The expression of toll-like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of toll-like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells

Authors


Abstract

Objective

To evaluate the expression of Toll-like receptors (TLRs) 3 and 7 in synovium and to study potential differences in the maturation and cytokine production mediated by TLR-2, TLR-3, TLR-4, and TLR-7/8 by dendritic cells (DCs) from rheumatoid arthritis (RA) patients and DCs from healthy controls.

Methods

Synovial expression of TLR-3 and TLR-7 in RA was studied using immunohistochemistry. Monocyte-derived DCs from RA patients and healthy controls were cultured for 6 days and subsequently stimulated for 48 hours via TLR-mediated pathways (lipoteichoic acid, Pam3Cys, and fibroblast-stimulating lipopeptide 1 for TLR-2, poly[I-C] for TLR-3, lipopolysaccharide and extra domain A for TLR-4, and R848 for TLR-7/8). Phenotypic DC maturation was measured using flow cytometry. The secretion of tumor necrosis factor α (TNFα), interleukin-6 (IL-6), IL-10, and IL-12 was measured using the Bio-Plex system. Cell lines expressing TLR-2 and TLR-4 were used for the detection of TLR-2 and TLR-4 ligands in serum and synovial fluid from RA patients.

Results

TLR-3 and TLR-7 were highly expressed in RA synovium. All TLR ligands elicited phenotypic DC maturation equally between DCs from RA patients and those from healthy controls. TLR-2– and TLR-4–mediated stimulation of DCs from RA patients resulted in markedly higher production of inflammatory mediators (TNFα and IL-6) compared with DCs from healthy controls. In contrast, upon stimulation of TLR-3 and TLR-7/8, the level of cytokine production was equal between DCs from RA patients and those from healthy controls. Remarkably, both TLR-3 and TLR-7/8 stimulation resulted in a skewed balance toward IL-12. Intriguingly, the combined stimulation of TLR-4 and TLR-3–7/8 resulted in a marked synergy with respect to the production of inflammatory mediators. As a proof of concept, TLR-4 ligands were increased in the serum and synovial fluid of RA patients.

Conclusion

TLRs are involved in the regulation of DC activation and cytokine production. We hypothesize that various TLR ligands in the joint trigger multiple TLRs simultaneously, favoring the breakthrough of tolerance in RA.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that leads to the destruction of synovial joints. Although the exact mechanisms that are responsible remain obscure, it is generally accepted that besides genetic factors, environmental triggers contribute to the disease pathogenesis (1, 2). An infectious etiology of RA has been a longstanding hypothesis, which has been examined with greater sophisticated scientific rigor recently. Currently, evidence points toward a potential role of Epstein-Barr virus (3–5), parvovirus B19 (6, 7), and cytomegalovirus (8, 9), all of which are found in RA synovium. The potential role of viruses in the pathogenesis of RA was further substantiated by the findings that human parvovirus B19–transgenic mice are highly susceptible to polyarthritis (10), the murine herpesvirus induces relapsing experimental autoimmune arthritis (11), and that double-stranded (viral) RNA (dsRNA) has overt arthritogenic properties (12). Several mechanisms have been attributed to the actual link between the presence of viral material and the observed immunohistopathologic changes during inflammatory synovitis; however, the precise mode of action has not been elucidated thus far.

Synovial inflammation is characterized by a massive influx of numerous inflammatory cells, including T cells, B cells, macrophages, and dendritic cells (DCs). Potentially, all of these contribute to the progressive breakdown of cartilage and destruction of the underlying bone (13–15). DCs are professional antigen-presenting cells that are highly adept at stimulating T cells and are therefore intricately involved in the balance between immunity and tolerance (16). DCs can produce large quantities of proinflammatory cytokines, chemokines, and metalloproteinases that potentially contribute to the detrimental processes during synovial inflammation. In fact, several observations indicate the potential role of DCs in RA disease pathogenesis. First, DCs are abundantly present at strategic locations in RA synovium (17–19). Second, models of experimental arthritis have shown that presentation of collagen-derived peptides by mature DCs is sufficient for the induction of arthritis (20), and finally, genetically modulated DCs were able to abrogate experimental arthritis (21).

The function of DCs is time and space dependent. In the immature state, DCs reside in the periphery and function as sentinels of the immune system, and specialize in antigen uptake and processing. To execute antigen uptake with utmost efficiency, DCs are equipped with several antigen uptake receptors, including Toll-like receptors (TLRs).

