Prevention of collagen-induced arthritis in mice by a polyphenolic fraction from green tea

Abstract

Identification of common dietary substances capable of affording protection or modulating the onset and severity of arthritis may have important human health implications. An antioxidant-rich polyphenolic fraction isolated from green tea (green tea polyphenols, GTPs) has been shown to possess anti-inflammatory and anticarcinogenic properties in experimental animals. In this study we determined the effect of oral consumption of GTP on collagen-induced arthritis in mice. In three independent experiments mice given GTP in water exhibited significantly reduced incidence of arthritis (33% to 50%) as compared with mice not given GTP in water (84% to 100%). The arthritis index also was significantly lower in GTP-fed animals. Western blot analysis showed a marked reduction in the expression of inflammatory mediators such as cyclooxygenase 2, IFN-gamma, and tumor necrosis factor alpha in arthritic joints of GTP-fed mice. Histologic and immunohistochemical analysis of the arthritic joints in GTP-fed mice demonstrated only marginal joint infiltration by IFN-gamma and tumor necrosis factor alpha-producing cells as opposed to massive cellular infiltration and fully developed pannus in arthritic joints of non-GTP-fed mice. The neutral endopeptidase activity was approximately 7-fold higher in arthritic joints of non-GTP-fed mice in comparison to nonarthritic joints of unimmunized mice whereas it was only 2-fold higher in the arthritic joints of GTP-fed mice. Additionally, total IgG and type II collagen-specific IgG levels were lower in serum and arthritic joints of GTP-fed mice. Taken together our studies suggest that a polyphenolic fraction from green tea that is rich in antioxidants may be useful in the prevention of onset and severity of arthritis.

Figure 1

Western blot analysis of (A) Cox-2, (B) TNF-α, (C) IFN-γ, and (D) IgG expression in arthritic joints of GTP-fed and non-GTP-fed DBA/1 mice. Arthritic joints were dissected free of soft tissue, and the cell-free extracts were prepared and analyzed as described in Materials and Methods. Results clearly show that the expression of Cox-2, TNF-α, IFN-γ, and mouse IgG was higher in the arthritic joints of non-GTP-fed DBA/1 mice and correlates with the clinical severity of the disease. Data from a representative experiment are shown. Similar results were obtained in a repeat Western blotting with samples obtained from a second independent experiment.

Figure 2

Immunohistochemical analysis of TNF-α and IFN-γ expression in arthritic joints of non-GTP-fed and GTP-fed DBA/1 mice. Cryosectioned joints from the non-GTP-fed and GTP-fed DBA/1 mice were used to determine the presence and frequency of (Upper) TNF-α- and (Lower) IFN-γ-producing cells. Arthritic joints of mice in the non-GTP-fed group had a higher number of TNF-α- and IFN-γ-producing cells in comparison to the arthritic joints of mice in the GTP-fed group. These results show that the arthritic joints of non-GTP-fed mice had robust Th1-like activity (IFN-γ, TNF-α producing), which would explain the severity of the disease observed in these mice. In contrast, joints obtained from the GTP-fed mice with mild arthritis showed highly reduced Th1-like activity indicated by the fewer number of TNF-α- and IFN-γ-producing cells. (Magnification: ×400.)

Figure 3

Titers of IgG2a antibodies reactive with CII in the arthritic joints and of non-GTP-fed and GTP-fed DBA/1 mice. Antibody titers were determined by an ELISA method using the mouse IgG isotyping kit (PharMingen) according to the manufacturer’s instructions. Joint extracts were prepared when clinical arthritis was evident, and the samples were run in triplicate for each analysis. Error bars indicate SEM.

Figure 4

Assay of NEP activity in the joints of control, non-GTP-fed, and GTP-fed DBA/1 mice with CIA. NEP activity was determined in the cell-free extracts of the joints prepared as described above. To estimate the basal level of enzyme activity, joints from the same lot of nonimmunized mice were used.