Increased expression of receptor for advanced glycation end products by synovial tissue macrophages in rheumatoid arthritis.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 54, No. 1, January 2006, pp 97–104 DOI 10.1002/art.21524 © 2006, American College of Rheumatology Increased Expression of Receptor for Advanced Glycation End Products by Synovial Tissue Macrophages in Rheumatoid Arthritis Katsue Sunahori, Masahiro Yamamura, Jiro Yamana, Koji Takasugi, Masanori Kawashima, and Hirofumi Makino Objective. The accumulation of advanced glycation end products (AGEs), S100A12, and high mobility group box chromosomal protein 1 has been associated with joint inflammation in rheumatoid arthritis (RA). This study was undertaken to determine the induction of the receptor for these proteins, termed receptor for AGEs (RAGE), in synovial tissue (ST) macrophages from RA patients. Methods. RAGE and CD68 expression in ST were determined by 2-color immunofluorescence labeling. Cell surface and messenger RNA (mRNA) expression of RAGE were examined by flow cytometry and reverse transcriptase–polymerase chain reaction (PCR) or realtime PCR, respectively. Results. CD68ⴙ lining macrophages, like the vasculature, expressed high levels of RAGE in inflamed ST from RA patients. RAGE mRNA expression was significantly higher in RA ST than in ST from patients with osteoarthritis. RAGE mRNA levels were significantly higher in ST macrophages and normal endothelial cells than in ST CD4ⴙ T cells and synovial fibroblasts stimulated with tumor necrosis factor ␣ and interleukin-1␤ (IL-1␤). Cell surface RAGE was highly induced on normal monocytes after a 24-hour incubation with a 20% concentration of RA ST cell culture supernatants. RAGE mRNA expression in adherent monocytes was augmented by various cytokines, most potently by IL-1␤. Conclusion. These results indicate that RAGE overexpression in lining macrophages may be induced, at least in part, by cytokines such as IL-1, leading to the amplification of inflammatory responses mediated by RAGE ligands that are abundant in RA joints. Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects multiple synovial joints. Synovial tissue (ST) macrophages are critical in the perpetuation of chronic inflammation and joint destruction by releasing proinflammatory cytokines, most notably, tumor necrosis factor ␣ (TNF␣) and interleukin-1 (IL-1), and matrix-degrading enzymes (1,2). TNF␣ is a master cytokine that governs the disease process by inducing a variety of inflammatory mediators through activation of the transcription factor NF-B and the MAP kinase cascade, but many other cytokines and molecules contribute importantly to the pathogenesis of RA. Recent studies have demonstrated that high mobility group box chromosomal protein 1 (HMGB-1), a member of the nonhistone chromatin-associated proteins, and S100A12, a member of the S100 family of calcium-binding proteins, may contribute to progression of the inflammatory response in RA (3–5). In the inflamed joint, HMGB-1 is actively secreted by ST macrophages after translocation from the nucleus to secretory lysosomes (3,4), and S100A12 is secreted via a tubulin-dependent pathway by activated neutrophils (5). Extracellular HMGB-1 and S100A12 proteins are thought to exert proinflammatory effects, at least in part, through their interaction with the receptor for advanced glycation end products (RAGE), although this multiligand receptor, a member of the immunoglobulin superfamily of cell surface molecules, was originally Katsue Sunahori, MD, Masahiro Yamamura, MD, Jiro Yamana, MD, Koji Takasugi, MD, Masanori Kawashima, MD, Hirofumi Makino, MD: Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan. Address correspondence and reprint requests to Masahiro Yamamura, MD, Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail: firstname.lastname@example.org. Submitted for publication November 7, 2004; accepted in revised form September 23, 2005. 