TLRs are involved in the detection of environmental signals and the regulation of DC function and are therefore the subject of investigations in autoimmunity. The signals that elicit TLRs are often called “danger signals” and include both exogenous ligands, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), flagellin, CpG motifs, and dsRNA, often referred to as pathogen-associated molecular patterns (22, 23), and endogenous ligands, such as heat-shock proteins (24), fibronectin (25), hyaluronic acid (26), and messenger RNA (mRNA) (27). Endogenous ligands are released upon normal cell turnover and stressful events and are likely to be abundant in the synovial compartment. The pivotal role of endogenous ligands was substantiated by the finding that such signals lead to primary immune responses under conditions of cell stress and precipitate autoimmunity in the presence of self antigens (28–31). Therefore, endogenous ligands are considered to be natural initiators of autoimmunity.

Evidence of a role of TLRs in RA pathogenesis originates from the findings that inducible Hsp70, generally accepted as a TLR-4 ligand, was increased in RA synovial fluid and on DCs isolated from RA synovial fluid (32). Expression of TLR-2 and TLR-4 is increased and regulated by proinflammatory cytokines that are present in the synovial compartment (33, 34) and by the identification of the TLR-4 (Asp299Gly) functional variant as a marker for RA disease susceptibility (35). In addition, the link between DNA and TLRs in RA, as recently demonstrated by Leadbetter and colleagues, further substantiated the potential role of TLRs in arthritis (36).

Taken together, it is tempting to speculate that TLRs play a role in the onset and/or severity of RA. This prompted us to investigate the expression of TLR pathways other than TLR-2 and TLR-4 in RA synovium and to study potential differences in TLR-mediated DC activation between RA patients and healthy controls using both exogenous and endogenous ligands. Furthermore, we tested the hypothesis that simultaneous stimulation of different TLRs resulted in a synergistic effect on cytokine production by DCs.

Here we demonstrate that TLR-3 and TLR-7 are increased in the synovial tissue of RA patients. Furthermore, we show that the cytokine repertoire produced by DCs depends heavily upon the TLR pathways triggered. Simultaneous stimulation of TLR-4, in combination with TLR-3–7/8 pathways, leads to a synergistic effect with regard to cytokine production, suggesting that the presence of different TLR agonists in the joint compartment, including viral RNA, favors a proinflammatory environment that leads to a disturbed balance between tolerance and immunity.

MATERIALS AND METHODS

Study population

A total of 22 patients with RA and 21 healthy volunteers were enrolled in this study. Patients attended Radboud University Nijmegen Medical Centre and fulfilled the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) criteria for RA (37). The treatment regimens of all patients were recorded before blood sampling, and patients who used high-dose prednisolone or anticytokine therapies (anti–tumor necrosis factor α [anti-TNFα] and/or interleukin-1 receptor antagonist) were excluded. All patients provided informed consent. For immunohistochemical analysis, percutaneous biopsies of the knee joint were performed with a Parker-Pearson needle following administration of local anesthesia. On average, 30 samples were obtained during each procedure. The synovial tissue from healthy individuals and from patients with osteoarthritis (OA) was isolated during arthroscopic procedures performed by the orthopedic surgeons. The Medical Ethics Committee of Radboud University Nijmegen Medical Centre approved the study protocol.

Antibodies used for TLR immunohistochemical analysis and cytokine detection

Antibodies against human TLR-2 (H-175), TLR-4 (H-80), TLR-3 (T-17), and TLR-7 (V-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Biotinylated swine anti-rabbit Ig affinity-isolated F(ab′)2 was obtained from Dako (Glostrup, Denmark). Biotinylated mouse anti-goat IgG was purchased from Jackson ImmunoResearch (West Grove, PA). Vectastain ABC reagent (Elite kit) was obtained from Vector (Burlingame, CA)–and streptavidin peroxidase was obtained from Dako. Diaminobenzidine (DAB) was obtained from Sigma (St. Louis, MO). Bio-Plex kits using Luminex bead array technology for the determination of interleukin-6 (IL-6), TNFα, IL-10, and IL-12 were purchased from Bio-Rad (Hercules, CA).

