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Abstract

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

Objective

Macrophages are the major source of inflammation mediators that are important in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to analyze macrophages obtained from the joints of RA patients in order to characterize the expression of Toll-like receptor 2 (TLR-2) and TLR-4 and the responses to TLR ligation.

Methods

Cells were isolated from the synovial fluid (SF) of RA patients or patients with other forms of inflammatory arthritis. Cell surface TLR-2 and TLR-4 expression and intracellular tumor necrosis factor α (TNFα) and interleukin-8 (IL-8) expression by CD14+ macrophages were determined by flow cytometry. Peptidoglycan (PG) and lipopolysaccharide (LPS) were used as ligands for TLR-2 and TLR-4, respectively.

Results

The expression of TLR-2 and TLR-4 was increased on CD14+ macrophages from the joints of RA patients compared with that on control in vitro–differentiated macrophages or control peripheral blood monocytes. Neither TLR-2 expression nor TLR-4 expression differed between RA and other forms of inflammatory arthritis. However, PG- and LPS-induced TNFα expression and IL-8 expression were greater with RA SF macrophages than with those obtained from the joints of patients with other forms of inflammatory arthritis or with control macrophages. PG-induced TNFα expression and IL-8 expression were highly correlated with TLR-2 expression in normal macrophages, but not with that in macrophages obtained from joints of RA patients or patients with other forms of inflammatory arthritis.

Conclusion

TLR-2 and TLR-4 ligation resulted in increased activation of RA synovial macrophages compared with those from patients with other forms of inflammatory arthritis or compared with control macrophages. Factors other than the level of TLR-2 and TLR-4 expression contributed to the increased activation of RA SF macrophages. These observations support the notion of a potential role for activation through TLR-2 and TLR-4 in the inflammation and joint destruction of RA.

The innate and adaptive immune systems are important in the pathogenesis of rheumatoid arthritis (RA). The presence of activated T cells and B cells, the rearrangement of their respective receptors, and their specificity for local antigens within the joint all support the role of the antigen-driven adaptive responses in the pathogenesis of RA (1). However, an increasing body of data supports the role of the innate immune system in RA and in experimental models of RA (2–4). Toll-like receptors (TLRs) may be critical for the generation of both innate and adaptive immunity. In response to pathogens, activation of the innate immune system through TLRs on macrophages and dendritic cells (DCs) results in the production of proinflammatory cytokines, including tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β). This activation promotes the development of the adaptive immune response involving T and B lymphocytes, which then contributes to clearance of the pathogenic stimulus and resolution of the inflammatory response. Despite the development of an adaptive immune response, expression of the early responders TNFα and IL-1β persists and may directly contribute to the pathogenesis of RA (2, 4–8).

At least 10 mammalian TLR family members have been identified. TLRs are pattern recognition receptors that may be found on a variety of cells and tissues (9), but they are particularly important on monocytes, macrophages, and DCs (10). Gene deletion studies have demonstrated that TLR-4 is principally responsible for lipopolysaccharide (LPS)–induced activation, while TLR-2–deficient mice were unresponsive to Staphylococcus aureus peptidoglycan (PG) (11, 12). TLR-2 and TLR-4 signaling lead to the activation of NF-κB (2) and the MAP kinases JNK (7) and p38 (13) through myeloid differentiation factor 88–dependent and –independent pathways (14, 15).

Recent studies have demonstrated the increased expression of TLR-2 and TLR-4 on peripheral blood (PB) monocytes from patients with RA (16, 17). In a study utilizing immunohistochemistry, both TLR-2 and TLR-4 were found to be expressed in the synovial tissue of patients with RA (16–18). Furthermore, in a study using reverse transcriptase–polymerase chain reaction (RT-PCR) and in situ hybridization, TLR-2 was found to be expressed in the rheumatoid joint and to be up-regulated in RA synovial fibroblasts by TNFα and IL-1β (8, 19).

While the expression of TLR-2 and TLR-4 on synovial tissue macrophages has been documented (16–18), quantitative studies have not been performed to examine the level of expression of TLR-2 and TLR-4 on synovial macrophages (the principal source of proinflammatory cytokines in RA) and to define the response to TLR ligands. These studies are important, because ligands for TLR-2 and TLR-4 have been identified in the rheumatoid joint. Bacterial PG was identified in RA synovial tissue by a monoclonal antibody, particularly in macrophages and antigen-presenting cells (20, 21). Additionally, endogenous mammalian TLR agonists, including fibrinogen, extra domain A (ED-A) of fibronectin, Hsp60 and Hsp70, low molecular weight fragments of hyaluronic acid, and high mobility group box chromosomal protein 1 (HMGB-1), a highly conserved nuclear protein that stabilizes nucleosome formation, are expressed in the RA joint, and each has been shown to activate NF-κB through TLR-4 and/or TLR-2 (22–29).