97 98 SUNAHORI ET AL identified by its ability to bind AGEs, the products of nonenzymatic glycation and oxidation of proteins (6). The activation of RAGE engages critical signaling pathways linked to proinflammatory responses, such as NF-B and MAP kinase pathways, in endothelium, monocyte/macrophages, and lymphocytes, resulting in activation of various inflammatory genes (7). In murine type II collagen–induced arthritis, in which RAGE expression is increased, ST inflammation and cartilage and bone destruction are decreased by treatment with soluble RAGE (sRAGE), the extracellular ligand-binding domain of the receptor, in parallel with diminished generation of TNF␣, IL-6, and matrix metalloproteinases (8). In addition, HMGB-1–mediated TNF␣ production in macrophages derived from RA synovial fluid (SF) were shown to be significantly inhibited by sRAGE (4). These findings suggest that cellular RAGE plays an important role in the activation of inflammatory effector cells such as macrophages by recognizing its pathogenic ligands in the joint. We therefore investigated RAGE expression by macrophage infiltrates and the mechanism of their RAGE induction in the ST lesion of RA. PATIENTS AND METHODS Patients and samples. The RA study patients were diagnosed according to the 1987 revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (9). All patients were receiving nonsteroidal antiinflammatory drugs (NSAIDs), prednisolone at ⱕ5 mg/day, and disease-modifying antirheumatic drugs such as methotrexate (MTX) and sulfasalazine. ST samples were obtained from 14 patients with longstanding RA (13 women and 1 man with a mean ⫾ SD age of 63.0 ⫾ 8.7 years and a mean ⫾ SD disease duration of ⱖ8 years) at the time of knee (n ⫽ 9) or elbow (n ⫽ 5) joint replacement and from 7 patients with osteoarthritis (OA) (6 women and 1 man with a mean ⫾ SD age of 68.3 ⫾ 4.1 years) at the time of knee replacement. RA patients had active disease, as indicated by multiple joint involvement (tender and/or swollen joint count ⱖ3) and increased acute-phase reactants (serum C-reactive protein [CRP] level 21 ⫾ 16 mg/liter), and 11 were positive for IgM class rheumatoid factor. OA patients exhibited no evidence of a systemic inflammatory response. SF samples were obtained from 3 female patients with active RA (mean ⫾ SD age 47.7 ⫾ 15.0 years with a tender and/or swollen joint count ⱖ5 and a CRP level of 55 ⫾ 20 mg/liter) despite treatment with NSAIDs, prednisolone, and MTX, showing an elevated white blood cell count (⬎10,000/l) with a predominance of neutrophils (⬎90%). Peripheral blood (PB) samples were collected from healthy volunteers. All patients and healthy individuals gave informed consent. Two-color immunofluorescence labeling. Cryostat sections (4 m) of ST samples were fixed in acetone. Double immunofluorescence was performed by serially incubating sections with 10 g/ml of mouse IgG1 anti-CD68 monoclonal antibody (mAb; Zymed, South San Francisco, CA) or isotypematched control mAb at 4°C overnight, followed by incubation with fluorescein isothiocyanate (FITC)–conjugated goat antimouse IgG1 mAb (ICN Pharmaceuticals, Costa Mesa, CA) for 30 minutes at room temperature, and then with 1% goat anti-RAGE polyclonal antibody (Chemicon, Temecula, CA) or control serum, followed by incubation with rhodamine-conjugated rabbit anti-goat IgG antibody (Jackson ImmunoResearch, Baltimore, PA). Double immunofluorescence of sections was examined with an LSM510 inverted laser-scanning confocal microscope (Zeiss, Jena, Germany) and illuminated with 488 nm and 568 nm of light. Images showing FITC and rhodamine staining were recorded simultaneously through separate optical detectors with a 530-nm band-pass filter and a 590-nm long-pass filter, respectively. Pairs of images were superimposed for colocalization analysis. Image analysis was performed using Optimas 6.5 software (Media Cybernetics, Silver Spring, MD). ST cells, synovial fibroblasts, and aortic endothelial cells. ST samples were fragmented and digested with collagenase and DNase for 1 hour at 37°C. After removing tissue debris, cells were resuspended in culture medium (RPMI 1640 medium [Life Technologies, Gaithersburg, MD] supplemented with 25 mM HEPES, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 g/ml streptomycin) with 10% heat-inactivated fetal calf serum (FCS; Life Technologies). RA ST cells were incubated at a density of 1 ⫻ 106/ml in 10% FCS/culture medium in 6-well plates. Culture supernatants were harvested 72 hours later and stored at ⫺30°C. CD4⫹ T cells and CD14⫹ macrophages were purified from RA ST cell suspensions by positive selection using anti-CD4 mAb–coated or anti-CD14 mAb–coated magnetic