Immunohistochemistry of synovial tissues

Tissue samples were immediately fixed with 4% formaldehyde and embedded in paraffin. For TLR-2 and TLR-4 staining, after dewaxing and dehydration, sections were blocked with normal swine serum followed by 60 minutes of incubation with the antibodies against TLR-2 and TLR-4 at concentrations of 10 μg/ml and 17 μg/ml, respectively. After this, endogenous peroxidase was blocked with 3% H2O2 in methanol for 15 minutes, and the secondary antibody, biotinylated swine anti-rabbit IgG, was incubated for 30 minutes. Slides were stained with streptavidin–peroxidase, developed with DAB, and counterstained with hematoxylin for 30 seconds. For TLR-3 and TLR-7 staining, sections were incubated with antibodies against human TLR-3 and human TLR-7 for 60 minutes. After this, endogenous peroxidase was blocked with 3% H2O2 in methanol for 15 minutes, and subsequently, the secondary antibody, biotinylated mouse anti-goat IgG, was incubated for 30 minutes. Vectastain ABC reagent was incubated for 30 minutes, developed with DAB, and counterstained with hematoxylin for 30 seconds. Staining of TLR-2, TLR-4, TLR-3, and TLR-7 at 200× magnification was scored semiquantitatively on a 5-point scale. A score of 0 represented no or minimal staining, 1 represented 10–20% positive cells, 2 represented 30–40% positive cells, 3 represented 50–60% positive cells, and a score of 4 represented staining of >60% of the cells.

Culture of monocyte-derived DCs

Monocyte-derived DCs were cultured using standardized protocols, as previously described (18). Briefly, peripheral blood mononuclear cells were isolated from heparinized venous blood by density gradient centrifugation over Ficoll-Paque (Amersham Biosciences, Roosendaal, The Netherlands). Low-density cells were collected and washed with citrated phosphate buffered saline and 5% fetal calf serum (FCS), after which the cells were allowed to adhere for 1 hour at 37°C in RPMI 1640 (Dutch modification) (Invitrogen, Carlsbad, CA) supplemented with 2% human serum (PAA Laboratories, Linz, Austria) in 6-well culture plates or 25-cm2 cell culture flasks (Corning, Corning, NY). Adherent monocytes were cultured in RPMI 1640 (Dutch modification) supplemented with 10% FCS and antibiotic/antimycotic (Life Technologies, Gaithersburg, MD) in the presence of IL-4 (500 units/ml; Schering-Plough, Amstelveen, The Netherlands) and granulocyte–macrophage colony-stimulating factor (800 units/ml; Schering-Plough) for 6 days. Fresh culture medium with the same supplements was added on day 3, and then immature DCs were harvested on day 6. To generate mature DCs, immature DCs were resuspended in fresh cytokine-containing culture medium and transferred to new culture plates at a concentration of 0.5 × 106/ml. To induce DC maturation, cells were stimulated as described below.

TLR stimulation of monocyte-derived DCs

Triggering via TLR-mediated pathways was achieved with various TLR-specific ligands. To trigger TLR-2, 10 μg/ml of LTA (Sigma) was used. To correct for potential LPS contamination in the LTA, Pam3Cys and fibroblast-stimulating lipopeptide 1 (FSL-1), which are known to be highly specific for the TLR-1/TLR-2 or TLR-2/TLR-6 pathways, were used. For stimulation of TLR-4, we used 2 μg/ml of Escherichia coli LPS (Sigma). As a known endogenous TLR-4 ligand, we used 1 μM recombinant extra domain A (EDA) proteins. As a control for this, recombinant extra domain B and III11 domain proteins (all kindly provided by Dr. J. Strauss, University of Pennsylvania, Philadelphia, PA) were used, which are also fibronectin fragments but lack TLR-4 binding capacity.

To trigger TLR-3 and TLR-7/8, we used poly(I-C) and R848, both obtained from InvivoGen (San Diego, CA). All TLR ligands that were used were checked for potential LPS contamination using a TLR-4 antagonist (E5564), which was kindly provided by Eisai Research Institute, Andover, MA (38). After culture for 48 hours at 37°C in the presence of 5% CO2, the supernatants were collected.

Simultaneous stimulation of different TLR pathways

To test whether simultaneous stimulation of different TLR pathways resulted in synergistic cytokine production, immature DCs were generated as described above. On day 6, cells were stimulated with combinations of TLR-2/TLR-3, TLR-2/TLR-4, TLR-2/TLR-7/8, TLR-3/TLR-4, TLR-3/TLR-7/8, and TLR-4/TLR-7/8 ligands and compared with cells stimulated with each of these substances alone. Furthermore, the combinations of TLR-2/TLR-3/TLR-7 and TLR-4/TLR-3/TLR-7/8 were used to test a potential additional effect of 3 TLR pathways. After culture for 24 hours at 37°C in the presence of 5% CO2, the supernatants were collected.

