with medium, cut into small pieces and minced, and the cells were released with collagenase digestion (Wako Pure Chemical). FLS were maintained in T75 flasks in DMEM medium supplemented with 10% FBS and antibiotics and used between passages 4 and 8.

All phosphospecific Abs and anti-Akt Ab were purchased from Cell Signaling Technologies. In addition, the following Abs were used: goat anti-actin (I-19) Ab (Santa Cruz Biotechnology); anti-CD11a mAb, anti-ICAM-2 (CBR-IC2/2) mAb (Abcam); mouse IgG1 control Ab and anti-ICAM-1 mAb (eBioscience); CD3-allophycocyanin Cy7, CD4-PerCP, CD8-PE Cy7, CD11a-FITC, CD28-PE, and CD45RA-allophycocyanin Abs (BD Biosciences). For cell separation, negative selection enrichment mixtures from StemCell Technologies and microbeads conjugated with Abs to CD4, CD8, CD14, and CD45RA were used (all obtained from Miltenyi Biotec). CD28-positive and -negative fractions were isolated using biotin-conjugated anti-CD28 Abs (ID Labs) and anti-biotin microbeads.

FLS were detached from the flask surface by brief trypsinization and washed with complete medium. A total of 1 × 105 to 2 × 105 FLS were mixed with T cells at a 1:8 ratio on ice, centrifuged at 400 rpm/3 min/4°C and transferred to 37°C water bath for indicated lengths of time. The reactions were stopped by addition of cold PBS (pH 7.4), and tubes were immediately shifted to ice. The cells were washed twice with cold PBS, and the cell pellets were lysed in cell extraction buffer (10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, and 0.5% deoxycholate (BioSource International)) supplemented with 1 mM PMSF and a protease inhibitor mixture (Sigma-Aldrich). Cell lysates were centrifuged at 14,000 × g for 10 min at 4°C, and the clear supernatant was collected. The protein content of the supernatants was estimated using the Bradford assay (Bio-Rad). For experiments with blocking Abs or signaling inhibitors, cells were preincubated with the Abs or the inhibitor for 30–60 min before mixing the cells together.

Equal amounts of cell lysate proteins (20–30 μg) were subjected to 10% SDS-PAGE under reducing conditions on precast gels (Bio-Rad). Separated proteins were transferred to a polyvinylidene fluoride membrane and the nonspecific sites blocked with 5% nonfat dried milk in 50 mM TBS (pH 7.6) containing 0.1% Tween 20 (TBST). Blots were incubated overnight at 4°C with optimally diluted primary Abs followed by washing and incubation with HRP-conjugated secondary Abs (Santa Cruz Biotechnology) for 1 h at room temperature. The blots were thoroughly washed and visualized by an Immobilon Western chemiluminescent detection system (Millipore). The membranes were stripped and reprobed for total protein or actin to ensure equal loading. In some experiments, phospho-Akt (S473) in cell lysates was quantified by ELISA (BioSource International).

FLS (1 × 104/ml) and T cells (8 × 104/ml) were cocultured in 24-well plates in DMEM supplemented with 10% FBS and antibiotics for up to 6 days. Cells were harvested at indicated times by using trypsin/EDTA, washed, resuspended in PBS containing 0.1% BSA, 0.01% sodium azide, and 25,000 beads (Spherotech), and counted on a LSRII flow cytometer. Supernatants were collected on day 3 and assayed for IL-6 by ELISA (BD Biosciences). For blocking studies, cells were incubated with control Abs or mAbs to ICAM-1, ICAM-2 (RA FLS), and CD11a (T cells) for 1 h at room temperature before coculturing. Results were expressed as percent inhibition, defined as the ratio of T cell-enhanced FLS proliferation in the presence of blocking Abs vs control Abs. T cell-enhanced FLS proliferation was defined as the difference of FLS proliferation in the presence of T cells minus baseline FLS proliferation.

PBMC (5 × 105/sample) from 20 healthy controls and 20 RA patients (demographics shown in Table I) were stained with an Ab panel consisting of anti-CD3-allophycocyanin Cy7, anti-CD4-PerCP, anti-CD8-PE Cy7, anti-CD11a-FITC, anti-CD28-PE, and anti-CD45RA-allophycocyanin. Data acquisition and analysis were done on an LSRII flow cytometer (BD Biosciences) with FACS DIVA software. Mean fluorescence intensity for CD11a on cell subpopulations and frequencies of CD28 T cells in healthy controls and RA patients were compared by Mann-Whitney U test.

Table I.

Demographic characteristics of RA and control populations

RAControlsp
n 20 20  
Age (years) 50.7 ± 16.1 45.6 ± 10.42 0.24 
Gender (F/M) 19/1 19/1 1.00 
Ethnicity (AA/C/Hisp)a 12/4/4 15/4/1 0.34 
RAControlsp
n 20 20  
Age (years) 50.7 ± 16.1 45.6 ± 10.42 0.24 
Gender (F/M) 19/1 19/1 1.00 
Ethnicity (AA/C/Hisp)a 12/4/4 15/4/1 0.34 
a

AA, African American; C, Caucasian; Hisp, Hispanic.

