In the present study, the induction of arthritis in transgenic mice, which over-express EC-SOD in synovial tissue, showed suppressed incidence and development of disease. These protective effects were not limited to the inhibition of key pro-inflammatory cytokines, such as IL-1β and TNF-α, but included significant protection of the cartilage and bone.
The importance of maintaining a balance between oxidants and antioxidants in inflammatory diseases has been established by the amelioration of collagen-induced arthritis (CIA) by administration of EC-SOD through gene transfer or by treatment with an SOD mimetic (Salvemini et al., 2001
). The EC-SOD deficient mice had shown much more severity in CIA (Ross et al., 2004
). An imbalance in a disease state may be secondary to enhanced production of oxidants, to the decreased presence of antioxidants, or to a combination of these abnormal conditions. It is possible that decreased levels of EC-SOD in the joints of rheumatoid arthritis patients may contribute to more severe inflammation and tissue destruction (Slot et al., 1986
). Although these studies suggested the importance of EC-SOD in inflammatory arthritis, there were no transgenic researches of EC-SOD in synovial tissue.
In a previous study, EC-SOD transgenic mouse reduced the incidence of tumor formation in the DMBA/TPA two-stage carcinogenesis model (Kim et al., 2005
). Furthermore, engraftment of the EC-SOD transgenic mouse embryonic fibroblast repressed inflammatory cytokines and the CII-specific T cells (Yu et al., 2008
). In this study, the transgenic mice over-expressed EC-SOD in joint tissue and this protein was located in the lining layer of the synovium and endotherial cells in the sub lining. These results mean that EC-SOD transgenic mice could be a candidate for inflammatory arthritis. This hypothesis was tested in transgenic mice by CIA. During the disease progression, EC-SOD transgenic mice showed delayed disease incidence and the suppressed disease severity in CIA. Interestingly, no difference could be found between IL-1β and TNF-α in peripheral blood from both transgenic and wild type mice. But these cytokines were down-regulated in fibroblast-like synoviocyte (FLS) in transgenic mice. In addition, other pro-inflammatory cytokines such as IL-2, IL-4, and IFN-γ were also decreased in FLS. This means the difference in disease severity was accompanied by changes in cytokine production, particularly in the joints. FLS play critical roles in normal embryogenesis and mature joint functioning. In RA, however, FLS take on both a different character and set of roles. The FLS in RA (RA-FLS) can be expended in cell culture over several passages and they escape from contact inhibition. These changes are often referred to as those of a tumor-like transformation since they result in aggressive and invasive behavior of RA-FLS in the adjacent cartilage and bone (Huber et al., 2006
). Activation of FLS by a broad array of soluble factors and cell surface interactions overrides homeostatic function, promoting synthesis of molecules that help mediate joint destruction and inflammation (Mor et al., 2005
). Inflammatory mediators that stimulate FLS include soluble, membrane-associated, and extracellular matrix-associated cytokines, chemokines, growth factors, and bioreactive lipids synthesized by both infiltlating leukocytes and FLS themselves. Several key cytokines that act on FLS such as TNF-α and IL-1β produced by macrophage (Bombara et al., 1993
; Nakahara et al., 2003
), IL-17 and IFN-γ produced by T cells (Tasi et al., 1996
; Kehlen et al., 2003
; Hwang et al., 2004
). Furthermore, in a recent study, the capacity of synovial cells to produce the superoxide anion was probed. A low level of constitutive superoxide production in RA cells was also observed in immortalized chondrocytes and endothelial cells (Moulton et al., 1997
; Ago et al., 2004
) and the superoxide anion in synovial cells was produced via an NADPH oxidase pathway (Chenevier-Gobeaux et al., 2006
). These factors suggest the over-expression of EC-SOD in FLS would provide a better approach to treat RA.
The histological change and the expression of matrix metalloproteinases (MMPs) in FLS from arthritic mice were also analyzed. As shown in these results, tissue damages and bone erosion were not shown in EC-SOD transgenic mice and the levels of MMPs were also depressed in FLS. MMPs are Zn-containing endopeptidases that are involved most prominently in tissue remodeling. Pro-inflammatory cytokines, growth factors and matrix molecules induce the expression of MMPs via transcriptional activation. In one model, patient derived RA-FLS co-implanted with human cartilage plugs under the renal capsule of SCID mice showed histologic evidence of FLS-mediated cartilage erosion. Additionally, this cartilage erosion was inhibited by transfection of constructs that blocked matrix-degrading enzymes such as MMP-1 or cathepsin L (Rutkauskaite et al., 2004
; Schedel et al., 2004
). Thus, histological protection in transgenic mice under in vivo
arthritic condition would be due to the suppression of MMPs by EC-SOD over-expression in FLS.
In conclusion, these data suggest that the over-expression of EC-SOD in FLS contribute to the activation of FLS and protection from joint destruction by depressing the production of the pro-inflammatory cytokine and MMPs. These results provide EC-SOD transgenic mice would be a useful animal model for further inflammatory arthritis research.