Phenotype analysis of monocyte-derived DCs

Flow cytometry was used to determine the expression of cell surface markers on both immature and mature DCs. First, DCs (1 × 105) were incubated with monoclonal antibodies against human CD14 (Dako), CD80 (Becton Dickinson, Mountain View, CA), CD83 (Beckman Coulter, Mijdrecht, The Netherlands), CD86 (PharMingen, San Diego, CA), type I major histocompatibility complex (MHCI) (clone W6/32), and MHCII HLA–DR/DP (clone Q1514) for 30 minutes at 4°C. Cells were then washed and incubated with fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG (Zymed, South San Francisco, CA) for 30 minutes at 4°C in complete darkness. Subsequently, cells were washed and analyzed using a fluorescence-activated cell sorter (FACSCalibur; Becton Dickinson) to determine the proportion of positive cells relative to cells stained with relevant IgG isotypes. For TLR-2 and TLR-4, expression cells were incubated with monoclonal antibodies against human TLR-2 (H-175) and TLR-4 (H-80), both obtained from Santa Cruz Biotechnology, and subsequently incubated with goat anti-rabbit IgG and goat anti-mouse IgG, respectively (both obtained from Zymed).

Measurement of cytokines in culture supernatant

TNFα, IL-6, IL-10, and IL-12 levels were measured in the supernatant of the DC cultures, using commercially available kits according to the manufacturer's instructions (Bio-Rad) (39). Cytokine levels were measured and analyzed using the Bio-Plex system (Bio-Rad). Data were analyzed using Bio-Plex Manager software (Bio-Rad).

Stimulation of TLR-2 and TLR-4 transgenic Chinese hamster ovary (CHO) cell lines

The CHO fibroblast CD14 reporter line (clone 3E10) is a stably transfected CD14-positive CHO cell line that expresses inducible membrane CD25 (Tac antigen) under transcriptional control of the human E-selectin promoter (40). The promoter fragment contains an essential NF-κB binding site, which, upon stimulation with LPS, results in a 3–10-fold increase in CD25 surface expression. CHO/CD14/human TLR-2 (HuTLR-2) reporter cell lines were constructed by stable cotransfection with complementary DNA for the human TLR-2 and pcDNA3 (Invitrogen). The CHO/CD14/HuTLR-4 reporter cell lines were derived in the same manner. (Both reporter cell lines were kindly provided by Dr. R. Ingalls and Dr. D. Golenbock from the Boston Medical Center, Boston, MA.) All of the experiments with the CHO cell lines were performed at least twice.

CHO/CD14 cells were cultured in Ham's F-12 medium with L-glutamine (BioWhittaker, Walkersville, MD), gentamicin (Centrafarm, Etten-Leur, The Netherlands), 2.5% FCS, and 400 units/ml hygromycin B (Calbiochem, Amsterdam, The Netherlands). G418 sulfate (0.5 mg/ml) and puromycin (50 μg/ml) (both obtained from Sigma) were added to the CHO/CD14/TLR-2 and CHO/CD14/TLR-4 cells, respectively. Cells at a concentration of 1 × 105/ml were stimulated with 10% serum or synovial fluid from RA patients, 10% serum from patients with systemic sclerosis (SSc) or systemic lupus erythematosus (SLE), or 10% normal human serum (PAA Laboratories). To check for potential endotoxin contamination of the synovial fluids used, some of the experiments were performed in the presence or absence of polymyxin B, a natural LPS antagonist. The synovial fluids were also measured for gram-negative bacterial endotoxins using the Limulus amebocyte lysate assay, performed according to the manufacturer's instructions (Pyrogent Plus multitest kit; BioWhittaker).

Analysis of CD25 surface expression by CHO cells

Flow cytometry was performed to determine the surface expression of CD25 on the transgenic cell lines. First, 1 × 105 cells were incubated with a monoclonal antibody against human CD25 (Dako) for 30 minutes at 4°C. Cells were then washed and incubated with FITC-conjugated goat anti-mouse IgG (Zymed) for 30 minutes at 4°C in complete darkness. Cells were washed and analyzed by FACSCalibur to determine the proportion of positive cells relative to cells stained with a relevant IgG isotype.