Macrophages, an important component of the innate immune system, are the principal source of TNFα, IL-1β, and other cytokines and chemokines, such as IL-8, that are pivotal in promoting inflammation and joint destruction in RA (30, 31). These observations demonstrate the critical role of macrophages in chronic synovitis, and they suggest that TLRs may be important not only for the initiation of this process, but also for its persistence and progression. The current study was undertaken to characterize the expression and function of TLR-2 and TLR-4 on CD14+ macrophages obtained from the joints of patients with RA. The expression of TLR-2 and TLR-4 was increased on macrophages isolated from the joints of patients with RA compared with control macrophages and monocytes; however, there was no significant difference in this expression between RA and other forms of inflammatory arthritis. Despite the lack of difference between cell surface TLR-2 and TLR-4 expression, the responses to PG and LPS were significantly greater with RA synovial macrophages compared with macrophages in other forms of inflammatory arthritis or compared with normal control in vitro–differentiated macrophages. Factors other than the level of cell surface expression of TLR-2 and TLR-4 were responsible for the increased response to TLR ligands by RA synovial macrophages.

PATIENTS AND METHODS

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

Reagents.

The TLR-2 agonist PG (from S aureus), the TLR-4 agonist LPS (from Escherichia coli 055:B5), polymyxin B sulfate, Histopaque-1077, and brefeldin A were from Sigma (St. Louis, MO). IL-1β and TNFα were from R&D Systems (Minneapolis, MN). Fluorescein isothiocyanate (FITC)–conjugated anti–TLR-4 monoclonal antibody (HTA 125) was obtained from Imgenex (San Diego, CA), and FITC-conjugated anti–TLR-2 (TL2.1) and isotype-matched control IgG monoclonal antibodies were obtained from eBioscience (San Diego, CA). Phycoerythrin (PE)–conjugated anti–IL-8 and anti-TNFα monoclonal antibodies, fluorescence-activated cell sorting (FACS) lysing and permeabilization solutions, and PE- and FITC-conjugated anti-CD14 were from BD PharMingen (San Diego, CA). TLR-2–specific and nonspecific control small interfering RNA (siRNA) were purchased from Dharmacon (Lafayette, CO).

Patients and controls.

The primary human macrophages used in this study were differentiated in vitro for 7 days from monocytes, which were purified by elutriation from the PB of healthy donors, as previously described (32–37). Purified monocytes were suspended in medium without fetal bovine serum (FBS) and allowed to adhere to cell culture dishes for 1 hour. Afterward, medium was changed to RPMI 1640 supplemented with 20% FBS, 100 units penicillin, 100 μg/ml streptomycin, and 1 μg/ml polymyxin B. Adherent cells were allowed to differentiate into normal control macrophages, as previously described (32–37).

Synovial fluid (SF) samples were obtained from the inflamed joints of patients with RA or with other forms of inflammatory arthritis, including psoriatic arthritis, ankylosing spondylitis, Crohn's disease, and polyarthritis associated with ulcerative colitis. The patients were recruited from the Northwestern Medical Faculty Foundation and the Rehabilitation Institute of Chicago. RA was diagnosed according to the 1987 revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (38). These studies were reviewed and approved by the Northwestern University Institutional Review Board.

With the exception of 1 patient with RA, all patients who donated SF were receiving therapy that included the nonbiologic disease-modifying antirheumatic drugs (DMARDs) methotrexate or leflunomide (n = 20), TNF inhibitors including etanercept, adalimumab, or infliximab (n = 12), nonsteroidal antiinflammatory drugs (NSAIDs) (n = 19), or low-dose prednisone (≤10 mg/day) (n = 13). All samples were collected with informed consent. SF samples were collected in tubes containing heparin and were centrifuged at 800g at room temperature for 10 minutes. The cells were resuspended in phosphate buffered saline (PBS) and used directly or fractionated by centrifugation on a Histopaque density gradient at 450g for 30 minutes to isolate mononuclear cells. PB was obtained from 7 patients with RA, and the mononuclear cells were harvested by Histopaque density-gradient centrifugation. The patients were receiving TNF inhibitors (n = 5), DMARDs (n = 5), NSAIDs (n = 3), or low-dose prednisone (≤10 mg/day) (n = 3).

Endotoxin.

Reagents not already tested for endotoxin were examined by the Limulus amebocyte cell lysate assay using the QCL-1000 kit (BioWhittaker, Walkersville, MD). At a concentration of PG 10-fold higher than that used in this study (50 μg/ml), the level of endotoxin was below the detection limit for this assay (<0.01 ng/ml LPS).