T cells communicate with FLS in a contact-dependent manner through a variety of receptor-ligand pairs, only a few of which have been defined. T cells cocultured with FLS enhanced FLS proliferation with a T cell to FLS ratio of 8:1 being optimal. After 6 days of culture, the number of FLS increases ∼6- to 8-fold in the presence of T cells compared with 4-fold for FLS cultured alone (Fig 1,a). To identify the mechanisms governing this T cell-mediated effect, we screened for signaling pathways in FLS activated under coculture conditions. Akt activation is known to have a central role in proliferation/survival in a variety of normal and tumor cells. To test whether T cells induce up-regulation of phospho-Akt, FLS were incubated with T cells for 0, 5, and 15 min and the cell lysates were analyzed for phospho-Akt (Ser473) by Western blotting. T cells up-regulated phospho-Akt (Fig. 1,b, lanes 5–7) compared with the baseline activity in FLS cultured alone (lanes 2–4). In addition to unfractionated polyclonal T cells, we tested three CD4 T cell clones established from RA patients, and all three were able to induce the Akt pathway (Fig. 1,c), confirming that this effect is truly mediated by T cells. Prior treatment of FLS with the PI3K inhibitor Ly294002 (50 μM) suppressed Akt phosphorylation; pretreatment of T cells had a less pronounced effect. In contrast, JNK inhibitor SP600125 (30 μM) did not influence Akt phosphorylation (Fig. 1 d).

FIGURE 1.

T cells enhance FLS proliferation and up-regulate phospho-Akt. a, RA FLS were cultured with (▦) or without (□) T cells, and FLS recovery was enumerated by flow cytometry on days 1 and 6. Data shown are representative of six independent experiments. b, Cell lysates from RA FLS (lanes 2–4) or RA FLS plus T cells (lanes 5–7) incubated for indicated lengths of time were probed with phospho-Akt (Ser473)-specific Abs. Lane 1, T cells alone incubated for 15 min. The membrane was reprobed with Abs against total Akt. c, Three CD4 T cell clones (A, B, C) from RA patients were incubated with RA FLS for 5 min, and the cell lysates were probed as in b. Lane 1, FLS alone. d, FLS preincubated with 50 μM of the PI3K inhibitor Ly294002 (lane 5) or 30 μM of JNK inhibitor SP600125 (lane 7) were brought into contact with T cells; conversely, T cells were preincubated with inhibitors (lanes 6 and 8) before coculture. Cell lysates were probed for phospho-Akt (Ser473) as in b and actin. Controls include T cells only (lane 1), FLS only (lane 2), FLS and T cells in the absence of any treatment (lane 3), or FLS and T cells in solvent control DMSO (lane 4).

FIGURE 1.

T cells enhance FLS proliferation and up-regulate phospho-Akt. a, RA FLS were cultured with (▦) or without (□) T cells, and FLS recovery was enumerated by flow cytometry on days 1 and 6. Data shown are representative of six independent experiments. b, Cell lysates from RA FLS (lanes 2–4) or RA FLS plus T cells (lanes 5–7) incubated for indicated lengths of time were probed with phospho-Akt (Ser473)-specific Abs. Lane 1, T cells alone incubated for 15 min. The membrane was reprobed with Abs against total Akt. c, Three CD4 T cell clones (A, B, C) from RA patients were incubated with RA FLS for 5 min, and the cell lysates were probed as in b. Lane 1, FLS alone. d, FLS preincubated with 50 μM of the PI3K inhibitor Ly294002 (lane 5) or 30 μM of JNK inhibitor SP600125 (lane 7) were brought into contact with T cells; conversely, T cells were preincubated with inhibitors (lanes 6 and 8) before coculture. Cell lysates were probed for phospho-Akt (Ser473) as in b and actin. Controls include T cells only (lane 1), FLS only (lane 2), FLS and T cells in the absence of any treatment (lane 3), or FLS and T cells in solvent control DMSO (lane 4).

Close modal

Because T cells interacting with FLS up-regulated the Akt pathway and enhanced FLS proliferation, we examined the phosphorylation status of targets downstream of Akt activation that have been shown to control cell proliferation and/or survival. As shown in Fig. 2, in addition to phospho-Akt (S473), T cells induced phospho-GSK-3α (S21)/GSK-3β (S9), phospho-FoxO1 (T24)/3a (T32), and phospho-MDM-2 (S166), suggesting that the Akt phosphorylation is functionally important and explains the effect of T cells on FLS proliferation and survival.

FIGURE 2.

Akt activation correlates with the phosphorylation of proproliferative mediators in FLS. Cell lysates from RA FLS incubated with T cells for indicated lengths of time were probed for downstream mediators of activated Akt using specific Abs. T cells only are in lane 1, and FLS only are in lane 2. Reprobing of the membrane for actin ensured equal loading.

FIGURE 2.

Akt activation correlates with the phosphorylation of proproliferative mediators in FLS. Cell lysates from RA FLS incubated with T cells for indicated lengths of time were probed for downstream mediators of activated Akt using specific Abs. T cells only are in lane 1, and FLS only are in lane 2. Reprobing of the membrane for actin ensured equal loading.