Statistical analysis

Differences in cytokine production between cells from healthy volunteers and those from RA patients were analyzed using the Mann-Whitney U test. P values were 2-sided, and P values less than 0.05 were considered significant.

RESULTS

Increased expression of TLR-3 and TLR-7 in RA synovium. To study the potential role of viral RNA in synovial inflammation, we tested the expression of TLR-3 and TLR-7 in RA synovium. To this end, we stained synovial tissue sections from 5 RA patients with antibodies against TLR-3 and TLR-7 and compared them with synovial tissue sections from 5 OA patients and 5 healthy controls (Figure 1). Intriguingly, both TLR-3 and TLR-7 were abundantly expressed in RA synovium. Whereas TLR-3 was predominantly located in the lining, sublining, and perivascular regions, TLR-7 was scattered throughout the lining and expressed in the lining of RA synovium (mean ± SEM TLR-3 score 0.7 ± 0.4, TLR-7 score 1.8 ± 0.7) as compared with synovium from OA patients (TLR-3 score 0.2 ± 0.2, TLR-7 score 0.5 ± 0.4) and healthy controls (TLR-3 score 0.2 ± 0.2, TLR-7 score 0.3 ± 0.2). Remarkably, TLR-3 and TLR-7 were hardly detectable in the sublining of synovial tissue from OA patients and healthy controls; in contrast, both TLR-3 and TLR-7 were abundant in the sublining of RA synovium (TLR-3 score 0.8 ± 0.4, TLR-7 score 1.8 ± 0.7).

Figure 1.

Immunohistochemical detection of Toll-like receptor 3 (TLR-3) and TLR-7 in synovial biopsy samples from patients with rheumatoid arthritis (RA) and healthy controls (HC). Top panels show staining with an irrelevant isotype control antibody. All tissues were counterstained with hematoxylin (original magnification × 400).

Maturation of DCs from RA patients and healthy controls upon stimulation with TLR-2, TLR-3, TLR-4, and TLR-7/8 agonists. Since TLR-2, TLR-3, TLR-4, and TLR-7 are highly expressed in RA synovium and important in DC maturation, we used monocyte-derived DCs to test the capacity of various TLR ligands to induce DC maturation (33). In addition, we studied potential differences between DCs from RA patients and those from healthy controls. Consistent with the literature, immature DCs were characterized by a low expression of CD14 (data not shown), intermediate expression of CD86 and MHCII, and low expression of the mature DC marker CD83 (Figure 2, top panel). TLR-mediated DC maturation was reflected by the up-regulation of CD83, CD86, and MHCII (Figure 2) combined with the down-regulation of CD14. Stimulation of the TLR-2 (LTA), TLR-3 (poly[I-C]), TLR-4 (LPS and EDA), and TLR-7/8 (R848) pathways resulted in full DC maturation, which was found to be equally efficient for DCs from RA patients (n = 10) and healthy controls (n = 8) (Figure 2). No effect was seen upon stimulation with EDB and III11, which were used as negative controls for EDA, which lacks the capacity to elicit TLR-4–mediated triggering (data not shown) (25).

Figure 2.

Expression of cell surface markers CD83, CD86, and type II major histocompatibility complex (MHCII) on dendritic cells (DCs) before and after Toll-like receptor (TLR)–mediated maturation with various TLR ligands. The indicated markers or isotype controls (gray peak) of DCs within the life gate were determined using fluorescence-activated cell sorting analysis. Each graph displays data from a representative rheumatoid arthritis (RA) patient (dotted line) and a healthy control (solid line). Top panel shows the expression of CD83, CD86, and type II MHC by immature DCs (iDCs) from an RA patient and a healthy control. Other panels show the expression of these markers upon stimulation with the TLR ligands indicated on the y-axis. LTA = lipoteichoic acid; LPS = lipopolysaccharide; EDA = extra domain A.

