Calgizzarin (S100A11): a novel inflammatory mediator associated with disease activity of rheumatoid arthritis

Abstract

Background: Calgizzarin (S100A11) is a member of the S100 protein family that acts in different tumors by regulating a number of biologic functions. Recent data suggest its association with low-grade inflammation in osteoarthritis (OA). The aim of our study is to compare S100A11 expression in the synovial tissues, synovial fluid and serum of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to characterize the potential association between S100A11 and disease activity.

Methods: S100A11 protein expression was detected in synovial tissue from patients with RA (n = 6) and patients with OA (n = 6) by immunohistochemistry and immunofluorescence. Serum and synovial fluid S100A11 levels were measured by ELISA in patients with RA (n = 40) and patients with OA (n = 34). Disease activity scores in 28 joints based on C-reactive protein (DAS28-CRP) were used to assess disease activity. Cytokine content in peripheral blood mononuclear cells (PBMCs), synovial fibroblasts (SFs) and synovial fluid was analysed by ELISA, western blotting or cytometric bead array.

Results: S100A11 expression was significantly up-regulated in the synovial lining and sublining layers (p < 0.01) and vessels (p < 0.05) of patients with RA compared to patients with OA, and was associated with fibroblasts and T cells. S100A11 was significantly increased in synovial fluid (p < 0.0001) but not in serum (p = 0.158) from patients with RA compared to patients with OA when adjusted for age and sex. Synovial fluid S100A11 correlated with DAS28 (r = 0.350, p = 0.027), serum CRP (r = 0.463, p = 0.003), synovial fluid leukocyte count (r = 0.677, p < 0.001), anti-cyclic citrullinated peptide antibodies (anti-CCP) (r = 0.424, p = 0.006) and IL-6 (r = 0.578, p = 0.002) and IL-8 (r = 0.740, p < 0.001) in synovial fluid from patients with RA. PBMCs and SFs isolated from patients with RA synthesized and spontaneously secreted higher levels of S100A11 in comparison with PBMCs and SFs from patients with OA (p = 0.011 and 0.03, respectively). S100A11 stimulated the production of the pro-inflammatory cytokine IL-6 by PBMCs (p < 0.05) and SFs (p < 0.01).

Conclusions: Our data provide the first evidence of S100A11 up-regulation and its association with inflammation and disease activity in patients with RA.

Keywords: Calgizzarin; Disease activity; Inflammation; Rheumatoid arthritis; S100 proteins.

Fig. 1

S100A11 protein in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissue assessed by immunohistochemical (IHC) analysis. Intensive staining for S100A11 was found in the synovial fibroblasts of the lining layer and within the inflammatory cell infiltrates in the RA synovial tissue (A1). The S100A11 expression was negligible or absent in OA synovium (B1). Mouse IgG was used as an isotype control (A2, B2). Representative images of IHC staining are shown at × 200 magnification. For the detailed view the × 400 magnification is shown (n = 6 patients with RA and n = 6 patients with OA)

Fig. 2

Cellular distribution of the S100A11 protein in rheumatoid arthritis synovial tissue. Representative images of immunofluorescence staining (a) for CD68 (macrophages), CD20 (B cells), CD3 (T cells) and vimentin (cells of mesenchymal origin) are shown at × 200 magnification (n = 4). DAPI 4',6-diamidino-2-phenylindole. b Average percentage of S100A11-positive cells presented as mean ± SEM

Fig. 3

S100A11 in serum and synovial fluid and its association with disease activity in rheumatoid arthritis (RA). Levels of S100A11 in the synovial fluid (a) but not in the serum (b) were up-regulated in patients with RA compared to patients with osteoarthritis (OA) when adjusted for age and sex. Levels of S100A11 in synovial fluid correlated with serum C-reactive protein (CRP) (c), disease activity score in 28 joints (DAS28) (d), synovial fluid leukocyte count (e), serum anti-cyclic citrullinated peptide antibodies (anti-CCP) (f), IL-6 (g) and IL-8 (h). The horizontal line represents the median

Fig. 4

Synthesis and release of S100A11 by peripheral blood mononuclear cells (PBMCs) and synovial fibroblasts (SFs). PBMCs and SFs from patients with rheumatoid arthritis (RA) synthesize (a, b) and spontaneously release (c, d) higher levels of S100A11 compared to the cells from patients with osteoarthritis (OA). Representative images of protein levels of S100A11 in PBMCs and SFs isolated from patients with RA (n = 4) and patients with OA (n = 4) by western blot

Fig. 5

Pro-inflammatory role of extracellular S100A11. S100A11 significantly enhances the release of IL-6 (a) and TNF-α (b) in peripheral blood mononuclear cells and IL-6 (c) in synovial fibroblasts (SFs). Levels of TNF-α were not changed in SFs treated with S100A11 protein (d). Protein levels in cell culture supernatants were measured after 24 h. The horizontal line represents the median. Ctrl control, LPS lipopolysaccharide