Close modal

To examine whether the T cell-mediated Akt phosphorylation in target cells was selective, T cells were incubated with FLS, dendritic cells, and B cells. As shown in Fig. 3,a, phospho-Akt was not detectable in the cell lysates from dendritic cells and B cells, irrespective of whether they had or had not been cocultured with T cells. This was in contrast to the rapid induction in FLS, suggesting that this effect was selective to FLS. When different FLS lines from RA and osteoarthritis patients were compared, they all showed the identical response pattern although quantitative differences in phospho-Akt were seen (Fig. 3 b).

FIGURE 3.

T cell-induced Akt activation is specific for RA FLS. a, T cells were incubated with RA FLS, dendritic cells (DC), and B cells as target cells for 5 min, and lysates were probed for phospho-Akt (Ser473). Akt phosphorylation was only seen in FLS. b, FLS lines from five RA patients were examined for phospho-Akt (Ser473) before and after 5 min of contact with T cells.

FIGURE 3.

T cell-induced Akt activation is specific for RA FLS. a, T cells were incubated with RA FLS, dendritic cells (DC), and B cells as target cells for 5 min, and lysates were probed for phospho-Akt (Ser473). Akt phosphorylation was only seen in FLS. b, FLS lines from five RA patients were examined for phospho-Akt (Ser473) before and after 5 min of contact with T cells.

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Assays so far used allogeneic combinations of T cells and FLS. Allorecognition can therefore not be excluded although the finding that T cell-dendritic cell coculture did not induce Akt phosphorylation argued against this interpretation. To directly address the issue of whether TCR-MHC interaction is involved, experiments were conducted with CD4 T cells in the absence or presence of blocking anti-HLA-DR Abs. Pretreatment of FLS with anti-HLA-DR Ab did not inhibit the T cell-induced Akt activation (Fig. 4 a).

FIGURE 4.

Stimulation of the Akt pathway is mediated by a common leukocyte molecule independent of Ag recognition. a, RA FLS were incubated with an anti-HLA-DR Ab for 1 h or left alone before coincubation with T cells for indicated lengths of time. Cell lysates were probed for phospho-Akt (Ser473). b, CD14+ monocytes, CD8+, CD4+CD45RA+, and CD4+CD45RA T cells, and NK/B cells isolated from PBMC were cocultured with RA FLS, and cell lysates were probed for phospho-Akt and total Akt.

FIGURE 4.

Stimulation of the Akt pathway is mediated by a common leukocyte molecule independent of Ag recognition. a, RA FLS were incubated with an anti-HLA-DR Ab for 1 h or left alone before coincubation with T cells for indicated lengths of time. Cell lysates were probed for phospho-Akt (Ser473). b, CD14+ monocytes, CD8+, CD4+CD45RA+, and CD4+CD45RA T cells, and NK/B cells isolated from PBMC were cocultured with RA FLS, and cell lysates were probed for phospho-Akt and total Akt.

Close modal

To define candidate molecules that mediate the T cell-FLS interaction and induce the activation of the PI3K pathway, we examined whether this activity is T cell specific. FLS were cocultured with positively selected CD14+, CD4+CD45RA+, CD4+CD45RA, CD8+, and negatively selected NK/B cells. Coculture with PBMC served as positive controls. All lymphocyte populations induced Akt activation (Fig. 4 b), indicating that a common leukocyte cell surface molecule stimulated FLS. Monocytes had less, but still appreciable, activity.

The finding that all leukocyte populations were able to mediate the activity raised the possibility of an integrin-mediated activity. FLS were preincubated with blocking Abs to ICAM-1 or 2; alternatively, T cells were blocked with anti-CD11a (results are shown in Fig. 5 a). Blocking ICAM-2 significantly inhibited the up-regulation of phospho-Akt (lanes 11 and 12) whereas ICAM-1 blocking had no effect (lanes 13 and 14). Conversely, LFA-1 (CD11a) blocking on T cells inhibited Akt phosphorylation (lanes 17 and 18). Control Abs or blocking ICAM-2 on T cells did not have any effect.

FIGURE 5.

T cells induce FLS activation via LFA-1 and ICAM-2. a, T cells and FLS were preincubated with anti-ICAM-1, anti-ICAM-2, anti-LFA-1, and control Abs as indicated and then mixed for 15 min. Cell lysates were probed for phospho-Akt and total Akt. b, Cell lysates from T cell-FLS cultured were probed for phosphoezrin (Tyr353). c, T cells and FLS were cocultured in the presence of anti-LFA-1, -ICAM-1, and -ICAM-2 Abs (left) or a combination of anti-ICAM-1 and -ICAM-2 Abs (right). FLS were enumerated after 6 days by flow cytometry. Results are given as percent inhibition of the T cell-induced enhanced FLS proliferation relative to the cultures with control Ig and represent mean ± SD of proliferation enhancement. d, Supernatants were harvested from FLS-T cell cocultures and assayed for the production of IL-6. Results are shown as mean ± SD of triplicate cultures.

FIGURE 5.