Different cytokine patterns upon DC stimulation via TLR-2, TLR-3, TLR-4, and TLR-7/8 pathways. The type of immune response is largely dependent on the production of cytokines. Therefore, we next investigated the production of TNFα, IL-6, IL-10, and IL-12 by monocyte-derived DCs from RA patients (n = 10) and healthy controls (n = 8) upon stimulation via TLR-2, TLR-3, TLR-4, and TLR-7/8 pathways. The cytokine levels, as shown in Figure 3, were corrected for cytokine production by unstimulated cells. For all stimuli used, cytokine production was enhanced compared with unstimulated DCs. Intriguingly, the production of TNFα by DCs from RA patients upon stimulation with the TLR-2 ligand LTA (P < 0.01) and the TLR-4 ligands LPS (P = 0.03) and EDA (P = 0.02) was significantly higher than that of DCs from healthy controls (Figure 3a). Similarly, the production of IL-6 was significantly increased by DCs from RA patients upon stimulation with LTA (P = 0.01), LPS (P = 0.03), and EDA (P = 0.03) (Figure 3b). In contrast to TLR-2/4 triggering, the level of cytokine production upon TLR-3 and TLR-7/8 stimulation was equal between DCs from RA patients and those from healthy controls (Figures 3a and b).

Figure 3.

Production of pro- and antiinflammatory cytokines by DCs after incubation for 48 hours with TLR-2–, TLR-3–, TLR-4–, and TLR-7/8–specific ligands. Production of a, tumor necrosis factor α (TNFα), b, interleukin-6 (IL-6), and c, IL-10 and IL-12 by DCs from healthy controls (n = 8) and from RA patients (n = 10) upon stimulation with LTA, poly(I-C), LPS, EDA, and R848 is shown. Values are the mean and SEM (corrected for cytokine production by unstimulated cells). The sensitivity of the cytokine assay was <5 pg/ml for each measured cytokine. ∗ P < 0.05. See Figure 2 for other definitions.

The balance between IL-10 and IL-12 largely determined the type of immune response. Upon stimulation of the TLR-2 and TLR-4 pathways, the IL-10 levels tended to be higher than the IL-12 levels, which occurred exclusively in DCs from RA patients. In contrast, TLR-3 and TLR-7/8 stimulation resulted in a skewed balance toward IL-12, which occurred in DCs from RA patients as well as healthy controls. Taken together, these data suggested that TLR-3– and TLR-7/8–mediated DC stimulation in RA results in a skewed balance toward IL-12 and strongly favors Th1-mediated responses.

Synergistic increase in cytokine production upon simultaneous stimulation of TLR-4 and TLR-3–7/8 pathways. To test the hypothesis that simultaneous stimulation of different TLR pathways results in an augmented DC activation, we stimulated DCs with combinations of several TLR ligands. To this end, we cocultured DCs from 5 healthy controls and 5 RA patients with combinations of TLR-3/4, TLR-4–7/8, TLR-3–7/8, and TLR-3–4–7/8 for 24 hours. As shown in Figure 4 and Table 1, the production of TNFα, IL-10, and IL-12 dramatically increased in a synergistic manner when 2 or more TLR pathways were stimulated simultaneously. Similar patterns of cytokine production were observed for DCs from RA patients and healthy controls (data not shown). Intriguingly, the highest absolute levels of TNFα (mean 10,152 pg/ml), IL-12 (3,089 pg/ml), and IL-10 (686 pg/ml), and the highest percentage increase in synergistic effect on TNFα (median 128%), IL-12 (478%), and IL-10 (428%) were reached upon stimulation using the combination of TLR-4 and TLR-7/8 agonists. Moreover, the addition of a third TLR stimulus resulted in a further increase in the absolute levels of TNFα, IL-10, and IL-12 compared with the combinations of 2 TLRs. However, the percentage increase in these cytokines was similar, or even lower, when DCs were stimulated with a combination of 3 TLR ligands as compared with 2 TLR ligands. Remarkably, the addition of TLR-2 ligands to any other TLR pathway did not result in a clear synergistic effect (data not shown). Taken together, these data underscore the potential role of simultaneous TLR stimulation in DC activation, with special emphasis on synergy between the TLR-4 and TLR-3–7/8 pathways.

Figure 4.

Production of A, tumor necrosis factor α (TNFα), B, interleukin-10 (IL-10), and C, IL-12 by DCs upon triggering of single and multiple TLR pathways. DCs were stimulated with TLR-3, TLR-4, and TLR-7/8 ligands alone (shaded bars; each individual shaded bar represents 1 TLR ligand as indicated) and with a combination of different TLR pathways simultaneously (solid bars). These values were compared with the cytokine production that would be estimated to occur if the combination of TLR ligands had an additive effect over the effect with one of the TLR pathways alone (open bars). TNFα, IL-10, and IL-12 were measured in the supernatants after 24 hours of coincubation. Each graph displays data from a representative RA patient. The sensitivity of the cytokine assay was <5 pg/ml for each measured cytokine. See Figure 2 for other definitions.