Flow cytometric analysis of TLR-2 and TLR-4 expression.

Cell surface expression of TLR-2 and TLR-4 was analyzed by 2-color flow cytometry. The cells were preincubated with 50% human serum and 0.5% bovine serum albumin (BSA) in PBS for 30 minutes. After washing with 0.5% BSA in PBS and centrifuging at 450g for 5 minutes, the cells were incubated for 30 minutes with 1 of the following FITC-conjugated monoclonal antibodies: anti–TLR-2, anti–TLR-4, or isotype-matched control IgG. After a second washing and centrifugation, cells were labeled with PE-conjugated anti-CD14 monoclonal antibodies for 30 minutes. All procedures were performed at room temperature. After staining, cells were fixed in 0.5 ml of 1% formaldehyde and 0.5% BSA in PBS for flow cytometric analysis, which was performed at the Northwestern University Flow Cytometry Core Facility using an Epics XL-MCL, System II instrument (Beckman Coulter, Fullerton, CA). The level of macrophage surface TLR expression was determined as the mean fluorescence intensity (MFI) of FITC of the CD14+ cell population.

Macrophage activation and cytokine expression.

RA PB mononuclear cells, control in vitro–differentiated macrophages, or cells from the joints of patients with RA or other forms of inflammatory arthritis were activated with PG (5 μg/ml) or with LPS (1 μg/ml) for 15–18 hours in the presence of brefeldin A (10 μg/ml) to inhibit the release of synthesized cytokine. Cytokine production by CD14+ cells was measured by intracellular staining. Harvested cells were blocked and incubated with FITC-conjugated anti-CD14 monoclonal antibody for 30 minutes, fixed with 0.5 ml FACS lysing solution for 10 minutes, and then permeabilized with FACS permeabilization solution for 10 minutes. The presence of intracellular IL-8 and TNFα was detected by PE-conjugated monoclonal antibodies or isotype control antibodies. Cells were resuspended in 0.5 ml of 1% formaldehyde and 0.5% BSA in PBS and analyzed by flow cytometry as described for TLR-2 staining. IL-8 and TNFα production by CD14+ cells was determined as the MFI of the double-positive cells.

For some experiments, RA SF macrophages were enriched by adherence to cell culture dishes, as previously described (39). The plates were then washed and the attached cells were incubated in medium containing 20% FBS for 24 hours. The cells were incubated with PG (1 μg/ml) for 4 hours and were then harvested and analyzed by quantitative real-time RT-PCR. Polymyxin B, which binds to endotoxin suppressing activation through TLR-4, effectively suppressed LPS-induced TNFα secretion, but resulted in no reduction of PG-induced TNFα secretion (data not shown), suggesting that endotoxin did not contribute to PG-induced macrophage activation.

Real-time RT-PCR.

Total cellular RNA was extracted using TRIzol according to the protocol of the manufacturer (Invitrogen, Carlsbad, CA). Reverse transcription was carried out with avian myeloblastosis virus transcriptase, using oligo(dT) primers. Real-time PCR was performed with the TaqMan gene expression assay system, using primer and probe mixes for TNFα, IL-8, and endogenous GAPDH control genes. PCRs were performed using the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). Relative gene expression was determined by the ΔΔCt method, where Ct = threshold cycle. All experiments were performed in duplicate or triplicate.

Inhibition of TLR-2 expression by siRNA.

Human TLR-2–specific and nonspecific control siRNA were transfected into control macrophages using Lipofectamine 2000 according to the protocol of the manufacturer (Invitrogen). Briefly, prior to transfection, the cultures were washed and medium was replaced by 400 μl/well (24-well plate) of RPMI 1640 (with no additives). Serial dilutions of siRNA were transfected at 10–60 nM with Lipofectamine 2000 (1 μl/well). The transfection reactions were supplemented with 100 μl of FBS after 4 hours and changed to fresh culture media containing 20% FBS the next morning. Forty-eight hours after transfection, the cells from some wells were harvested and examined for surface TLR-2 and TLR-4 expression by flow cytometry. The cells from the remaining wells were stimulated with PG (1 μg/ml) in the presence of 10 μg/ml polymyxin B for 4 hours. Cell culture supernatants were collected to measure TNFα using enzyme-linked immunosorbent assay DuoSets (BD PharMingen) according to the manufacturer's instructions.

Statistical analysis.

Analysis of variance was used for comparison between groups using Stairview version 5.0 (SAS Institute, Cary, NC). The paired 2-tailed t-test was used to analyze differences before and after stimulation within each group. The relationship of patient demographics and medications was analyzed by the generalized linear model. Linear regression analysis was used to determine the relationship between TLR-2 expression and PG-induced cytokine production. All tests used the 5% significance level.