T cells induce FLS activation via LFA-1 and ICAM-2. a, T cells and FLS were preincubated with anti-ICAM-1, anti-ICAM-2, anti-LFA-1, and control Abs as indicated and then mixed for 15 min. Cell lysates were probed for phospho-Akt and total Akt. b, Cell lysates from T cell-FLS cultured were probed for phosphoezrin (Tyr353). c, T cells and FLS were cocultured in the presence of anti-LFA-1, -ICAM-1, and -ICAM-2 Abs (left) or a combination of anti-ICAM-1 and -ICAM-2 Abs (right). FLS were enumerated after 6 days by flow cytometry. Results are given as percent inhibition of the T cell-induced enhanced FLS proliferation relative to the cultures with control Ig and represent mean ± SD of proliferation enhancement. d, Supernatants were harvested from FLS-T cell cocultures and assayed for the production of IL-6. Results are shown as mean ± SD of triplicate cultures.

Close modal

Ezrin, a member of the ezrin/radixin/moesin family of cytoplasmic linker proteins, has previously been shown to associate with clustered ICAMs and becomes phosphorylated on serine, threonine, and tyrosine residues (27). When bound to ICAM2/3, but not with ICAM1, ezrin is phosphorylated at Tyr353 to interact with the p85 subunit of PI3K and to activate the PI3K–Akt pathway (27, 28). To examine whether T cell-FLS interaction-mediated Akt activation was mediated through the LFA1–ICAM-2–ezrin–PI3K pathway, we probed cell lysates for phosphoezrin (Tyr353). As shown in Fig. 5 b, T cells and FLS had no detectable basal level of phosphoezrin (lanes 1 and 2). Upon contact, the levels increased and stayed high for at least up to 30 min (lanes 3–6).

Functional studies confirmed that the LFA-1–ICAM-2 interaction was largely responsible for the T cell-mediated enhanced FLS proliferation and survival. Inclusion of anti-ICAM-2 and anti-LFA-1 (CD11a) Abs in the T cell-FLS coculture inhibited the T cell-induced enhanced FLS growth by 50% (p = 0.005) compared with ∼25% by anti-ICAM-1 Abs (Fig. 5,c). The combination of anti-ICAM-1 and anti-ICAM-2 Abs was only slightly better than anti-ICAM-2 mAb alone (Fig. 5 c).

T cells have been shown to activate FLS to produce IL-6, IL-8, and stromelysin (14). To examine the role of LFA-1–ICAM-2 interaction in FLS cytokine production, supernatants from FLS-T cell cocultures were assayed for IL-6 by ELISA. Inclusion of anti-ICAM-2 Abs in the cocultures significantly (p = 0.006) inhibited the T cell-induced IL-6 secretion by FLS (Fig. 5 d).

To examine whether T cells from patients with RA have higher expression of LFA-1 and are therefore more effective in activating ICAM-2 on FLS, we quantified CD11a expression on T cell subsets by flow cytometry. As shown in Fig. 6, CD28 CD4 and CD8 memory T cells had the highest CD11a expression, followed by CD28+ memory and naive T cells. Previous studies have reported increased frequencies of CD28 CD4+ T cells in patients with erosive and severe RA (29, 30). This was also the case in the current flow cytometric study of 20 RA patients and 20 age-, sex-, and ethnicity-matched healthy controls. In particular, the frequencies of CD8+CD28 T cells were increased in the RA population (p = 0.03) while the frequencies of CD4+CD28 T cells showed a trend in the same direction.

FIGURE 6.

CD28 T cells expanded in RA express higher levels of the CD11a chain of LFA-1. PBMCs from 20 healthy controls and 20 RA patients were stained for CD3, CD4, CD8, CD11a, CD28, and CD45RA. Upper panel, The expression of CD11a on CD28+CD45RA+, CD28+CD45RA, and CD28 cells in CD4 (a) and CD8 (b) populations. The frequencies of CD28 T cells within the memory CD4 (c) and CD8 (d) compartments of RA patients and controls are compared in the lower panel.

FIGURE 6.

CD28 T cells expanded in RA express higher levels of the CD11a chain of LFA-1. PBMCs from 20 healthy controls and 20 RA patients were stained for CD3, CD4, CD8, CD11a, CD28, and CD45RA. Upper panel, The expression of CD11a on CD28+CD45RA+, CD28+CD45RA, and CD28 cells in CD4 (a) and CD8 (b) populations. The frequencies of CD28 T cells within the memory CD4 (c) and CD8 (d) compartments of RA patients and controls are compared in the lower panel.

Close modal

To test whether the increased frequencies of T cells with high CD11a expression in RA patients is of functional importance for FLS proliferation, we compared CD28+ and CD28 T cells purified from PBMC for their ability to induce Akt phosphorylation in FLS. Purified T cell subsets and FLS were cocultured, and the amount of phosphorylated Akt in the cell lysates was quantified by ELISA. As shown in Fig. 7, CD28 T cells were found to be more potent in inducing phospho-Akt than CD28+ T cells.

FIGURE 7.

CD28 T cells more competently induce Akt activation in FLS than CD28+ T cells. CD28+ and CD28 T cells isolated from PBMCs were incubated with RA FLS for 15 min. Phospho-Akt (Ser473) in the cell lysates was quantified by ELISA.

FIGURE 7.

CD28 T cells more competently induce Akt activation in FLS than CD28+ T cells. CD28+ and CD28 T cells isolated from PBMCs were incubated with RA FLS for 15 min. Phospho-Akt (Ser473) in the cell lysates was quantified by ELISA.

Close modal

Hyperplasia of the synovial lining is the principal hallmark of RA with pseudotumor proliferation and insufficient apoptosis of FLS considered the major mechanisms (31). Data in this manuscript identify the LFA-1–ICAM-2 receptor-ligand interaction as a major pathway that mediates antiapoptotic and proliferative signals in FLS. It is of particular interest that ICAM-2-mediated activation of the PI3K/Akt pathway rather than ICAM-1 was the major mechanism.

Synovial fibroblast proliferation and survival are controlled by a variety of intrinsic and extrinsic factors. Among the exogenous factors are growth factors that are secreted in the inflammatory milieu and stimuli that activate the PI3K/Akt pathway (7, 8, 9, 22). Akt is a serine/threonine kinase that plays a central role in supporting proliferation and survival in a variety of cells. Akt activity is induced upon its recruitment to the plasma membrane and phosphorylation on its serine and threonine residues by regulatory kinases. This recruitment and subsequent activation of Akt is accomplished through its binding to the PI3K product phosphoinositide-3,4,5-triphosphate (25). The Akt pathway is stimulated by soluble factors and cell-to-cell contact. It has been shown that phosphorylated Akt is overexpressed in the rheumatoid synovial tissue (24).

In this manuscript, we showed that contact with mononuclear cells, in particular T cells, activates the PI3K–Akt pathway in synovial fibroblasts and the phosphorylation of the downstream targets such as GSK-3α/GSK-β, FoxO3a and FoxO1, and MDM-2 that promote proliferation and prevent apoptosis. This activation of the Akt pathway was associated with increased cell recovery of FLS, likely due to the combination of increased survival and increased proliferation. Signals that can activate the Akt pathway are numerous and include a multitude of receptors, such as costimulatory receptors or G protein-linked receptors (32). The finding that virtually all leukocyte subsets, albeit to varying degrees, had the ability to activate the Akt pathway in FLS suggested integrins such as CD11a-CD18 (LFA-1) and CD11b-CD18 (Mac-1) that are expressed on all leukocytes as likely candidates. The LFA-1 ligands, ICAM-1, −2, and −3, differentially bind to LFA-1 and govern the adhesion of leukocytes. ICAM-2 has recently been shown to mediate cell survival signals in B lymphocytes and fibroblasts. Upon binding to LFA-1, ICAM-2 activates the PI3K/Akt pathway through a mechanism that involves ezrin activation by Src and Rho kinases. Activated ezrin recruits PI3K to the plasma membrane, thereby initiating the activation of the PI3K/Akt pathway (27). Our studies showed that this mechanism is operative in T cell-FLS interaction and nearly solely responsible for the increased Akt phosphorylation and increased survival of and IL-6 production by FLS after contact with T cells. Surprisingly, other receptor-ligand interactions including LFA-1–ICAM-1 did not make major contributions.

In the context of autoimmune disease, LFA-1 is primarily considered a molecule that facilitates adhesion of leukocytes to endothelial cells and initiates the process of transmigration (33). Oppenheimer-Marks et al. (34) have demonstrated the importance of integrin-mediated T cell migration for RA in the SCID mouse-human synovium model. Abs to inhibit the recruitment of leukocytes to inflammatory sites have been introduced into clinical testing (35). Efalizumab is an Ab to CD11a that has been proven to be successful in plaque psoriasis (36). Although LFA-1 blocking also had efficacy in animal models of arthritis, anti-CD11a Abs did not suppress inflammation in moderate and severe RA at the doses used, and the clinical studies were discontinued in 2003.

Adhesion receptors do not only play a major role in leukocyte migration: they have a broad function in mediating cell-to-cell communication. Although the FLS-FLS contact in the synovial lining is predominantly governed by cadherins (11, 12), integrins appear to control the interaction of FLS with cells of the myeloid and lymphocyte lineages (33, 37). LFA-1 clusters at the contact point between T cells and FLS, and T cell adherence to FLS is inhibited by blocking Ab to LFA-1. β2 integrins collaborate with other cell surface molecules to form a signaling complex. A classical example is the formation of the T cell recognition platform where the LFA-1–ICAM interaction facilitates the formation of a signaling synapse (38). Synovial fibroblasts also express MHC class II molecules, and it has previously been shown that they can function as APCs for TCR recognition (13, 39). However, Ab blocking of MHC class II molecules did not abrogate the T cell-induced Akt activation in FLS, clearly showing that the TCR-MHC class II molecule receptor-ligand interaction was not involved in the proliferative effects that T cells had on FLS. This interpretation is further supported by the finding that LFA-1 expressed on non-T cells was also effective in triggering Akt phosphorylation in FLS, clearly indicating that LFA-1 was functional in different contexts and most likely solely responsible for the observed effect. Interestingly, monocytes that express mostly Mac-1 as a β2 integrin and, relatively minimally, LFA-1, had reduced ability to activate FLS. Mac-1 and LFA-1 both bind to ICAMs, but their binding activities are different.

