Study patients with RA and control patients with osteoarthritis (OA) were diagnosed according to the revised 1987 criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) 2021. All patients with RA were receiving prednisolone at no more than 5 mg/day and various disease-modifying antirheumatic drugs. Paired serum and SF samples were obtained from 17 patients with RA (14 women, 3 men; mean ± standard deviation (SD) age, 63 ± 9 years) and 17 patients with OA (12 women, 5 men; 65 ± 6 years); SF samples were aspirated from the knee joint during therapeutic arthrocentesis. Most patients with RA were active; they had multiple joint tenderness and swelling, systemic inflammatory responses (mean ± SD, serum C-reactive protein [CRP, 53 ± 53 mg/liter] and erythrocyte sedimentation ratio [63 ± 42 mm/hour]), and elevated serum IgM class rheumatoid factor titer (110 ± 133 units/ml), whereas patients with OA did not show evidence of a systemic inflammatory response. ST samples were obtained from patients with RA and patients with OA at the time of total knee joint replacement. PB samples were collected from healthy volunteers. All patients and healthy individuals gave informed consent.

Fresh ST samples were fragmented and digested with collagenase and DNase in RPMI 1640 medium (Life Technologies, Gaithersburg, MD, USA) for 1 hour at 37°C. After removal of tissue debris, cells were washed with medium. The resultant single-cell suspensions were adjusted at a density of 1 × 10⁶ cells per ml in culture medium (RPMI 1640 medium supplemented with 25 mM HEPES, 2 mM L-glutamine, 2% nonessential amino acids, 100 IU/ml penicillin, and 100 μg/ml streptomycin; Life Technologies) with 10% heat-inactivated fetal calf serum (FCS; Life Technologies). The cells were incubated in the wells of six-well plates (Corning, Corning, NY, USA) at 37°C in a humidified atmosphere containing 5% CO₂. Culture supernatants were harvested 72 hours later and stored at -30°C until the S100A8/A9 assay.

Concentrations of S100A8, S100A9, and S100A8/A9 were measured in duplicate by the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using commercially available kits (BMA Biomedicals AG, Augst, Switzerland) according to the manufacturer's instructions. The detection limits for S100A8, S100A9, and S100A8/9 were 0.69, 0.31, and 4.69 ng/ml, respectively.

Concentrations of TNF-α, IL-1-β, IL-6, IL-8, IL-10, and IL-12 p70 in monocyte culture supernatants were measured by cytometric beads array (CBA) with a series of anticytokine monoclonal antibody (mAb)-coated beads and phycoerythrin-conjugated anticytokine mAbs followed by flow cytometric analysis performed on a FACScan flow cytometer using the CBA kit and CBA software (all three obtained from Becton, Dickinson and Company, San Jose, CA, USA). TNF-α concentrations in some experiments were measured using the ELISA kits (Becton, Dickinson and Company). The detection limits for cytokines were 20 pg/ml.

Cryostat sections (4 μm) from ST samples were fixed in acetone and blocked with 10% donkey serum for 30 minutes. Double immunofluorescence was performed by serially incubating sections with 1 μg/ml of mouse IgG1 mAb against human S100A8 (8-5C2), S100A9 (S36.48), or S100A8/A9 (27E10, which exclusively recognizes the heterodimer but not the homodimers 22; BMA Biomedicals AG) or isotype-matched control mAb (BMA Biomedicals AG) at 4°C over night, followed by incubation with rhodamine-conjugated donkey anti-mouse IgG1 mAb (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 30 minutes at room temperature, and subsequently with 1 μg/ml of fluorescein isothiocyanate (FITC)-conjugated anti-CD68 mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or control mAb (Santa Cruz Biotechnology) for 30 minutes at room temperature. The double immunofluorescence of sections was examined with an LSM510 inverted laser-scanning confocal microscope (Carl Zeiss, Jena, Germany) and illuminated with 488 and 568 nm of light. Images decorated with FITC and rhodamine 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.