RESULTS

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

Increased expression of TLR-2 and TLR-4 on RA SF macrophages.

The expression of TLR-2 and TLR-4 on synovial macrophages was examined. For these experiments, control normal PB monocytes were used shortly after isolation, and control macrophages were used after in vitro differentiation from monocytes for 7 days, as previously described (34, 36, 37). Macrophages and monocytes were identified by the cell surface marker CD14. Essentially all the control macrophages and those from the joints of patients with RA or other forms of inflammatory arthritis expressed both TLR-2 and TLR-4 (Figure 1A). TLR-2 was more strongly expressed on control monocytes compared with control macrophages (P < 0.03), while there was no difference in TLR-4 expression between these cell types (Figures 1B and C). The mean expression of TLR-2 on RA synovial macrophages was increased 3.4-fold compared with that on in vitro–differentiated control macrophages (P < 0.001) and 1.8-fold compared with that on control monocytes (P < 0.001) (Figure 1B). The expression of TLR-4 on RA synovial macrophages was increased 2-fold compared with that on in vitro–differentiated macrophages (P < 0.01) and 2.8-fold compared with that on normal monocytes (P < 0.001) (Figure 1C).

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Figure 1. Increased expression of Toll-like receptor 2 (TLR-2) on synovial macrophages (MΦ) from patients with rheumatoid arthritis (RA) and other forms of inflammatory arthritis (OIA). Two-color flow cytometry was performed with phycoerythrin-labeled anti-CD14 and fluorescein isothiocyanate (FITC)–labeled anti–TLR-2 or anti–TLR-4 to examine TLR expression on CD14+ cells. A, Flow cytometry overlay graphs showing TLR-2 and TLR-4 expression on the surface of CD14+ control macrophages or macrophages from the joints of representative RA patients or patients with other forms of inflammatory arthritis. B and C, Summary of TLR-2 and TLR-4 expression on the surface of CD14+ normal control monocytes (Mono) (diamonds; n = 20), in vitro–differentiated control macrophages (triangles; n = 29), synovial fluid (SF) macrophages from RA patients (circles; n = 25), and SF macrophages from patients with other forms of inflammatory arthritis (squares; n = 9). Horizontal lines and vertical bars represent the mean ± SEM for each group. ∗ = P < 0.03; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001. MFI = mean fluorescence intensity.

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Similar to SF macrophages from patients with RA, SF macrophages from patients with other forms of inflammatory arthritis also demonstrated significantly increased TLR-2 expression compared with in vitro–differentiated macrophages (P < 0.001) and control monocytes (P < 0.01) (Figure 1B). There was no difference between RA and other forms of inflammatory arthritis in the expression of either TLR-2 or TLR-4 on the surface of SF macrophages. There was no relationship observed between TLR-2 or TLR-4 expression and age or therapeutic regimen in the patients examined.

Increased TLR-2–induced cytokine production by RA synovial macrophages.

Since the expression of TLR-2 was increased on RA SF macrophages, the next experiments were performed to determine whether TLR-2 engagement resulted in increased cytokine expression. Cells were activated with PG, and the intracellular production of TNFα by CD14+ cells was determined by flow cytometry (Figure 2A). In the absence of stimulation, low levels of TNFα were detected in macrophages from the SF of patients with RA, and these levels were increased compared with those in macrophages from the SF of patients with other forms of inflammatory arthritis or compared with those in RA PB monocytes (P < 0.005) (Figure 2B). Following activation with PG, TNFα expression determined by MFI increased in all groups compared with unstimulated cells within each group (P < 0.03 to P < 0.01) (Figure 2B). PG-induced TNFα expression (Figure 2B) was significantly greater in RA synovial macrophages than in RA PB monocytes, in vitro–differentiated macrophages, or macrophages from the joints of patients with other forms of inflammatory arthritis (P < 0.03 to P < 0.005).

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Figure 2. Increased expression of tumor necrosis factor α (TNFα) in RA synovial macrophages following TLR-2 ligation with peptidoglycan (PG). A, Representative flow cytometry histograms showing intracellular production of TNFα by RA SF CD14+ cells stimulated for 18 hours with control medium (none) or with peptidoglycan (5 μg/ml) containing brefeldin A (10 μg/ml). TNFα expression was determined by intracellular staining with phycoerythrin (PE)–labeled anti-TNFα or isotype control antibodies. In each histogram, the CD14+ population is indicated on the x-axis (FITC), and intracellular TNFα production is indicated on the y-axis (PE). Numbers indicate the percentage of cells in each quadrant; upper right quadrant represents double-positive cells. B, Summary of intracellular expression of TNFα in CD14+ cells. Monocytes from RA peripheral blood (PB) (n = 7), control macrophages (n = 16), SF macrophages from the joints of RA patients (n = 15), or SF macrophages from the joints of patients with other forms of inflammatory arthritis (n = 8) were stimulated as described in A with control medium or peptidoglycan. Values are the mean ± SEM MFI. # = P < 0.03; ## = P < 0.005 between the indicated groups. ∗ = P < 0.03; ∗∗ = P < 0.01, versus control medium within each group. See Figure 1 for other definitions.