beads (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer’s instructions. The anti-CD4 mAb– and antiCD14 mAb–selected cell populations consisted of ⬎95% CD3⫹ T cells and ⬎90% nonspecific esterase-stained macrophages, respectively. Synovial fibroblasts were isolated from RA ST cell suspensions. Briefly, ST cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO) with 10% FCS in a humidified atmosphere containing 5% CO2. Adherent cells were split weekly after primary cultures had reached confluence. These synovial fibroblast cell lines (passage 4–5) were incubated at a density of 1 ⫻ 105 cells/ml in 1% FCS/DMEM, with or without 10 ng/ml of TNF␣ (Sigma) or 1 ng/ml of IL-1␤ (Sigma), in 6-well plates (Costar, Cambridge, MA) at 37°C for 3 hours. Human aortic endothelial cells (HAECs) were purchased from Clontech (Palo Alto, CA). Activation of adhered monocytes. PB mononuclear cells (PBMCs) were prepared by Ficoll-Hypaque densitygradient centrifugation of heparinized PB samples obtained from healthy individuals. Monocytes were purified from PBMCs by negative selection using a cocktail of antibodies against CD3, CD7, CD16, CD19, CD56, CD123, and glycophorin A (monocyte isolation kit II; Miltenyi Biotec) according to the manufacturer’s instructions. After incubation at a density of 1 ⫻ 106/ml in 10% FCS/culture medium in 6-well plates for 24 hours, adherent monocytes were stimulated for 3 hours in fresh 1% FCS/culture medium with or without a 20% concentration of RA ST culture supernatants, 10 ng/ml of TNF␣, 1 ng/ml of IL-1␤, 1 ng/ml of interferon-␥ (IFN␥; PeproTech, Rocky Hill, NJ), or 10 ng/ml of IL-10 (R&D Systems, Minneapolis, MN). RAGE EXPRESSION IN RA MACROPHAGES Reverse transcriptase–polymerase chain reaction (RTPCR) and real-time PCR. Total cellular RNA was extracted from fresh RA and OA ST cells, RA ST CD14⫹ macrophages and CD4⫹ T cells, RA synovial fibroblasts, RA SF cells, HAECs, and activated normal monocytes using an RNA isolation kit (RNeasy Mini kit; Qiagen, Valencia, CA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from total RNA with Moloney murine leukemia virus reverse transcriptase (US Biochemical, Cleveland, OH) and oligo(dT)15 primers (Promega, Madison, WI). Samples of cDNA were PCR-amplified for 25 cycles for GAPDH and for 40 cycles for RAGE with rTaq DNA polymerase (Promega) with specific primers in a thermal cycler (iCycler; Bio-Rad, Hercules, CA). Each cycle consisted of 1 minute of denaturation at 95°C and 2 minutes of annealing/ extension at 60°C for GAPDH or at 63°C for RAGE. PCR products were electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining. The sequences of oligonucleotide primers were as follows: for RAGE, 5⬘-TGGAACCGTAACCCTGACCT-3⬘ (forward) and 5⬘-CGATGATGCTGATGCTGACA-3⬘ (reverse); and for GAPDH, 5⬘-TGGTATCGTGGAAGGACTCATGAC-3⬘ (forward) and 5⬘-ATGCCAGTGAGCTTCCCGTTCAGC-3⬘ (reverse). To ascertain differences in RAGE messenger RNA (mRNA) expression in different cell types, we normalized cDNA concentrations to yield equivalent GAPDH PCR products and then performed PCR in triplicate for these cDNA samples, confirming that repeated PCR analysis of the same samples yielded reproducible results. To semiquantify the amounts of RAGE mRNA in RA and OA ST cells, synovial fibroblasts, and activated monocytes, real-time PCR was performed with a LightCycler instrument (Roche Diagnostics, Penzberg, Germany) in glass capillary tubes. The reaction mixture containing double-stranded DNA– specific SYBR Green I dye (Roche Diagnostics) and specific primers for human ␤-actin and RAGE (Roche Diagnostics) was added to cDNA dilutions. The cDNA samples were amplified and analyzed according to the manufacturer’s instructions. RAGE expression was determined by normalization against ␤-actin expression. Flow cytometry. Purified monocytes were incubated in polypropylene tubes (Becton Dickinson, San Jose, CA) at a density of 1 ⫻ 106/ml in 10% FCS/culture medium with or without a 20% concentration of RA ST cell culture supernatants. Cells were harvested 24 hours later, and cell surface expression of RAGE was analyzed by flow cytometry. Monocyte suspensions in phosphate buffered saline with 1% FCS were incubated with goat anti-RAGE polyclonal antibody or control goat serum, followed by incubation with FITCconjugated donkey anti-goat IgG antibody (Jackson ImmunoResearch). Analysis was performed on a FACScan flow cytometer (Becton Dickinson), and the monocytes were specifically analyzed by selective gating based on parameters of forward and side light scatter. Coexpression of RAGE and the CD4 or CD14 antigen on fresh RA ST cells was also determined by flow cytometry. ST cell suspensions were stained with anti-RAGE antibody and then incubated with peridin chlorophyll protein– conjugated anti-CD4 mAb (Becton Dickinson), phycoerythrinconjugated anti-CD14 mAb (eBioscience, San Diego, CA), or control mAb (Becton Dickinson). 99 Figure 1. A, Expression of CD68 and receptor for advanced glycation end products (RAGE) in synovial tissue (ST) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). ST sections from RA and OA patients were stained with anti-CD68 antibody and anti-RAGE antibody, followed by incubation with isotype-specific fluorescein isothiocyanate– and rhodamine-conjugated secondary antibodies. Two-color immunofluorescence confocal images were obtained for CD68 and RAGE expression (green and red staining, respectively). The 2 images were superimposed, and double-positive cells are shown in yellow (original magnification ⫻ 200). B, Quantification of CD68⫹ and RAGE⫹ cells in RA and OA ST. The proportion of CD68⫹ area in the whole tissue section and the proportion of RAGE⫹ area in the CD68⫹ area were measured using Optimas 6.5 software. Values are the mean and SEM of n samples evaluated. Statistical analysis. The statistical significance of differences between 2 groups was determined by the MannWhitney U test or the Wilcoxon signed rank test. 100 SUNAHORI ET AL Figure 2. A, RAGE mRNA expression in RA and OA ST. Total cellular RNA was extracted from ST cells obtained from RA and OA patients. RAGE mRNA expression was analyzed by reverse transcriptase–polymerase chain reaction (PCR) and real-time PCR as described in Patients and Methods. For real-time PCR analysis, levels of RAGE mRNA were normalized against ␤-actin expression. Values are the mean and SEM; n is the number of samples evaluated. B, RAGE expression in ST CD14⫹ macrophages and CD4⫹ T cells. CD14⫹ and CD4⫹ cells were purified from RA ST cells by positive selection with anti-CD14 antibody– and anti-CD4 antibody–coated magnetic beads. RAGE mRNA expression in CD14⫹ and CD4⫹ ST cells, synovial fluid (SF) cells, and human aortic endothelial cells (HAECs) is shown. Results are representative of 3 individual experiments with ST cells and SF cells from different RA patients. Coexpression of cell surface RAGE, CD4, and CD14 was determined by flow cytometry. Surface RAGE expression was calculated as the ratio of the mean fluorescence intensity (MFI) of staining with anti-RAGE antibody to the MFI of staining with control antibody. Values are the mean and SEM; n is the number of samples evaluated. C, RAGE mRNA expression in RA synovial fibroblasts (SFb). Synovial fibroblast cell lines (fourth to fifth passage; 1 ⫻ 105 cells/ml in Dulbecco’s modified Eagle’s medium with 1% fetal calf serum) were incubated for 3 hours with 10 ng/ml tumor necrosis factor ␣ (TNF␣), 1 ng/ml interleukin-1␤ (IL-1␤), or a 20% concentration of RA ST cell culture supernatants. Similar results were obtained in additional experiments with 2 synovial fibroblast cell lines from different patients. See Figure 1 for other definitions. RESULTS Increased RAGE expression on CD68ⴙ synovial lining macrophages in RA. The expression of RAGE by macrophage infiltrates in ST samples from RA and OA patients was examined by 2-color immunofluorescence labeling. Although staining patterns of RAGE expres- sion were similar in RA and OA lesions, the RAGE antigen was more extensively expressed in RA lesions in association with cellular infiltration (Figure 1A). The major cellular constituents of the vasculature, endothelial cells and smooth muscle cells, showed strong staining for RAGE, a finding consistent with the previous obser- RAGE EXPRESSION IN RA MACROPHAGES 101 Figure 3. A, Induction of cell surface RAGE expression on normal monocytes by RA ST cell culture supernatants. Peripheral blood monocytes from normal subjects (1 ⫻ 106/ml in RPMI 1640 medium with 10% fetal calf serum [FCS]) were incubated for 24 hours with or without a 20% concentration of RA ST cell culture supernatants in polypropylene tubes. Cells were stained with anti-RAGE antibody and control antibody, followed by incubation with fluorescein isothiocyanate–conjugated secondary antibody. Flow cytometric analysis was performed by gating on monocytes according to the light scatter profile. Representative histographic patterns of RAGE expression on monocytes with or without supernatants are shown (top); replacement of supernatants with human albumin had no effect on RAGE induction. RAGE induction was expressed as the ratio of the mean fluorescence intensity (MFI) of staining with anti-RAGE antibody to the MFI of staining with control antibody (bottom). Values are the mean and SEM; n is the number of samples evaluated. B, Induction of RAGE mRNA expression in monocytes by tumor necrosis factor ␣ (TNF␣), interleukin-1␤ (IL-1␤), interferon-␥ (IFN␥), and IL-10. After 24 hours of incubation in plastic, adherent monocytes (1 ⫻ 106/ml in RPMI 1640 medium with 1% FCS) were stimulated for 3 hours with or without a 20% concentration of RA ST cell culture supernatants, 10 ng/ml TNF␣, 1 ng/ml IL-1␤, 1 ng/ml IFN␥, or 10 ng/ml IL-10. RAGE mRNA expression was analyzed by real-time polymerase chain reaction. Cytokine-mediated RAGE induction was expressed as the ratio of the level of ␤-actin–normalized RAGE expression with cytokine stimulation to the level with no stimulation. Values are the mean and SEM; n is the number of samples evaluated. See Figure 1 for other definitions. vation (10). In RA, the lining layer contained a large number of RAGE-stained cells that were mostly positive for the CD68 antigen. RAGE⫹ cells were also inter- spersed throughout the sublining layer, including both CD68⫹ and CD68⫺ cells. Quantification of CD68 and RAGE staining by image analysis indicated that RA ST 102 samples were more densely infiltrated with CD68⫹ macrophages (mean ⫾ SEM CD68⫹ area/ST area 9.2 ⫾ 1.4%) than were OA samples (3.2 ⫾ 0.5%) and that RA CD68⫹ cells more strongly expressed RAGE (RAGE⫹ area/CD68⫹ area 38.0 ⫾ 5.7%) than did OA CD68⫹ cells (15.3 ⫾ 1.0%) (Figure 1B). These results suggest that CD68⫹ macrophages (particularly the cells in the lining layer) and vascular cells are activated to express high levels of RAGE under inflammatory conditions in RA. RAGE mRNA expression in macrophages, CD4ⴙ T cells, and fibroblasts from RA ST. The synthesis of RAGE transcripts in ST samples from RA and OA patients was measured by RT-PCR and real-time PCR. RAGE mRNA expression was markedly greater in RA than in OA lesions (Figure 2A), suggesting that transcription of the RAGE gene may be enhanced by the particular stimuli abundant in RA ST, such as cytokines. To identify which inflammatory cell types were responsible for the local RAGE overexpression, the levels of RAGE mRNA in purified CD14⫹ macrophages and CD4⫹ T cells from RA ST samples, as well as in SF cells from RA patients (⬎90% of which were neutrophils) and normal endothelial cells (HAECs), were determined by RT-PCR. RAGE mRNA levels were significantly higher in CD14⫹ macrophages and endothelial cells than in CD4⫹ T cells and SF cells (Figure 2B). To confirm the high-level expression of RAGE in macrophages, the intensity of cell surface RAGE antigen on CD14⫹ and CD4⫹ RA ST cell populations was compared by flow cytometric analysis. Surface RAGE expression (calculated as the ratio of the mean fluorescence intensity of staining with anti-RAGE antibody to that with control antibody) was always increased in CD14⫹ macrophages (mean ⫾ SEM ratio 7.6 ⫾ 4.8) compared with CD4⫹ T cells (3.7 ⫾ 2.3) in 4 different RA patients. In addition, synovial fibroblast cell lines (fourth to fifth passage) from 3 different RA patients were examined for RAGE mRNA expression by RT-PCR. These fibroblasts showed a low level of constitutive RAGE mRNA expression, and the RAGE mRNA levels were increased after TNF␣ and IL-1␤ stimulation (Figure 2C). However, the ratios of RAGE mRNA expression normalized against ␤-actin mRNA expression in cytokine-activated fibroblasts, as determined by realtime PCR, were limited (⬍0.10) in comparison with those in CD14⫹ macrophages (mean ⫾ SEM 0.50 ⫾ 0.34; n ⫽ 4). Therefore, despite RAGE expression by various cell types, activated macrophages and vascular SUNAHORI ET AL cells appear to be responsible for RAGE overexpression in the ST lesion. RAGE