Recombinant human S100A8 and S100A9 proteins were prepared as described previously 2324. Briefly, competent Escherichia coli strain BL21 (DE3) cells (Novagen, Madison, WI, USA) were transformed with the pET1120-MRP8wt and pET1120 S100A9wt vectors. The transformed cells were grown at 37°C in (2× YT) media supplemented with 100 μg/ml ampicillin for 24 hours; the cells produced the proteins as inclusion bodies. The harvested cells were solubilized with B-PER™ Bacterial Protein Extraction Reagent (Pierce, Rockford, IL, USA). The inclusion bodies were solubilized with Inclusion Body Solubilization Reagent (Pierce), and the proteins were refolded according to the manufacturer's protocol. The proteins were purified by reverse-phase column chromatography (Resource™ RPC; Amersham Bioscience, Buckingamshire, UK) furnished in a BioLogic HR system (Bio-Rad, Hercules, CA, USA), followed by UNO-Q anion exchange chromatography (Bio-Rad). The buffer systems used were the same as those described previously 23. The purity of the S100A8 protein was found to be greater than 95% by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electophoresis) and silver-staining, followed by densitometric analysis using the ImageJ program (Sun Microsystems, Santa Clara, CA, USA). Because the S100A9 showed weak silver-staining, its purity was estimated by Coomasie Brilliant Blue staining and densitometric analysis to be greater than 95%. The endotoxin content was measured using an endotoxin-specific assay kit (Endospecy; Seikagaku Kogyo, Tokyo, Japan), and the 10 μM solutions of recombinant S100A8 and S100A9 used in the present study contained endotoxin at 0.255 and 0.467 ng/ml, respectively. The mixture of equal amounts of S100A8 and S100A9 solutions was found by its specific ELISA to mostly form the heterodimer.

PB mononuclear cells (PBMCs) were prepared from heparinized PB samples of healthy individuals by Ficoll-Hypaque density gradient centrifugation. Monocytes were purified from PBMCs by negative selection using monocyte isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The monocyte suspensions were adjusted at a density of 1 × 10⁶ cells per ml in culture medium with 10% FCS and were incubated in the wells of 48-well plates (Corning) with or without 0.1–10 μM of S100A8, S100A9, and S100A8/A9 in the presence of 100 μg/ml polymyxin B sulfate (ICN Pharmaceuticals, Costa Mesa, CA, USA). Culture supernatants were harvested 24 hours later and stored at -30°C until the cytokine assay. Because the S100 preparations contained as much as 0.722 ng of endotoxin, we determined whether TNF-α induction in monocytes stimulated by 1.0 ng/ml lipopolysaccharide (LPS; Sigma, St. Louis, MO, USA) was blocked by 100 μg/ml polymyxin B.

Monocytes were incubated at a density of 1 × 10⁶ cells per ml in culture medium with 10% FCS for 7 days with or without 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) or 1 ng/ml interferon gamma (IFN-γ). The in vitro-differentiated macrophages were stimulated for 24 hours with 2 μM S100A8/A9 in the presence of polymyxin B, and culture supernatants were measured for TNF-α concentrations.

Recombinant human endogenous secretory RAGE (esRAGE) was prepared as described previously 25. Briefly, COS-7 cells stably were transformed with pCI-neo (Promega, Madison, WI, USA) carrying the esRAGE cDNA. The esRAGE protein was purified from conditioned media of the transfectant with an AKTA purifier system using HiTrap-Heparin column, RESOURCE S column, and HiTrap desalting column (Amersham Bioscience), sequentially. The purified material largely consisted of the 50-kDa esRAGE, as evidenced by immunoreactivity with the antibody against the peptide unique to the C truncated-type RAGE 25.

To examine the possibility of CD36 or RAGE as a putative receptor for S100A8/A9 on monocytes, the monocyte suspensions, at a density of 1 × 10⁶ cells per ml in culture medium with 10% FCS in 48-well plates, were stimulated for 24 hours by 10 μM S100A8/A9 with or without 2% vol/vol goat anti-RAGE polyclonal Ab (Chemicon International, Temecula, CA, USA) or control goat serum (Chemicon International), 25 μg/ml of esRAGE or human serum albumin (Sigma), and 20 μg/ml of mouse IgG2a/κ anti-CD36 mAb (NeoMarkers, Fremont, CA, USA) or mouse isotype-matched control mAb (NeoMarkers) in the presence of polymyxin B. Culture supernatants were measured for cytokine concentrations.

To examine the involvement of MAPK and NF-κB activation in S100A8/A9-stimulated monocyte cytokine production, monocytes, at a density of 1 × 10⁶ cells per ml in culture medium with 10% FCS in 48-well plates, were preincubated with 1 μM of the cell-permeable MAPK inhibitors PD98059, SB202190, and SB203580 and the negative control SB202474 (Calbiochem, San Diego, CA, USA), or with or without 1 μM of the inhibitors of NF-κB activation, 1-pyrrolidinecarbodithioic acid (PDTC; Calbiochem), N^α-tosyl-phenylalanylchloromethylketone (TPCK; Affiniti Research Products, Mamhead Castle, UK), and BAY 11-7082 (Calbiochem) for 2 hours and were then stimulated for 24 hours with or without 0.1–10 μM S100A8/A9 in the presence of polymyxin B. Culture supernatants were measured for cytokine concentrations.

To detect the activation of p38 MAPK by S100A8/A9, monocytes, at a density of 1 × 10⁶ cells per ml in culture medium with 10% FCS in 48-well plates, were stimulated for 30 minutes with 10 μM S100A8/A9 and were immediately placed in the lysing buffer (Passive Lysis Buffer; Promega). The activation of p38 MAPK in cell lysates was determined using ELISA kits for p38 MAPK protein and p38 MAPK protein phosphorylated on threonine 180/tyrosine 182 (pThr¹⁸⁰/pTyr¹⁸²) (Sigma) according to the manufacturer's instructions.

To examine the effects of S100A8/A9 on NF-κB activation, monocytes, at a density of 1 × 10⁶ cells per ml in culture medium with 1% FCS in polypropylene tubes (Becton, Dickinson and Company), were stimulated with or without 0.1–10 μM S100A8/A9 in the presence of polymyxin B. Cells were collected 30 minutes later, and nuclear proteins were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's instructions. In the experiments of p38 MAPK inhibition, monocytes were preincubated with the MAPK inhibitors (PD98059, SB202190, and SB203580) and the control SB202474, then stimulated for 30 minutes by 10 μM S100A8/A9, and nuclear proteins were prepared. The nuclear protein content was measured by the Lowry method using a DC protein assay kit (Bio-Rad). Electrophoretic mobility shift assay (EMSA) was performed using the Nushift NF-κB p65 kit (Active Motif) according to the manufacturer's instructions. Nuclear extracts were incubated with [α-³²P] dATP (Amersham, Little Chalfont, UK)-labeled double-stranded oligonucleotide probe in binding buffer for 20 minutes. Samples and positive controls (nuclear proteins prepared from monocytes stimulated with 100 ng/ml LPS) were electrophoresed on 5% polyacrylamide gel, followed by autoradiography. To verify the specificity of NF-κB protein binding, competition and supershift analysis was performed by adding an excess of unlabeled competitor or mutant oligonucleotides and rabbit polyclonal anti-NF-κB p65 Ab (Active Motif) to the incubation on ice for 20 minutes before the binding reaction.

Samples with values below the detection limit for the assay were regarded as negative and assigned a value of 0. Data were expressed as the mean value ± standard error of the number of samples evaluated. The statistical significance of differences between two groups was determined by the Mann-Whitney U test or the Wilcoxon signed rank test; p values less than 0.05 were considered significant. The correlation coefficient was obtained by the Spearman rank correlation test.

Concentrations of S100A8, S100A9, and the S100A8/A9 heterodimer in paired serum and SF samples obtained from 17 patients with RA and 17 patients with OA were compared by ELISA. The levels of S100A8, S100A9, and S100A8/A9 in serum and SF were all significantly increased in patients with RA (20.4 ± 7.8 ng/ml, 2.9 ± 0.3 ng/ml, and 38.9 ± 6.0 mg/ml in serum and 65.3 ± 23.4 ng/ml, 27.9 ± 4.5 ng/ml, and 54.8 ± 7.2 μg/ml in SF, respectively) as compared with patients with OA (7.0 ± 3.0 ng/ml, 0.9 ± 0.1 ng/ml, and 16.8 ± 4.8 μg/ml in serum and 5.1 ± 2.2 ng/ml, 3.6 ± 0.4 ng/ml, and 7.3 ± 4.5 μg/ml in SF, respectively) (Figure 1). The amounts of S100A8/A9 heterodimers detected by ELISA were approximately 1,000-fold greater than those of S100A8 and S100A9 homodimers. S100A8/A9 was present at higher concentrations in SF than in serum in patients with RA, but not in patients with OA, and the serum levels correlated positively with serum CRP levels in patients with RA (r = 0.802, p < 0.0001). These results suggest that S100A8 and S100A9 proteins may be secreted mainly as the heterodimer from inflammatory cell infiltrates, such as neutrophils and monocytes/macrophages, in the joints of patients with active RA; however, these proteins are thought to be secreted also as the homodimer, because the expression pattern of S100A8 and S100A9 has been shown to differ in vivo, depending on cell types, cell differentiation, and inflammatory conditions 62226.

S100A8/A9 proteins are expressed by infiltrating tissue macrophages during inflammation but not by resident tissue macrophages 23. To determine S100A8/A9 expression at the site of macrophage infiltration, the proliferative ST samples from three patients with RA were analyzed by two-color immunofluorescence labeling with anti-S100A8/A9 Ab and anti-CD68 Ab. Figure 2 shows representative staining patterns of the S100A8/A9 protein and the CD68 antigen in the ST. These tissues were characterized by the extensive infiltration of CD68-expressing macrophages. Colocalization analysis revealed that the heterodimeric S100A8/A9 was highly expressed by CD68⁺ macrophages, in particular the cells localized to the lining layer. However, S100A8/A9 staining was negligible in CD68-negative cells in the sublining layer, including lymphocytes and vascular endothelial cells. Immunostaining of the S100A8 and S100A9 homodimers shows similar staining patterns, but the intensity and number of S100A8 and S100A9 staining were less prominent (data not shown). These results indicate that S100A8 and S100A9 are expressed predominantly in the form of a heterodimeric complex by highly activated macrophages in RA ST.

To confirm the local production of S100A8/A9 proteins at the site of chronic inflammation in RA, isolated cells from ST samples of nine patients with RA and six patients with OA were incubated for 72 hours without any stimulation and culture supernatants were measured for the heterodimer by ELISA. As shown in Figure 3, ST cells from patients with RA spontaneously secreted larger amounts of S100A8/A9 proteins (1.20 ± 0.25 μg/ml) than did the cells from patients with OA (0.45 ± 0.17 μg/ml). Lactate dehydrogenase activity in these culture supernatants was undetectable or detectable only at negligible levels, indicating that S100A8/A9 is released as a consequence of active secretion but not of cellular injury.

Although the cellular composition of ST cells was not determined in this study, we had previously measured the frequencies of nonspecific esterase-staining macrophages and CD3-positive T cells in the ST cell population isolated in the same manner and found that RA ST samples consisted of 31.7 ± 5.3% (mean ± SD) macrophages and 44.7 ± 6.6% T cells (n = 7) and that OA ST samples consisted of 30.3 ± 4.9% macrophages and 28.8 ± 3.3% T cells (n = 4) (unpublished data). Additionally, we could not detect significant neutrophil contamination (≤1%) in two of the studied ST samples. Therefore, it would seem that the increased S100A8/A9 secretion from RA ST cells may be due to the presence of in vivo-activated macrophages in the culture. This notion is likely consistent with the fact that tissue macrophages are more activated in RA than in OA 1516.

Purified blood monocytes were stimulated for 24 hours by diluted concentrations (0.1–10 μM) of recombinant S100A8 and S100A9 proteins in the presence of 100 μg/ml polymyxin B, and culture supernatants were measured for cytokine concentrations by immunoassay. Polymyxin B was able to effectively block TNF-α production by 1 ng/ml LPS (1,259 ± 125 pg/ml versus 36 ± 4 pg/ml; n = 3), but TNF-α production by 10 μM S100A8/A9 decreased from 1,874 ± 51 pg/ml to 955 ± 40 pg/ml with polymyxin B. The results indicate that endotoxin contamination in the S100 proteins was not completely neutralized by polymyxin B and that low levels of endotoxin act in synergy with S100 proteins.

As shown in Figure 4, S100A9 and S100A8/A9 induced the production of TNF-α, IL-1-β, IL-6, and IL-8, but not of IL-10 and IL-12 p70, in a dose-dependent manner; all values for the complex were lower than those for the A9 homodimer, although not statistically significant. In contrast, S100A8 failed to induce significant levels of cytokine production. It is thus likely that the S100A9 subunit in the heterodimer is critical in activating the proinflammatory signaling.

Next, in vitro-differentiated macrophages, prepared by incubating monocytes for 7 days with or without GM-CSF or IFN-γ, were stimulated for 24 hours by 2 μM S100A8/A9 (50 μg/ml; equivalent to approximately the mean concentration in RA SF), and TNF-α production was determined. As shown in Figure 5, these macrophages, particularly when treated with cytokines, could respond well to S100A8/A9 by producing TNF-α. These results suggest that the secreted S100A8/A9 heterodimer may amplify the local expression of proinflammatory cytokines in joints of patients with RA.

To examine whether S100A8/A9 used RAGE and CD36 as signal transducing receptors on monocytes, monocytes were stimulated for 24 hours by 10 μM S100A8/A9 with or without anti-RAGE Ab, esRAGE (the splice variant lacking the transmembrane-spanning domain), and anti-CD36 Ab, and culture supernatants were measured for cytokine concentrations by immunoassay. The S100A8/A9-stimulated cytokine response was not significantly reduced by blockade of the RAGE or CD36 receptor (percentage of inhibition, < 30%). Therefore, neither RAGE nor CD36 appears to be relevant to S100A8/A9 stimulation in monocytes.