In RA, several mechanisms support FLS proliferation and survival; their relative contributions to this effect are currently unclear. FLS are mainly found in the synovial lining where they intermingle with macrophage-like synoviocytes. Whether ICAM-2 stimulation by β2 integrins expressed on macrophage-like cells is quantitatively relevant remains to be determined; however, our data would suggest that monocytes are less effective than LFA-1-expressing lymphocytes. Mononuclear cells are mostly found in the sublining. Soluble mediators secreted by these cells in the sublining can also influence FLS survival in the lining layer (7, 8, 9). These mediators include inflammatory cytokines as well as growth factors, and a recent study has indeed suggested that blocking of the signaling pathway of the PDGFR ameliorates arthritis in animal models (40). Our group has shown that T cells can induce an autocrine loop in synovial fibroblasts mediated by the secretion of fractalkine that acts as a growth factor on FLS (10). In addition to these growth factors, contact-dependent mechanisms control FLS proliferation and survival, and T cells may interact with isolated FLS that migrate through the sublining layers. Among these contact-dependent mechanisms, the LFA-1–ICAM-2 interaction appears to have a prominent role in supporting FLS survival. Obviously, cell contact and soluble factors act in concert in the rheumatoid inflammation; however, even the inhibition of one of them may have a major impact if their contribution is synergistic or if they induce an autocrine amplification loop by inducing cytokine or fractalkine production in FLS. ICAM-2 is able to stimulate autocrine cytokine production; however, the efficacy of ICAM-2 blockade in vivo cannot be predicted in these in vitro studies.

Implicating the LFA-1 molecule in mediating FLS activation is of particular interest because the expression of this molecule is not static and changes with T cell differentiation. Naive T cells have a relatively low expression of LFA-1, while expression is increased in memory and effector T cells and, in particular, in those cells that have lost the expression of the CD28 molecule. Previous studies have shown that the frequency of such an effector T cell population is increased in patients with RA and has prognostic predictive value for severity of disease (30, 41). Here, we showed that the different expression levels of LFA-1 have functional consequences in the synovium. CD28 T cells more potently activate the Akt pathway in FLS than do CD28+ T cells.

Our findings suggest that LFA-1–ICAM-2-mediated stimulation plays an important role in the synovial hyperplasia in RA. The selective blockade of this interaction may be a promising target for influencing FLS survival while keeping the vital functions of the LFA-1–ICAM-1 receptor-ligand interaction intact. This intervention would target synovial hyperplasia and, therefore, primarily not influence the synovial inflammation. However, recent experiments in the cadherin knockout mouse have shown that interfering with the synovial hyperplasia has secondary benefits and eventually controls the inflammatory response (11). The ICAM-2–ezrin–Akt pathway emerges as a pharmacological target that is more selective than the broad PI3K inhibition that is efficacious in animal models of RA (42).

We thank Tamela Yeargin for manuscript editing and Chloe Rivera for technical support.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by grants from the National Institutes of Health (RO1 AR 41974, RO1 AI 44142, and RO1 AR 42527).

3

Abbreviations used in this paper: RA, rheumatoid arthritis; FLS, fibroblast-like synoviocyte; PTEN, phosphatase and tensin homolog; MDM-2, mouse double minute-2.