expression in monocytes activated by cytokines. To determine RAGE induction at the primary site of disease, normal monocytes were stimulated for 24 hours with a 20% concentration of RA ST cell culture supernatants, and the expression of surface RAGE was analyzed by flow cytometry. RAGE expression on the cell surface of normal monocytes was negligible, but high levels of surface RAGE were induced on monocytes cultured in the presence of the supernatants (Figure 3A). To determine the involvement of cytokines in RAGE induction, RAGE mRNA expression in adherent monocytes stimulated with TNF␣, IL-1␤, IFN␥, or IL-10 for 3 hours was measured by real-time PCR. The mean ⫾ SEM stimulation index value of RA ST cell culture supernatants was 39.3 ⫾ 16.0 (Figure 3B). These cytokines were all able to induce RAGE mRNA expression (3.2 ⫾ 1.0 for TNF␣, 10.6 ⫾ 4.8 for IL-1␤, 2.3 ⫾ 0.8 for IFN␥, and 5.4 ⫾ 2.6 for IL-10); IL-1␤ was the most effective. It thus seems that a range of cytokines produced in RA joints contribute to increased RAGE expression in tissue macrophages, and IL-1 may be one of the potent RAGE inducers. DISCUSSION It has been proposed that the multiligand receptor RAGE functions as a progressive factor in chronic inflammatory conditions in which its ligands accumulate, by amplifying proinflammatory pathways (6). RAGE interaction with AGEs stimulates vascular cell expression of adhesion molecules and cytokines, which contributes to the pathogenesis of diabetic vascular complications (10). The accumulation of natural RAGE ligands, such as HMGB-1 and S100A12 polypeptides, has been associated with chronic cellular activation and tissue injury (6), although the role of RAGE in HMGB-1– mediated macrophage activation remains a subject of controversy. It has been shown that Toll-like receptor 2 (TLR-2) and TLR-4 are mainly responsible for HMGB1–induced NF-B activation that involves the p38 MAP kinase pathway (11) and that HMGB-1 induces growth inhibition and apoptosis in macrophages through Rac1 and JNK signaling despite NF-B activation (12). However, recent studies using macrophages from RAGE-, IL-1 receptor type I–, and TLR-2–deficient mice have demonstrated that RAGE is the major functional receptor that mediates HMGB-1–induced activation through both NF-B and MAP kinase activation (13). Thus, the RAGE EXPRESSION IN RA MACROPHAGES significance of other, disparate receptors, such as TLR-2 and TLR-4, in HMGB-1 signaling and the mechanisms for regulating the balance of proinflammatory and apoptotic signals are unknown at present, but RAGE interaction with HMGB-1, like that with S100A12, is believed to induce proinflammatory responses in macrophages under pathologic conditions such as RA. In inflamed RA joints, levels of HMGB-1 and S100A12, as well as those of AGEs, are strikingly increased (3–5,14). In mice with type II collagen– induced arthritis, both joint inflammation and destruction can be prevented by sRAGE administration (8). The RAGE 82S allele (a glycine-to-serine change at position 82 in the RAGE gene; allelic variation within the ligand-binding domain of the receptor), identified as being associated with enhanced binding and signaling, was reported to contribute to RA disease susceptibility (8), although further studies will be required to establish the relevance of this polymorphism (15). In addition, TNF␣ production in RA SF–derived macrophages stimulated with HMGB-1 was shown to be significantly reduced by sRAGE (4). Our immunofluorescence analysis of RAGE in tissues demonstrated that expression of the receptor was markedly increased in RA lesions compared with OA lesions, suggesting that RAGE overexpression is associated with the enhanced inflammatory reaction. In contrast, Drinda et al (16) previously reported no significant difference in immunohistochemical staining for RAGE between RA and OA lesions. Although the reason for this discrepancy is uncertain, the notion of RAGE up-regulation by RA inflammation has been supported by other studies. High levels of RAGE and proinflammatory adhesion molecules were found to be expressed in the RA ST endothelium (10). In addition, macrophages established from RA SF cells were shown to contain large amounts of RAGE protein (4). In RA, the site of inflammatory infiltration was characterized by strong expression