Table 1. Median level of synergy of TNFα, IL-12, and IL-10 production by dendritic cells from rheumatoid arthritis patients upon stimulation with a combination of TLR pathways*
CombinationTNFαIL-12IL-10
  • *

    TNFα = tumor necrosis factor α; IL-12 = interleukin-12; TLR = Toll-like receptor. Synergy is expressed as the median percentage increase compared with the value that would be expected when the combination of TLR pathways was considered to have an additive effect.

TLR-3/4232095
TLR-4–7/8128478428
TLR-3–7/8116116299
TLR-3–4–7/837211210

Presence of TLR-4 ligands in RA serum and synovial fluid. Since we demonstrated that the combination of TLR-4 ligands with TLR-3 and/or TLR-7/8 resulted in a synergistically elevated production of cytokines, we tested whether TLR-4 agonists are present in RA serum and synovial fluid. To this end, we used CHO reporter cell lines that were stably transfected with CD14 alone (CHO/CD14), with CD14 and human TLR-2 (CHO/CD14/HuTLR-2), and with CD14 and human TLR-4 (CHO/CD14/HuTLR-4). These cells were incubated overnight with 10% serum and/or synovial fluid from RA patients.

Whereas incubation of the TLR-4–expressing CHO cells with normal human serum did not result in an up-regulation of CD25, incubation of the cells with serum from RA patients with low levels of disease activity (Disease Activity Score <3.0 [41]) (n = 5) resulted in increased expression of CD25 surface expression (18%) (Figure 5). Intriguingly, the serum from RA patients with active disease (n = 7) even further increased the expression of CD25 to 35%, which was similar to that found after incubation with synovial fluid (36%) from the same patients. A comparison with serum samples from patients with SSc (n = 5) and SLE (n = 5) demonstrated that, although at a lower level than in RA, serum from SSc patients (CD25 expression level 11%) also contained TLR-4 ligands, whereas SLE serum (1%) did not. In contrast, incubation of the CHO/CD14 and CHO/CD14/HuTLR-2 cell lines with the same set of sera did not result in increased CD25 expression, suggesting that the stimulation was TLR-4 specific. The addition of polymyxin B in these experiments had no effect on CD25 expression, and results of the Limulus amebocyte lysate assay were negative for all synovial fluid samples measured. Stimulation with LPS, included as a positive control, resulted in a potent activation response, as seen by the increased expression of CD25 (66%).

Figure 5.

Expression of CD25 on Chinese hamster ovary (CHO) CD14/human TLR-4 (HuTLR-4) cell lines. CHO CD14/HuTLR-4 cells were activated upon coculturing with serum from patients with inactive RA (n = 5) and with serum and synovial fluid from patients with active RA (n = 7). In contrast, serum from patients with systemic sclerosis (SSc) (n = 5) and systemic lupus erythematosus (SLE) (n = 5) failed to induce CHO CD14/HuTLR-4 cell activation. As expected, LPS, a potent inducer of TLR-4, was highly capable of activating TLR-4–expressing cells. Values are the mean and SEM. See Figure 2 for other definitions.

DISCUSSION

In this study, we present novel data regarding the increased expression of TLR-3 and TLR-7 in synovium from RA patients compared with that of healthy donors. Since TLR-3 and TLR-7 are receptors that recognize single-stranded and double-stranded viral mRNA, these findings shed new light on the possible involvement of viruses in RA pathogenesis. Currently, a large body of evidence points toward the involvement of DNA viruses in the pathogenesis of RA. In the synovial tissue of RA patients, parvovirus B19, Epstein-Barr virus, and cytomegalovirus were found to be frequently present (3, 4, 7, 42–45). Although not fully investigated, it is also very likely that RNA viruses occur in the synovial compartment. In light of this, it is interesting that local TLR-3 and TLR-7 expression is increased in the synovium of RA patients. Although the mechanisms behind this increased expression of “viral recognition molecules” have not been elucidated thus far, it is tempting to speculate that the recognition of viral RNA and/or mRNA from the host itself leads to the activation of antigen-presenting cells (APCs) that are abundant in the RA synovial compartment, thereby amplifying the inflammatory reaction.

