Upregulation of tumor necrosis factor receptor-associated factor 6 correlated with synovitis severity in rheumatoid arthritis

Abstract

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory disease leading to joint destruction and disability. Focal bone erosion is due to excess bone resorption of osteoclasts. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the critical mediators both in inflammatory signal pathway and differentiation and resorption activity of osteoclasts. Here we aimed to investigate TRAF6 expression in RA synovium and its correlation with histological synovitis severity and radiological joint destruction in RA.

Methods: Synovitis score was determined in needle biopsied synovium from 44 patients with active RA. Synovium from nine patients with osteoarthritis (OA) and seven with orthopedic arthropathies (Orth.A) were enrolled as "less inflamed" disease controls. Serial sections were stained immunohistochemically for TRAF6 as well as CD68 (macrophage), CD3 (T cell), CD20 (B cell), CD38 (plasmocyte), CD79a (B lineage cells from pre-B cell to plasmocyte stage), and CD34 (endothelial cell). Double immunofluorescence staining of TRAF6 and CD68 were tested. Densities of positive staining cells were determined and correlated with histological disease activity (synovitis score) and radiographic joint destruction (Sharp score).

Results: TRAF6 expression was found in the intimal and subintimal area of RA synovium, with intense staining found in the endochylema and nucleus of intimal synoviocytes and subintimal inflammatory cells. Double immunofluorescence staining showed TRAF6 was expressed in most of the intimal cells and obviously expressed in CD68+ cells and some other CD68- cells in subintimal area. Synovial TRAF6 was significantly over-expressed in the RA group compared with the OA and Orth.A group (2.53 ± 0.94 vs. 0.72 ± 0.44 and 0.71 ± 0.49, P < 0.0001). Synovial TRAF6 expression in RA correlated significantly with synovitis score (r = 0.412, P = 0.006), as well as the inflammatory cell infiltration (r = 0.367, P = 0.014). Significant correlation was detected between synovial TRAF6 expression and intimal CD68+ cells, as well as the cell density of subintimal CD68+ cells, CD3+ cells, CD20+ cells, CD38+ cells, and CD79a+ cells (all P < 0.05).

Conclusions: Elevated synovial TRAF6 expression correlated with synovitis severity and CD68+ cell density in RA. It is, therefore, hypothesized that synovial TRAF6 is involved in the pathogenesis of synovial inflammation and osteoclast differentiation in RA.

Figure 1

Representative immunohistochemical findings of synovial TRAF6 expression. A, Mild synovial TRAF6 expression in an OA patient; B, Intensive synovial TRAF6 expression in a RA patient; C, Expression of TRAF6 in intimal layer of the synovium in a RA patient. The arrows point to TRAF6+ synoviocytes located at intimal layer of the synovium; D, Expression of TRAF6 in subintimal layer of the synovium in a RA patient. The arrows point to TRAF6+ plasmocytes located at the subintimal layer of the synovium. (Anti-TRAF6 immunostaining, original magnification: A and B ×100, C and D ×400. OA, osteoarthritis; RA, rheumatoid arthritis; TRAF6, Tumor necrosis factor receptor-associated factor 6).

Figure 2

Representative confocal microscopic images showed TRAF6 and CD68 by indirect double immunofluorescence staining in RA synovium. A, original magnification, ×400; B, original magnification ×1,200. (a, TRAF6 (red); b, CD68 (green); c, DAPI (blue); d, merged a, b with c. The white arrows point to intimal and subintimal CD68/TRAF6 double+ cells. DAPI, diamidino-phenyl-indole; RA, rheumatoid arthritis; TRAF6, Tumor necrosis factor receptor-associated factor 6).

Figure 3

Synovial expression of TRAF6 in RA, OA and Orth.A. Scatterplots showing synovial TRAF6 expression scores in RA, OA and Orth.A patients as measured by immunohistochemistry. Synovial expression of TRAF6 was scored semiquantitatively on a five-point scale [29]. Each circle represents the determination for an individual with open circles indicating RA patients (n = 44), filled circles indicating OA patients (n = 9), and open diamonds indicating Orth.A patients (n = 7). Horizontal lines show the median for each population examined. The P-values were calculated using the Kruskal-Wallis one way analysis. (OA, osteoarthritis; Orth.A, orthopedic arthropathies; RA, rheumatoid arthritis; TRAF6, Tumor necrosis factor receptor-associated factor 6).

Figure 4

Spearman's rank correlation analysis for synovial TRAF6 expression and synovitis score in RA. A, Correlation between synovial TRAF6 expression and synovitis score; B, Correlation between synovial TRAF6 expression and hyperplasia of intima; C, Correlation between synovial TRAF6 expression and cellular density of synovial stroma; D, Correlation between synovial TRAF6 expression and inflammatory cell infiltration. (RA, rheumatoid arthritis; TRAF6, Tumor necrosis factor receptor-associated factor 6; n = 44).