1
Firestein, G. S..
2003
. Evolving concepts of rheumatoid arthritis.
Nature
423
:
356
-361.
2
Liu, H., P. Eksarko, V. Temkin, G. K. Haines, 3rd, H. Perlman, A. E. Koch, B. Thimmapaya, R. M. Pope.
2005
. Mcl-1 is essential for the survival of synovial fibroblasts in rheumatoid arthritis.
J. Immunol.
175
:
8337
-8345.
3
Tak, P. P., N. J. Zvaifler, D. R. Green, G. S. Firestein.
2000
. Rheumatoid arthritis and p53: how oxidative stress might alter the course of inflammatory diseases.
Immunol. Today
21
:
78
-82.
4
Franz, J. K., T. Pap, K. M. Hummel, M. Nawrath, W. K. Aicher, Y. Shigeyama, U. Muller-Ladner, R. E. Gay, S. Gay.
2000
. Expression of sentrin, a novel antiapoptotic molecule, at sites of synovial invasion in rheumatoid arthritis.
Arthritis Rheum.
43
:
599
-607.
5
Pap, T., J. K. Franz, K. M. Hummel, E. Jeisy, R. Gay, S. Gay.
2000
. Activation of synovial fibroblasts in rheumatoid arthritis: lack of expression of the tumour suppressor PTEN at sites of invasive growth and destruction.
Arthritis Res.
2
:
59
-64.
6
Yamasaki, S., N. Yagishita, K. Nishioka, T. Nakajima.
2007
. The roles of synoviolin in crosstalk between endoplasmic reticulum stress-induced apoptosis and p53 pathway.
Cell Cycle
6
:
1319
-1323.
7
Granet, C., W. Maslinski, P. Miossec.
2004
. Increased AP-1 and NF-κB activation and recruitment with the combination of the proinflammatory cytokines IL-1β, tumor necrosis factor α and IL-17 in rheumatoid synoviocytes.
Arthritis Res. Ther.
6
:
R190
-R198.
8
Krause, A., N. Scaletta, J. D. Ji, L. B. Ivashkiv.
2002
. Rheumatoid arthritis synoviocyte survival is dependent on Stat3.
J. Immunol.
169
:
6610
-6616.
9
Lacey, D., A. Sampey, R. Mitchell, R. Bucala, L. Santos, M. Leech, E. Morand.
2003
. Control of fibroblast-like synoviocyte proliferation by macrophage migration inhibitory factor.
Arthritis Rheum.
48
:
103
-109.
10
Sawai, H., Y. W. Park, X. He, J. J. Goronzy, C. M. Weyand.
2007
. Fractalkine mediates T cell-dependent proliferation of synovial fibroblasts in rheumatoid arthritis.
Arthritis Rheum.
56
:
3215
-3225.
11
Lee, D. M., H. P. Kiener, S. K. Agarwal, E. H. Noss, G. F. Watts, O. Chisaka, M. Takeichi, M. B. Brenner.
2007
. Cadherin-11 in synovial lining formation and pathology in arthritis.
Science
315
:
1006
-1010.
12
Valencia, X., J. M. Higgins, H. P. Kiener, D. M. Lee, T. A. Podrebarac, C. C. Dascher, G. F. Watts, E. Mizoguchi, B. Simmons, D. D. Patel, et al
2004
. Cadherin-11 provides specific cellular adhesion between fibroblast-like synoviocytes.
J. Exp. Med.
200
:
1673
-1679.
13
Tran, C. N., M. J. Davis, L. A. Tesmer, J. L. Endres, C. D. Motyl, C. Smuda, E. C. Somers, K. C. Chung, A. G. Urquhart, S. K. Lundy, et al
2007
. Presentation of arthritogenic peptide to antigen-specific T cells by fibroblast-like synoviocytes.
Arthritis Rheum.
56
:
1497
-1506.
14
Yamamura, Y., R. Gupta, Y. Morita, X. He, R. Pai, J. Endres, A. Freiberg, K. Chung, D. A. Fox.
2001
. Effector function of resting T cells: activation of synovial fibroblasts.
J. Immunol.
166
:
2270
-2275.
15
Goronzy, J. J., G. Henel, H. Sawai, K. Singh, E. B. Lee, S. Pryshchep, C. M. Weyand.
2005
. Costimulatory pathways in rheumatoid synovitis and T-cell senescence.
Ann. NY Acad. Sci.
1062
:
182
-194.
16
Sawai, H., Y. W. Park, J. Roberson, T. Imai, J. J. Goronzy, C. M. Weyand.
2005
. T cell costimulation by fractalkine-expressing synoviocytes in rheumatoid arthritis.
Arthritis Rheum.
52
:
1392
-1401.
17
Snyder, M. R., M. Lucas, E. Vivier, C. M. Weyand, J. J. Goronzy.
2003
. Selective activation of the c-Jun NH2-terminal protein kinase signaling pathway by stimulatory KIR in the absence of KARAP/DAP12 in CD4+ T cells.
J. Exp. Med.
197
:
437
-449.
18
Vallejo, A. N., H. Yang, P. A. Klimiuk, C. M. Weyand, J. J. Goronzy.
2003
. Synoviocyte-mediated expansion of inflammatory T cells in rheumatoid synovitis is dependent on CD47-thrombospondin 1 interaction.
J. Immunol.
171
:
1732
-1740.
19
Groh, V., A. Bruhl, H. El-Gabalawy, J. L. Nelson, T. Spies.
2003
. Stimulation of T cell autoreactivity by anomalous expression of NKG2D and its MIC ligands in rheumatoid arthritis.
Proc. Natl. Acad. Sci. USA
100
:
9452
-9457.
20
Inoue, T., D. Hammaker, D. L. Boyle, G. S. Firestein.
2005
. Regulation of p38 MAPK by MAPK kinases 3 and 6 in fibroblast-like synoviocytes.
J. Immunol.
174
:
4301
-4306.
21
Miagkov, A. V., D. V. Kovalenko, C. E. Brown, J. R. Didsbury, J. P. Cogswell, S. A. Stimpson, A. S. Baldwin, S. S. Makarov.