Moreover, the finding that DC stimulation mediated by TLR-3 and TLR-7/8 results in a markedly skewed balance toward IL-12 is interesting, since we recently demonstrated that IL-12 is indirectly involved in the up-regulation of TLR-2 and TLR-4, both of which are highly expressed in RA synovium (33). These data suggest that TLR-3/7 signaling results in an up-regulation of TLR-2/4, subsequently leading to a higher sensitivity for endogenous ligands, favoring a vicious inflammatory circle. In addition, we produced novel data showing that the stimulation of multiple TLR pathways simultaneously results in clear synergistic effects with respect to the production of inflammatory mediators. The combination of TLR-4 and TLR-7/8 pathways especially resulted in synergistic effects, further substantiating the idea that the combination of endogenous ligands and viral mRNA might contribute to the proinflammatory environment and propel a chronic inflammatory response in the synovial compartment.

Interestingly, we found that DCs from RA patients secrete higher levels of TNFα and IL-6 compared with DCs from healthy controls upon triggering with specific TLR-2 (LTA) and TLR-4 (LPS and EDA) ligands. Since much controversy exists about the specificity of LTA, we also tested other TLR-1/TLR-2 and TLR-2/TLR-6 heterodimer-specific ligands (FSL-1 and Pam3Cys) (46, 47). Although the absolute levels of proinflammatory cytokines by stimulation with FSL-1 and Pam3Cys were lower compared with LTA, clear differences between RA patients and healthy controls remained, indicating that this phenomenon is specific for TLR-2 and TLR-4 pathways. The fact that TLR-4 ligands were increased in the serum and synovial fluid from RA patients further substantiates a potential role of TLR-4 in the onset and/or perpetuation of this condition. Every reaction that is started has to be terminated; the same is true for the immune response. Therefore, the innate immune system is subjected to sophisticated regulation, and there is increasing evidence that an imbalance between pro- and antiinflammatory pathways might give rise to autoimmunity. Some intracellular adapters (e.g., TIRAP) (48) and inhibitory molecules (e.g., SIGIRR) (49) are involved in TLR-2 and TLR-4 signaling. However, it is likely that many more, yet unidentified, molecules are implicated. Seen in this light, the increased TLR-2 and TLR-4 responses found in RA might be caused by an alteration in one of these molecules.

DCs are professional APCs that are unrivaled in their capacity for activating naive and effector T cells. DCs initiate immunity and uphold tolerance, 2 critical processes necessary to successfully combat pathogenic invaders and prevent autoimmunity (16). Autoimmunity is attributed to the breakdown of tolerance when lymphocyte clones fail to discriminate between self and nonself, resulting in destructive responses directed to multiple organs. The mechanisms by which autoimmunity is triggered are not fully understood, but DCs and TLRs are likely to play a role in this process. Recently, Ichikawa and colleagues (30) and Waldner and associates (31) provided a conceptual framework demonstrating that TLRs are key molecules for the breakthrough of tolerance. Our data show that triggering different TLRs at the same time resulted in marked synergy with respect to the production of proinflammatory mediators, indicating that simultaneous stimulation of different TLR pathways facilitates an environment in which the breakthrough of tolerance is likely to occur.

Taken together, these data suggest that TLRs are involved in the regulation of inflammatory responses in the synovial compartment. TLRs recognize many exogenous and host-derived molecules; the latter are present in RA serum and synovial fluid. In this light, it could be envisaged that, upon the occurrence of a certain event, a variety of TLR ligands that lead to the activation of DCs are released in the synovial compartment. Perhaps a trivial joint trauma or viral infection leads to such an event. As a result, cell activation and the subsequent release of proinflammatory mediators result in cartilage breakdown and the subsequent release of more endogenous TLR ligands. At the same time, the abundance of proinflammatory cytokines leads to an increased surface expression of TLRs and an increased capability to sense and react to TLR ligands. Normally, every proinflammatory response should lead to the activation of a counteractive mechanism to restore the immunologic balance. However, in RA, such a mechanism seems to be insufficient, resulting in a vicious circle of cell influx and activation, ending up in total joint destruction.

In conclusion, this is the first study to show that TLR-3 and TLR-7 expression is increased in RA synovium and that the simultaneous triggering of multiple TLR pathways results in the augmentation of the proinflammatory response, favoring a proinflammatory environment and the breakthrough of tolerance in RA.

Acknowledgements

We are indebted to Drs. M. C. de Waal Malefijt and B. W. Schreurs of the Department of Orthopedic Surgery for their contribution in obtaining synovial tissue.

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