of the RAGE antigen on CD68⫹ macrophages and the vasculature, but RAGE was also expressed on other cell types. We thus compared the levels of RAGE expression in CD4⫹ T cells and synovial fibroblasts, the major cellular constituents of RA ST, with those in macrophages. CD4⫹ T cells expressed cell surface RAGE, but at a lower level than in CD14⫹ macrophages. Previous studies have demonstrated that lining fibroblasts constitutively express RAGE in ST lesions from patients with dialysisrelated amyloidosis, and AGE-modified ␤ 2 microglobulin stimulates fibroblast production of the chemokine monocyte chemoattractant protein 1 through 103 its binding to the receptor (17). However, synovial fibroblast cell lines isolated from RA patients showed relatively low expression of RAGE mRNA as compared with macrophages. The different expression of RAGE in fibroblasts from patients with these 2 diseases may be explained by differences in disease pathogenesis. Thus, despite RAGE expression by various cells, activated macrophages represent the major source of RAGE expression in RA ST. RAGE immunoreactivity was particularly increased on CD68⫹ macrophages localized to the lining layer. CD68⫹ lining cells express high levels of proinflammatory cytokines and cell surface molecules, thus illustrating the highly activated phenotype of these macrophages (1,2). We showed that RAGE-inducing factors were secreted spontaneously from RA ST cells, and RAGE mRNA expression in monocytes was augmented by various cytokines, including the 2 essential proinflammatory cytokines IL-1␤ and TNF␣, the Th1 cytokine IFN␥, and, of interest, the antiinflammatory cytokine IL-10. It is noteworthy that despite its inhibitory effect on cytokine synthesis, IL-10 stimulates monocyte maturation toward the CD16 (IgG Fc␥ receptor type IIIA)– positive proinflammatory type with high TLR-2 expression (18). Of these cytokines, IL-1 appears to be the most effective. However, RAGE induction by RA ST cell culture supernatants in monocytes was not significantly inhibited by neutralizing anti–IL-1 antibody (data not shown). We speculate that the supernatants also contained endogenous RAGE ligands capable of RAGE induction, such as HMGB-1 and S100A12, because these intracellular proteins can be released from cultured cells during activation and cell death. In fact, ligand accumulation in the tissues is crucial for enhanced RAGE expression in diverse situations (6). Taken together, these findings suggest that high concentrations of both cytokines and ligands in the lining may contribute to the local enrichment of RAGE. Studies of the RAGE gene identified the presence of at least 2 functional NF-B sites in the promoter (19), and RAGE expression is thereby regulated at the transcriptional level by NF-B activation (19,20). The NF-B pathway is believed to play a key role in RAGE induction by both RAGE ligands and the proinflammatory cytokines IL-1 and TNF␣. These cytokines also stimulate HMGB-1 and S100A12 secretion from macrophages and neutrophils (3–5). Thus, RAGE–ligand interactions appear to be quantitatively enhanced in RA joints, where TNF␣ and IL-1 are abundant. On the other hand, RAGE signaling activates the synthesis of inflammatory mediators, including these cytokines, in macro- 104 SUNAHORI ET AL phages and lymphocytes (7). There seems to be an intimate relationship between the RAGE–ligand system and the cytokine cascade in RA, forming a positivefeedback loop that leads to the propagation of the disease process. In summary, enhanced expression of RAGE in tissue macrophages is induced by overexpression of cytokines such as IL-1, as well as of RAGE ligands, in RA joints, leading to the amplification of inflammatory responses through RAGE–ligand interactions. In addition, high levels of RAGE expression in the vasculature facilitate ST infiltration of monocytes and lymphocytes (6). Therefore, blockade of RAGE–ligand interactions may have implications for reducing disease severity in RA patients. ACKNOWLEDGMENTS The authors thank Drs. H. Inoue and K. Nishida (Okayama University, Okayama, Japan) for providing clinical samples. REFERENCES 1. Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 1996;14:397–440. 2. Burmester GR, Stuhlmuller B, Keyszer G, Kinne RW. 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