1998
. NF-κB activation provides the potential link between inflammation and hyperplasia in the arthritic joint.
Proc. Natl. Acad. Sci. USA
95
:
13859
-13864.
22
Morel, J., R. Audo, M. Hahne, B. Combe.
2005
. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rheumatoid arthritis synovial fibroblast proliferation through mitogen-activated protein kinases and phosphatidylinositol 3-kinase/Akt.
J. Biol. Chem.
280
:
15709
-15718.
23
Sundarrajan, M., D. L. Boyle, M. Chabaud-Riou, D. Hammaker, G. S. Firestein.
2003
. Expression of the MAPK kinases MKK-4 and MKK-7 in rheumatoid arthritis and their role as key regulators of JNK.
Arthritis Rheum.
48
:
2450
-2460.
24
Zhang, H. G., Y. Wang, J. F. Xie, X. Liang, D. Liu, P. Yang, H. C. Hsu, R. B. Ray, J. D. Mountz.
2001
. Regulation of tumor necrosis factor α-mediated apoptosis of rheumatoid arthritis synovial fibroblasts by the protein kinase Akt.
Arthritis Rheum.
44
:
1555
-1567.
25
Datta, S. R., A. Brunet, M. E. Greenberg.
1999
. Cellular survival: a play in three Akts.
Genes Dev.
13
:
2905
-2927.
26
Namekawa, T., M. R. Snyder, J. H. Yen, B. E. Goehring, P. J. Leibson, C. M. Weyand, J. J. Goronzy.
2000
. Killer cell activating receptors function as costimulatory molecules on CD4+CD28null T cells clonally expanded in rheumatoid arthritis.
J. Immunol.
165
:
1138
-1145.
27
Perez, O. D., S. Kinoshita, Y. Hitoshi, D. G. Payan, T. Kitamura, G. P. Nolan, J. B. Lorens.
2002
. Activation of the PKB/AKT pathway by ICAM-2.
Immunity
16
:
51
-65.
28
Gautreau, A., P. Poullet, D. Louvard, M. Arpin.
1999
. Ezrin, a plasma membrane-microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway.
Proc. Natl. Acad. Sci. USA
96
:
7300
-7305.
29
Schmidt, D., P. B. Martens, C. M. Weyand, J. J. Goronzy.
1996
. The repertoire of CD4+ CD28 T cells in rheumatoid arthritis.
Mol. Med.
2
:
608
-618.
30
Goronzy, J. J., E. L. Matteson, J. W. Fulbright, K. J. Warrington, A. Chang-Miller, G. G. Hunder, T. G. Mason, A. M. Nelson, R. M. Valente, C. S. Crowson, et al
2004
. Prognostic markers of radiographic progression in early rheumatoid arthritis.
Arthritis Rheum.
50
:
43
-54.
31
Pope, R. M..
2002
. Apoptosis as a therapeutic tool in rheumatoid arthritis.
Nat Rev. Immunol.
2
:
527
-535.
32
Downward, J..
1998
. Mechanisms and consequences of activation of protein kinase B/Akt.
Curr. Opin. Cell Biol.
10
:
262
-267.
33
Agarwal, S. K., M. B. Brenner.
2006
. Role of adhesion molecules in synovial inflammation.
Curr. Opin. Rheumatol.
18
:
268
-276.
34
Oppenheimer-Marks, N., R. I. Brezinschek, M. Mohamadzadeh, R. Vita, P. E. Lipsky.
1998
. Interleukin 15 is produced by endothelial cells and increases the transendothelial migration of T cells In vitro and in the SCID mouse-human rheumatoid arthritis model In vivo.
J. Clin. Invest.
101
:
1261
-1272.
35
Nicolls, M. R., R. G. Gill.
2006
. LFA-1 (CD11a) as a therapeutic target.
Am. J. Transplant.
6
:
27
-36.
36
Leonardi, C. L..
2003
. Efalizumab: an overview.
J. Am. Acad. Dermatol.
49
:
S98
-S104.
37
Watts, G. M., F. J. Beurskens, I. Martin-Padura, C. M. Ballantyne, L. B. Klickstein, M. B. Brenner, D. M. Lee.
2005
. Manifestations of inflammatory arthritis are critically dependent on LFA-1.
J. Immunol.
174
:
3668
-3675.
38
Wulfing, C., M. D. Sjaastad, M. M. Davis.
1998
. Visualizing the dynamics of T cell activation: intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels.
Proc. Natl. Acad. Sci. USA
95
:
6302
-6307.
39
Tsai, C., L. A. Diaz, Jr, N. G. Singer, L. L. Li, A. H. Kirsch, R. Mitra, B. J. Nickoloff, L. J. Crofford, D. A. Fox.
1996
. Responsiveness of human T lymphocytes to bacterial superantigens presented by cultured rheumatoid arthritis synoviocytes.
Arthritis Rheum.
39
:
125
-136.
40
Paniagua, R. T., O. Sharpe, P. P. Ho, S. M. Chan, A. Chang, J. P. Higgins, B. H. Tomooka, F. M. Thomas, J. J. Song, S. B. Goodman, et al
2006
. Selective tyrosine kinase inhibition by imatinib mesylate for the treatment of autoimmune arthritis.
J. Clin. Invest.
116
:
2633
-2642.
41
Schmidt, D., J. J. Goronzy, C. M. Weyand.
1996
. CD4+ CD7 CD28 T cells are expanded in rheumatoid arthritis and are characterized by autoreactivity.
J. Clin. Invest.
97
:
2027
-2037.
42
Camps, M., T. Ruckle, H. Ji, V. Ardissone, F. Rintelen, J. Shaw, C. Ferrandi, C. Chabert, C. Gillieron, B. Francon, et al
2005
. Blockade of PI3Kγ suppresses joint inflammation and damage in mouse models of rheumatoid arthritis.
Nat. Med.
11
:
936
-943.