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Next, we examined the induction of IL-8 by PG. IL-8 expression in CD14+ cells (Figures 3A and B) was spontaneously increased in RA synovial macrophages compared with that in RA PB monocytes, in vitro–differentiated control macrophages, or synovial macrophages from patients with other forms of inflammatory arthritis (P < 0.01 to P < 0.001). Following stimulation with PG, the expression of IL-8 in CD14+ cells increased in all groups (P < 0.05 to P < 0.01) (Figure 3B). The response to PG was significantly greater in RA synovial macrophages than in control macrophages or in macrophages from the joints of patients with other forms of inflammatory arthritis (P < 0.02) (Figure 3B). There was no difference between RA SF and RA PB in the induction of IL-8 in response to PG (Figure 3B). Together, these observations demonstrate increased TLR-2–mediated activation of macrophages from the joints of patients with RA compared with macrophages from the joints of patients with other forms of inflammatory arthritis or compared with control in vitro–differentiated macrophages.

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Figure 3. Increased intracellular interleukin-8 (IL-8) expression in RA synovial macrophages following TLR-2 ligation with peptidoglycan (PG). RA peripheral blood (PB) monocytes, control macrophages, SF macrophages from the joints of RA patients, or SF macrophages from the joints of patients with other forms of inflammatory arthritis were stimulated as described in Figure 2. A, Spontaneous and peptidoglycan-stimulated expression of IL-8 in CD14+ macrophages from a representative SF specimen from an RA patient. B, Summary of IL-8 expression in CD14+ cells in the 4 groups examined. The results following stimulation with control medium (none) and with peptidoglycan are presented as the mean ± SEM MFI from RA PB monocytes (n = 7), control macrophages (n = 16), SF macrophages from the joints of RA patients (n = 15), or SF macrophages from the joints of patients with other forms of inflammatory arthritis (n = 8). # = P < 0.02; ## = P < 0.01; ### = P < 0.001 between the indicated groups. ∗ = P < 0.05; ∗∗ = P < 0.01, versus control medium within each group. PE = phycoerythrin (see Figure 1 for other definitions).

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Increased TLR-4–induced cytokine production by RA synovial macrophages.

Since TLR-4 expression was also increased on RA synovial macrophages compared with that on in vitro–differentiated control macrophages, the response to the TLR-4 ligand LPS was also examined. Intracellular TNFα expression was significantly (P < 0.05 to P < 0.01) increased following treatment with LPS in each group (Figure 4A). The LPS-induced TNFα expression was significantly (P < 0.002) greater with RA synovial macrophages than with those from patients with other forms of inflammatory arthritis, in vitro–differentiated control macrophages, or RA PB monocytes (Figure 4A). Following incubation with LPS, a significant (P < 0.05 to P < 0.01) increase of IL-8 expression was observed within each group except for the macrophages from the joints of patients with other forms of inflammatory arthritis (Figure 4B). The LPS-induced IL-8 response was significantly greater with RA synovial macrophages than with those from patients with other forms of inflammatory arthritis (P < 0.01), in vitro–differentiated control macrophages (P < 0.01), or RA PB monocytes (P < 0.03) (Figure 4B). In summary, similar to the results observed for PG, RA synovial macrophages were more responsive to the TLR-4 ligand LPS than were in vitro–differentiated macrophages or those from the joints of patients with other forms of inflammatory arthritis.

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Figure 4. Increased expression of tumor necrosis factor α (TNFα) and interleukin-8 (IL-8) in RA synovial macrophages following TLR-4 ligation with lipopolysaccharide (LPS). RA peripheral blood (PB) monocytes, control macrophages, SF macrophages from the joints of RA patients, or SF macrophages from the joints of patients with other forms of inflammatory arthritis were stimulated as described in Figures 2 and 3, except that LPS (1 μg/ml) was used. A, Expression of TNFα in CD14+ cells. B, Expression of IL-8 in CD14+ cells. RA PB monocytes (n = 7), control macrophages (n = 16), SF macrophages from the joints of RA patients (n = 15), or SF macrophages from the joints of patients with other forms of inflammatory arthritis (n = 9) were incubated with control medium (none) or LPS. The data for the control medium are the same as those presented in Figures 2 and 3. Values are the mean ± SEM MFI. # = P < 0.03; ## = P < 0.01 between the indicated groups. ∗ = P < 0.05; ∗∗ = P < 0.01, versus control medium within each group. See Figure 1 for other definitions.

