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

Abstract

S100A8 and S100A9, two Ca2+-binding proteins of the S100 family, are secreted as a heterodimeric complex (S100A8/A9) from neutrophils and monocytes/macrophages. Serum and synovial fluid levels of S100A8, S100A9, and S100A8/A9 were all higher in patients with rheumatoid arthritis (RA) than in patients with osteoarthritis (OA), with the S100A8/A9 heterodimer being prevalent. By two-color immunofluorescence labeling, S100A8/A9 antigens were found to be expressed mainly by infiltrating CD68+ macrophages in RA synovial tissue (ST). Isolated ST cells from patients with RA spontaneously released larger amounts of S100A8/A9 protein than did the cells from patients with OA. S100A8/A9 complexes, as well as S100A9 homodimers, stimulated the production of proinflammatory cytokines, such as tumor necrosis factor alpha, by purified monocytes and in vitro-differentiated macrophages. S100A8/A9-mediated cytokine production was suppressed significantly by p38 mitogen-activated protein kinase (MAPK) inhibitors and almost completely by nuclear factor kappa B (NF-kappaB) inhibitors. NF-kappaB activation was induced in S100A8/A9-stimulated monocytes, but this activity was not inhibited by p38 MAPK inhibitors. These results indicate that the S100A8/A9 heterodimer, secreted extracellularly from activated tissue macrophages, may amplify proinflammatory cytokine responses through activation of NF-kappaB and p38 MAPK pathways in RA.

Figure 1

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.

Figure 2

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.

Figure 3

Secretion of S100A8 and S100A9 proteins from isolated synovial tissue (ST) cells of rheumatoid arthritis (RA) and osteoarthritis (OA). Freshly isolated ST cells (1 × 10⁶ 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.

Figure 4

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 × 10⁶ 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.

Figure 5

Induction of tumor necrosis factor alpha (TNF-α) production by in vitro-differentiated macrophages after S100A8/A9 stimulation. Monocytes (1 × 10⁶ 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.

Figure 6

Effects of mitogen-activated protein kinase (MAPK) inhibitors on S100A8/A9-mediated cytokine production. Monocytes from healthy individuals (1 × 10⁶ 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.

Figure 7

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 × 10⁶ 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.

Figure 8

Effects of nuclear factor kappa B (NF-κB) inhibitors on S100A8/A9-mediated cytokine production. Monocytes (1 × 10⁶ 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.