Journal: Arthritis Research & Therapy

Article Title: The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis

doi: 10.1186/ar1939

Figure Lengend Snippet: Concentrations of S100A8 and S100A9 proteins in the serum and synovial fluid (SF) of rheumatoid arthritis (RA) and osteoarthritis (OA). Paired serum and SF samples were prepared from patients with RA and patients with OA. Concentrations of the S100A8 and S100A9 homodimer and the S100A8/A9 heterodimer were measured in duplicate by enzyme-linked immunosorbent assay. Values are the mean ± standard error of the mean. n , number of samples tested.

Article Snippet: 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 [ ]; 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.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis

doi: 10.1186/ar1939

Figure Lengend Snippet: Expression of CD68 and the S100A8/A9 heterodimer in the synovial tissue (ST) of rheumatoid arthritis (RA). Cryostat sections of ST samples from patients with RA were incubated with mouse immunoglobulin (Ig) G1 monoclonal antibody (mAb) against the S100A8/A9 complex or control mAb, followed by incubation with rhodamine-conjugated donkey anti-mouse IgG1 mAb, and were then incubated with fluorescein isothiocyanate-conjugated anti-CD68 mAb. Two-color immunofluorescence confocal images were obtained for CD68 and S100A8/A9 expression (green and red staining, respectively). The two images were superimposed; double-positive cells are shown in yellow. No isotype-matched control mAb was observed. Similar staining patterns were obtained in additional analyses with two synovial tissue samples from different patients.

Article Snippet: 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 [ ]; 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.

Techniques: Expressing, Incubation, Control, Immunofluorescence, Staining

Journal: Arthritis Research & Therapy

Article Title: The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis

doi: 10.1186/ar1939

Figure Lengend Snippet: Secretion of S100A8 and S100A9 proteins from isolated synovial tissue (ST) cells of rheumatoid arthritis (RA) and osteoarthritis (OA). Freshly isolated ST cells (1 × 10 6 cells per ml in culture medium with 10% fetal calf serum) were incubated without any stimulation for 72 hours. Culture supernatants were measured in duplicate for the S100A8/A9 heterodimer by enzyme-linked immunosorbent assay. Values are the mean ± standard error of the mean. n , number of samples tested.

Article Snippet: 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 [ ]; 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.

Techniques: Isolation, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis

doi: 10.1186/ar1939

Figure Lengend Snippet: Induction of proinflammatory cytokine production by monocytes after stimulation with S100A8, S100A9, and S100A8/9. Monocytes were purified by negative selection from peripheral blood mononuclear cells of healthy individuals. The monocyte suspensions (1 × 10 6 cells per ml in culture medium with 10% fetal calf serum) were incubated with or without S100A8, S100A9, and S100A8/A9 (0.1–10 μM) in the presence of polymyxin B (100 μg/ml). Culture supernatants were harvested 24 hours later. Cytokine concentrations were measured by cytometric beads array (using anticytokine monoclonal antibody [mAb]-coated beads and phycoerythrin-conjugated anticytokine mAbs). Values are the mean ± standard error of the mean. IL, interleukin; n , number of samples tested; TNF-α, tumor necrosis factor alpha.

Article Snippet: 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 [ ]; 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.

Techniques: Purification, Selection, Incubation

Journal: Arthritis Research & Therapy

Article Title: The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis

doi: 10.1186/ar1939

Figure Lengend Snippet: Induction of tumor necrosis factor alpha (TNF-α) production by in vitro -differentiated macrophages after S100A8/A9 stimulation. Monocytes (1 × 10 6 cells per ml in culture medium with 10% fetal calf serum) were incubated 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 then stimulated for 24 hours with 2 μM S100A8/A9 in the presence of polymyxin B (100 μg/ml). Culture supernatants were measured for TNF-α concentrations by enzyme-linked immunosorbent assay. Values are the mean ± standard error of the mean. n , number of samples tested.

Article Snippet: 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 [ ]; 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.

Techniques: In Vitro, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis

doi: 10.1186/ar1939

Figure Lengend Snippet: Effects of mitogen-activated protein kinase (MAPK) inhibitors on S100A8/A9-mediated cytokine production. Monocytes from healthy individuals (1 × 10 6 cells per ml in culture medium with 10% fetal calf serum) were preincubated for 2 hours in the presence of the MAPK inhibitors PD98059, SB202190, and SB203580 and the negative control SB202474 (1 μM) and were then stimulated for 24 hours with or without S100A8/A9 (0.1–10 μM) in the presence of polymyxin B (100 μg/ml). Culture supernatants were measured for cytokines by cytometric beads array. Values are the mean ± standard error of the mean. IL, interleukin; n , number of samples tested; TNF-α, tumor necrosis factor alpha.

Article Snippet: 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 [ ]; 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.

Techniques: Negative Control

Journal: Arthritis Research & Therapy

Article Title: The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis

doi: 10.1186/ar1939

Figure Lengend Snippet: S100A8/A9-induced nuclear factor kappa B (NF-κB) activation and its independence of p38 mitogen-activated protein kinase (MAPK) activation. (a) Activation of the transcriptional factor NF-κB in S100A8/A9-stimulated monocytes. Monocytes (1 × 10 6 cells per ml in culture medium with 1% fetal calf serum) were stimulated for 30 minutes with or without S100A8/A9 (0.1–10 μM) in the presence of polymyxin B (100 μg/ml). Nuclear proteins were extracted from the cells, and electophoretic mobility shift assay (EMSA) was performed, as described in Materials and methods. Nuclear proteins prepared from monocytes stimulated with 100 ng/ml of lipopolysaccharide were used as positive controls. The specificity of NF-κB protein binding was verified by inhibition and supershift experiments with unlabeled NF-κB consensus or mutant oligonucleotides and anti-NF-κB p65 antibody. (b) Effects of p38 MAPK inhibition on S100A8/A9-induced NF-κB activation. Monocytes, after two-hour pretreatment with or without the MAPK inhibitors (PD98059, SB202190, and SB203580) and the control compound SB202474, were stimulated for 30 minutes with or without S100A8/A9 (10 μM), and nuclear proteins were prepared. The DNA-binding activity of NF-κB was determined by EMSA.

Article Snippet: 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 [ ]; 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.

Techniques: Activation Assay, Mobility Shift, Protein Binding, Inhibition, Mutagenesis, Control, Binding Assay, Activity Assay

Journal: Arthritis Research & Therapy

Article Title: The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis

doi: 10.1186/ar1939

Figure Lengend Snippet: Effects of nuclear factor kappa B (NF-κB) inhibitors on S100A8/A9-mediated cytokine production. Monocytes (1 × 10 6 cells per ml in culture medium with 10% fetal calf serum), after two-hour pretreatment with or without the inhibitors of NF-κB activation (1 μM), 1-pyrrolidinecarbodithioic acid (PDTC), and N α -tosyl-phenylalanylchloromethylketone (TPCK), were stimulated for 24 hours with or without S100A8/A9 (1 or 10 μM) in the presence of polymyxin B. Culture supernatants were measured for tumor necrosis factor alpha (TNF-α) concentrations by enzyme-linked immunosorbent assay. Values are the mean ± standard error of the mean. n , number of samples tested.

Article Snippet: 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 [ ]; 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.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay