Protective effect of eotaxin-2 inhibition in adjuvant-induced arthritis

Abstract

Eotaxin-2 is a potent chemoattractant for eosinophils, basophils and T helper type 2 (Th2) lymphocytes. The eotaxin-2/CCL24 receptor CCR3 is expressed in human brain, skin, endothelium and macrophages. The aim of the current study was to evaluate the protective effect of a monoclonal anti-eotaxin-2 antibody on the development of adjuvant-induced arthritis in rats (AIA). Adjuvant arthritis was induced in Lewis rats by intradermal injection of incomplete Freund's adjuvant +Mycobacterium tuberculosis. Rats were treated by intraperitoneal (i.p.) injection with three monoclonal antibodies against eotaxin-2 (G7, G8, D8) three times per week. Controls were treated with total mouse immunoglobulin G (IgG), methotrexate (MTX) or phosphate-buffered saline (PBS). Arthritis severity was evaluated by measuring ankle swelling, arthritic score, whole animal mobility and body weight. Sample joints were obtained for pathological evaluation and postmortem X-ray of ankle joints was performed to document erosions. Significant inhibition of arthritis was observed in rats treated with anti-eotaxin-2 antibodies compared to those treated with immunoglobulin or PBS. Inhibition was manifest in ankle diameter, arthritic score and mobility score. The antibody marked D8 showed the greatest efficacy. The effect was observed both in animals treated before the appearance of arthritis and in those where treatment was begun after development of joint inflammation. Combined treatment with D8 and MTX caused additional protection. Significant reduction of inflammation in D8-treated animals was also demonstrated in pathological and X-ray examinations. Inhibition of eotaxin-2 by monoclonal antibodies has a significant protective effect in adjuvant arthritis. These results may introduce a novel therapeutic target in rheumatoid arthritis and additional inflammatory joint disorders.

Fig. 1

(a) Percentage of adhesion to fibronectin (FN) of D8 (50 mg)-treated compared to immunoglobulin G (IgG)-treated lymphocytes. D8 inhibits adhesion of human, rat and murine lymphocytes to fibronectin. (b) D8 inhibits adhesion of human, rat and murine lymphocytes to fibronectin D8 inhibits migration of lymphocytes towards vascular endothelial growth factor (VEGF).

Fig. 2

(a) Effect of treatment with anti-eotaxin-2 monoclonal antibodies, immunoglobulin G (IgG) and phosphate-buffered saline (PBS) on the arthritic score (AS) of rats (± standard errors, percentage). Statistically significant differences (P < 0·05) were obtained at every determination, from days 13 to 21, when comparing rats treated with D8 both to rats treated with PBS and to rats treated with IgG. (b) Effect of treatment with anti-eotaxin-2 monoclonal antibodies, IgG and PBS on the mobility score of rats (± standard errors, percentage). Statistically significant differences (P < 0·05) were obtained at every determination, from days 13 to 21, when comparing rats treated with D8 both to rats treated with PBS and to rats treated with IgG. (c) Effect of treatment with anti-eotaxin-2 monoclonal antibodies, IgG and PBS on ankle diameter (± standard errors, percentage). Statistically significant difference (P < 0·05) was obtained between D8 and PBS as of day 19. The difference between D8 and IgG did not reach statistical significance.

Fig. 3

Histological results (a) and representative appearance of joints from rats treated with phosphate-buffered saline (PBS), immunoglobulin G (IgG), D8, G7 and G8 antibodies. (a) Results of histological scoring in rats treated with PBS, IgG, D8, G7 and G8, respectively. An intense inflammatory infiltrate is obvious in the synovial tissue in rats treated with PBS (b) and is absent in rats treated with D8200 µg (c) (haematoxylin and eosin staining).

Fig. 4

Effect of anti-eotaxin-2 treatment on weight of rats (grams) with adjuvant-induced arthritis (AIA) (± standard errors). A statistically significant difference in weight (P < 0·05) between D8 and phosphate-buffered saline (PBS) was obtained on day 17. The difference between D8 and immunoglobulin G (IgG) did not reach statistical significance.

Fig. 5

(a) Effect of D8 anti-eotaxin-2 antibody prevention at three doses (20 µg, 100 µg and 1000 µg) on arthtitic score (AS) in adjuvant-induced arthritis (AIA) (prevention group) (± standard error, percentage). Statistically significant differences (P < 0·05) were obtained at every determination from days 17 to 24 when comparing rats treated with D8100 prevention to rats treated with phosphate-buffered saline (PBS). (b) Effect of treatment with D8 antibody at the appearance of arthritis on arthritic score (± standard error, percentage) (treatment group). Statistically significant differences (P < 0·05) were obtained between rats treated with D8 at a dose of 100 µg at the appearance of arthritis on day 12 as of day 13 and to the end of the experiment.

Fig. 6

Protective effect of D8 (100 µg), methotrexate (MTX) (0·25 mg/kg) and MTX combined with D8100 µg, compared with phosphate-buffered saline (PBS) controls, on the arthritic score in adjuvant-induced arthritis (AIA) (± standard error, percentage). Both D8100 µg and MTX treatment caused a statistically significant reduction of the arthritis score (P < 0·05) compared with PBS as of day 13. By the end of the experiment on day 24, a statistically significant reduction in the arthritis score was observed in rats treated with MTX + D8100 µg compared with rats treated with MTX alone. A significant difference was also observed on day 24 in the mean ankle width between these groups (not shown).

Fig. 7

Post-mortem X-ray analysis of joints from rats treated with phosphate-buffered saline (PBS) (a) and D8 (b). (a) Intense periarticular soft tissue swelling is evident in the PBS-treated rats (star), as well as decalcification and early bony erosion at the ankle joint (arrow head); these findings are not evident in the D8-treated rats (b) (representative figures). Similar results were seen in X-rays of the forefeet (not shown).