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Relationship of TLR-2 and TLR-4 with ligand-induced cytokine expression.

Linear regression was performed to determine the relationship between TLR expression and response to TLR ligands. When we used control macrophages, there were significant positive correlations between cell surface TLR-2 expression and both PG-induced TNFα expression (r = 0.75, P < 0.01) (Figure 5A) and PG-induced IL-8 expression (r = 0.74, P < 0.01) (Figure 5B) in CD14+ cells. However, when we used macrophages from RA SF or SF from patients with other forms of inflammatory arthritis, there were no significant correlations between expression of TLR-2 and PG-induced expression of TNFα or IL-8. In contrast to PG, there was no relationship between the expression of TLR-4 and the response to LPS when we used in vitro–differentiated macrophages or macrophages from the joints of patients with RA or other forms of inflammatory arthritis.

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Figure 5. Correlation of TLR-2 expression level with peptidoglycan-induced cytokine expression in control macrophages. A and B, Correlation of cell surface expression of TLR-2 with production of tumor necrosis factor α (TNFα) (A) and interleukin-8 (IL-8) (B) following peptidoglycan activation in control macrophages (n = 14). C and D, Forced reduction of TLR-2 expression, resulting in decreased activation by peptidoglycan. Control macrophages were transfected with control small interfering RNA (siRNA) or TLR-2–specific siRNA and examined for cell surface expression of TLR-2 (C) or stimulated with peptidoglycan and examined for secreted TNFα (D). The expression of TLR-2 on the surface of macrophages is presented as the MFI, and TNFα concentration was determined by enzyme-linked immunosorbent assay. Results are presented as the percent of TLR-2 expression and the percent of TNFα production normalized to the nontransfected control (100%). Values are the mean ± SEM of 3 individual experiments. ∗ = P < 0.05; ∗∗ = P < 0.002; # = P < 0.001, versus nontransfected controls. See Figure 1 for other definitions.

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RA patients receiving nonbiologic DMARDs (n = 10) demonstrated decreased (P < 0.05) PG-induced IL-8 expression, but not decreased spontaneous or LPS-induced IL-8 expression, compared with those not receiving these agents (data not shown). There was no effect of nonbiologic DMARDs on PG- or LPS-induced TNFα expression, and there was no effect of anti-TNFα therapy on the expression of TNFα or IL-8, either spontaneously or following stimulation with PG or LPS. However, an association was observed between age and spontaneous IL-8 expression in RA patients and those with other forms of inflammatory arthritis. Increasing age was positively associated with increased IL-8 expression (r = 0.62, P < 0.01). No association was observed between age and spontaneous TNFα expression or between age and PG- or LPS-mediated expression of either TNFα or IL-8. Therefore, no correlation between TLR expression and response to TLR ligands was observed with RA synovial macrophages.

Diminished activation by PG resulting from forced reduction of TLR-2 expression.

Since the relationship of TLR-2 expression and activation by PG was not consistent between control and RA SF macrophages, experiments were performed to directly determine the effects of the forced reduction of TLR-2 expression on PG-induced activation. Forty-eight hours after introducing siRNA into macrophages, cell surface TLR-2 expression was significantly (P < 0.05 and P < 0.002) reduced by TLR-2–specific siRNA, but not by control siRNA (Figure 5C). As a control, TLR-4 expression showed no change with TLR-2–specific or nonspecific siRNA (data not shown). PG-induced TNFα production demonstrated a significant dose-dependent reduction (P < 0.001 at concentrations of ≥20 nM) following the introduction of TLR-2–specific siRNA into control macrophages, but no reduction was detected in the nonspecific control siRNA group (Figure 5D). These results demonstrate that the forced reduction of TLR-2 expression in control macrophages resulted in diminished PG-mediated secretion of TNFα.

Increased PG-induced TNFα and IL-8 messenger RNA (mRNA).

Additional experiments were performed to determine whether cells other than macrophages may have contributed to the increased activation of RA synovial macrophages by PG, resulting in the lack of correlation of TLR-2 expression with response to PG. RA synovial macrophages were enriched by adherence as previously described (39) and stimulated with PG. In the absence of stimulation, RA synovial macrophages expressed increased TNFα or IL-8 mRNA (P < 0.01) compared with in vitro–differentiated control macrophages (Figures 6A and B). Following activation with PG, the expression of TNFα and IL-8 mRNA by RA synovial macrophages was significantly increased (P < 0.02) compared with that by control macrophages (Figures 6A and B). These observations confirmed that RA synovial macrophages are more responsive to the TLR-2 ligand PG compared with in vitro–differentiated control macrophages.

