Interleukin 6 is required for the development of collagen-induced arthritis

Abstract

Interleukin-6 (IL-6) is overproduced in the joints of patients with rheumatoid arthritis (RA) and, based on its multiple stimulatory effects on cells of the immune system and on vascular endothelia, osteoclasts, and synovial fibroblasts, is believed to participate in the development and clinical manifestations of this disease. In this study we have analysed the effect of ablating cytokine production in two mouse models of arthritis: collagen-induced arthritis (CIA) in DBA/1J mice and the inflammatory polyarthritis of tumor necrosis factor alpha (TNF-alpha) transgenic mice. IL-6 was ablated by intercrossing an IL-6 null mutation into both arthritis-susceptible genetic backgrounds and disease development was monitored by measuring clinical, histological, and biochemical parameters. Two opposite responses were observed; while arthritis in TNF-alpha transgenic mice was not affected by inactivation of the IL-6 gene, DBA/1J, IL-6(-/-) mice were completely protected from CIA, accompanied by a reduced antibody response to type II collagen and the absence of inflammatory cells and tissue damage in knee joints. These results are discussed in the light of the present knowledge of cytokine networks in chronic inflammatory disorders and suggest that IL-6 receptor antagonists might be beneficial for the treatment of RA.

Figure 1

Serum levels of IL-6 in DBA/1J mice with CIA correlated with the arthritis index. Type II collagen immunized mice were bled 6 wk after CII immunization. IL-6 activity was measured by hybridoma growth assay and the arthritis index evaluated as described in Materials and Methods. Results were analyzed using the Spearman correlation coefficient. Rs = 0.694; P = 0.008.

Figure 2

Effects of antibody treatment on arthritis index of CII-immunized DBA/1J mice. (A) Mice (n = 9 for each group) were treated once per week with 1 mg/mouse (white symbols) or 0.5 mg/mouse (black symbols) of either anti–IL-6Rα antibody 15A7 (circles), anti–dinitrophenyl hapten antibody LO-DNP (triangles), or left untreated (squares). *P <0.05 either 15A7 or LO-DNP versus none. (B) Mice were treated with 1 mg/ wk/mouse of either anti–IL6-Rα antibody 15A7 (circles; n = 9), rat total IgG antibodies (diamonds; n = 8), or left untreated (squares; n = 8). *P < 0.02 15A7 versus none; ^# P <0.04 total IgG versus none. Results were reported as mean ± SD. The weeks after the first immunization with CII and the duration of antibody treatment are indicated on the abscissa.

Figure 3

IL-6 is absolutely required for the development of CIA. DIL-6^+/+ (squares; n = 17) and DIL-6^−/− (diamonds; n = 16) mice were observed for arthritis lesions and the percentage of mice that developed CIA is reported. The figure represents two independent experiments. Weeks after the first immunization with CII are indicated.

Figure 4

DIL-6^−/− mice do not show any histological lesion of the joints. Representative examples of six knee joints for each genotype are shown: DIL-6^−/− (A) and DIL-6^+/+ (B). In A no signs of inflammatory lesions are detectable. In B severe signs of inflammatory process are depicted; the pannus completely infiltrated the articular cartilage (arrowheads); severe signs of cartilage and bone destruction and mononuclear cells infiltration are shown. Mice were killed 7 wk after first immunization with CII. Paraffin sections were stained with Villanueva staining. T, Tibia; F, femur. Bars: 50 μm.

Figure 5

DIL-6^−/− mice have abnormal humoral response to type II collagen. DIL-6^+/+ (black bars; n = 9) and DIL-6^−/− (white bars; n = 8) mice were bled 10 wk after the first immunization with CII, and levels of anti-CII antibodies were measured by ELISA. (A) Total IgG anti-CII antibodies were reported as mean ± SD (*P <0.02). (B) Isotype percentages of total IgG antibodies anti-CII were reported as mean ± SD. IgG isotypes are indicated.

Figure 6

The lack of IL-6 activity does not influence the onset and the development of arthritis in TNF-α transgenic mice. TIL-6^−/− (squares; n = 16) and TIL-6^+/+ (diamonds; n = 11) mice were scored for arthritic lesions as described in Materials and Methods. Arthritis index is expressed as mean values ± SD. The differences were not statistically significant.