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Figure 6. Increased peptidoglycan (PG)–mediated activation of RA synovial macrophages. RA SF macrophages enriched by adherence (n = 7) or in vitro–differentiated control macrophages (n = 6) were incubated with peptidoglycan (5 μg/ml) for 4 hours or without peptidoglycan (none). The cells were harvested and examined for expression of tumor necrosis factor α (TNFα) (A) or interleukin-8 (IL-8) (B) mRNA employing quantitative real-time reverse transcriptase–polymerase chain reaction. Results are reported as the mean ± SEM fold change compared with unstimulated control macrophages. # = P < 0.02; ## = P < 0.01 between the indicated groups. ∗ = P < 0.05; ∗∗ = P < 0.02, versus the absence of peptidoglycan within each group. See Figure 1 for other definitions.

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DISCUSSION

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

Previous studies demonstrated that TLR-2 and TLR-4 are expressed in RA synovial tissue (8, 16, 18) and that this expression was increased compared with that in osteoarthritis or normal synovial tissue (18). TLR-2 was detected by in situ hybridization primarily in cells expressing fibroblast markers (8), while 2-color immunohistochemistry showed that TLR-2 colocalized with CD16+ macrophages in the lining (17). We have extended these observations, demonstrating that the expression of both TLR-2 and TLR-4 is increased on RA SF macrophages compared with that on normal in vitro–differentiated macrophages or normal monocytes. There was no difference in the expression of TLR-2 or TLR-4 on synovial macrophages from patients with RA and those from patients with other forms of inflammatory arthritis. Nonetheless, activation by both TLR-2 and TLR-4 ligands was greater with RA synovial macrophages than with those from patients with other forms of inflammatory arthritis or with control in vitro–differentiated macrophages. These observations suggest that activation through TLR-2 and TLR-4 might contribute to the ongoing inflammation in RA.

The mechanism contributing to the increased expression of TLR-2 and TLR-4 on macrophages obtained from the joints of patients with RA might be related to local factors, since there was no difference between RA and other forms of inflammatory arthritis. TLR-2 expression was increased on PB monocytes in response to LPS and IL-1β (40, 41). Further, treatment of monocytes with interferon-γ (IFNγ) resulted in increased cell surface expression of both TLR-2 and TLR-4 (18, 42), and IFNγ sensitized the monocytes to respond to LPS (43). Although the levels of IFNγ detected in SF of patients with established RA were low (44), IFNγ mRNA was present in synovial tissue lymphocytes (45), and it is possible that IFNγ contributes to sensitizing macrophages to express increased levels of TLR-2 and TLR-4 in vivo. Other cytokines expressed locally, including IL-10 and macrophage colony-stimulating factor, may also contribute to the increased TLR-2 expression in the RA joint (17). In summary, local factors may contribute to the increased expression of TLR-2 and TLR-4 in RA.

The effect of age and therapy on TLR expression was examined. We observed no effect of age or medications, including nonbiologic DMARDs or TNF inhibitors, on the expression of TLR-2 or TLR-4 in joints of RA patients or patients with other forms of inflammatory arthritis. A recent study demonstrated a decrease of TLR-2 and TLR-4 expression on PB monocytes and in the synovial tissue following therapy with TNFα blockers, supporting a role for inflammation in effecting the expression of TLRs (16). We did not observe an effect of therapy on TLR expression, probably because we obtained SF samples from patients with active disease despite therapy and did not examine the samples before and after the initiation of therapy, when disease activity was improved.

Prior studies have not examined the ability of RA synovial macrophages to respond to TLR ligands. A key observation in this study is that RA synovial macrophages demonstrated increased activation, employing both TLR-2 and TLR-4 ligands, compared with macrophages from the joints of patients with other forms of inflammatory arthritis and compared with control in vitro–differentiated macrophages. There are a number of potential explanations for the differences between these groups. We first explored the possibility that the level of TLR may control the response. When we used control macrophages, the cell surface expression of TLR-2 was highly associated with the level of cytokine induced by stimulation with the TLR-2 ligand PG. Further, the forced reduction of TLR-2 expression resulted in a decreased response to PG. These observations support the hypothesis that increased expression of TLR-2 may result in an enhanced response to TLR-2 ligands. Concordant with such a relationship for TLR-4, the level of TLR-4 expression in mice determined the responsiveness to the TLR-4 ligand LPS (46). In contrast, in the present study, no relationship between the cell surface expression of TLR-2 and response to PG, or between the cell surface expression of TLR-4 and response to LPS, was observed with SF macrophages in RA or other forms of inflammatory arthritis. This lack of association may be explained by several factors.

The effects of therapy may have contributed to these results, since PG-induced IL-8 expression by synovial macrophages of RA patients receiving nonbiologic DMARDs was reduced (P < 0.05) compared with that by synovial macrophages of RA patients who were not receiving these medications. There was no reduction of the response to LPS in patients receiving nonbiologic DMARDs. It is possible that a reduction of PG-induced IL-8 expression by RA synovial macrophages in patients receiving nonbiologic DMARDs may explain why this response was not significantly greater than that observed with RA PB monocytes. Also, in the absence of stimulation, IL-8 mRNA expression was 50-fold greater in normal monocytes than in normal in vitro–differentiated macrophages (data not shown), which may contribute to an increased PG-induced expression of IL-8 by monocytes. This explanation seems less likely, since the LPS-induced IL-8 expression by RA synovial macrophages was significantly greater than that observed with RA PB monocytes. Further, no effect on activation by PG or LPS was associated with anti-TNFα therapy. Therefore, although therapy may have modulated the degree of activation, the increased response to TLR ligands by RA synovial macrophages was not associated with the type of therapy.

It is possible that the RA synovial macrophages were inherently more responsive than those obtained from patients with other forms of inflammatory arthritis, due to conditioning within the joint. It is possible that activation through an endogenous TLR ligand, such as heat-shock proteins (HSPs) (17, 47), may predispose the macrophages to heightened activation by subsequent exposure to microbial TLR ligands (48, 49). Alternatively, it is also feasible that prior in vivo exposure to potential endogenous TLR-2 or TLR-4 ligands may have induced tolerance to repeat stimulation (50, 51), partially reducing the response expected for the level of TLR-2 or TLR-4 expression in some RA patients, and possibly accounting for the lack of association between TLR expression and response to TLR ligand in RA patients. In support of this possibility, the PG-induced responses by RA synovial macrophages, although increased compared with those by control in vitro–differentiated macrophages, were less than would have been expected by the control macrophages at comparable levels of TLR-2. This was also the case for macrophages from the joints of patients with other forms of inflammatory arthritis. Further, it is possible that the reduced response by macrophages in other forms of inflammatory arthritis (compared with that by RA macrophages) may be due to greater prior in vivo activation of these macrophages in response to endogenous TLR ligands, resulting in tolerance to a repeat challenge by microbial TLR ligands (50–52).

Differences between RA synovial macrophages and control in vitro–differentiated macrophages, examined by intracellular staining for cytokines, may relate to the fact that the control macrophages were purified, while the macrophages present in the RA SF samples contained a mixture of cells including T lymphocytes. It is possible that additional cytokines, such as IFNγ, IL-6, or granulocyte–macrophage colony-stimulating factor, secreted from other cell types may have increased the responsiveness of the macrophages obtained from the SF of RA patients compared with that of the control in vitro–differentiated macrophages. However, the SF from patients with other forms of inflammatory arthritis and the RA PB also contained a mixture of cell types, and their response to PG and LPS was generally reduced compared with the results obtained with RA synovial macrophages. The fact that purified RA synovial macrophages also demonstrated an increased response to PG compared with that of control macrophages supports the notion of a primary role for the RA synovial macrophage.

How might activation through TLR-2 or TLR-4 contribute to the pathogenesis of RA? We suggest that once inflammation within the joint causes joint destruction, a variety of molecules are released, such as ED-A of fibronectin, HSPs, and HMGB-1. Each of these molecules is highly expressed in the rheumatoid joint, and each is capable of activating TLR-2 and/or TLR-4 (22–29). Therefore, once joint damage occurs, a self-perpetuating process, mediated by TLR ligation by endogenous ligands, may be established that promotes the chronic, progressive destruction mediated by the continued activation of macrophages. In support of the notion of the role of potential endogenous TLR ligands, a recent study demonstrated that SF from RA patients activated a TLR-4–expressing line, suggesting that TLR-4 ligands may be present in RA SF (53). Further studies with animal models will be required to fully elucidate the potential mechanisms by which activation of macrophages through endogenous TLRs may contribute to the progression of inflammatory synovitis.

AUTHOR CONTRIBUTIONS

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

Dr. Pope 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 design. Huang, Pope.

Acquisition of data. Huang, Ma, Adebayo.

Analysis and interpretation of data. Huang, Pope.

Manuscript preparation. Huang, Pope.

Statistical analysis. Huang, Pope.

REFERENCES

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