Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis

Abstract

Two distinct IL-18 neutralizing strategies, i.e. a rabbit polyclonal anti-mouse IL-18 IgG and a recombinant human IL-18 binding protein (rhIL-18BP), were used to treat collagen-induced-arthritic DBA/1 mice after clinical onset of disease. The therapeutic efficacy of neutralizing endogenous IL-18 was assessed using different pathological parameters of disease progression. The clinical severity in mice undergoing collagen-induced arthritis was significantly reduced after treatment with both IL-18 neutralizing agents compared to placebo treated mice. Attenuation of the disease was associated with reduced cartilage erosion evident on histology. The decreased cartilage degradation was further documented by a significant reduction in the levels of circulating cartilage oligomeric matrix protein (an indicator of cartilage turnover). Both strategies efficiently slowed disease progression, but only anti-IL-18 IgG treatment significantly decreased an established synovitis. Serum levels of IL-6 were significantly reduced with both neutralizing strategies. In vitro, neutralizing IL-18 resulted in a significant inhibition of TNF-alpha, IL-6, and IFN-gamma secretion by macrophages. These results demonstrate that neutralizing endogenous IL-18 is therapeutically efficacious in the murine model of collagen-induced arthritis. IL-18 neutralizing antibody or rhIL-18BP could therefore represent new disease-modifying anti-rheumatic drugs that warrant testing in clinical trials in patients with rheumatoid arthritis.

Figure 1

Neutralization of endogenous IL-18 decreases disease severity in CIA mice. (a and b) Changes in clinical scores over time in DBA/1 mice with type II CIA. CIA mice were treated intraperitoneally when the first clinical signs of arthritis appeared with: (a) control IgG (2 mg/mouse) (squares), or anti–mIL-18 IgG (2 mg/mouse) (triangles) (n = 9, for each dose); and (b) with saline (squares) (n = 16) or rhIL-18BP: 0.25 mg/kg (circles), 0.5 mg/kg (diamonds) (n = 7, for each dose), 1 mg/kg (inverted triangles), and 3 mg/kg (triangles)(n = 16, for each dose). Disease development was monitored as described in Methods. (c and d) The same results are expressed as the mean area under the curve (AUC) ± SEM of the clinical scores from the first to the last day of the experiment after treatment with anti–IL-18 IgG (c) and rhIL-18BP (d). *P < 0.05, **P < 0.001, ***P < 0.0001, treated versus control groups.

Figure 2

IL-18 neutralization decreases inflammation and cartilage degradation. At the end of the experiment, the paw that first developed arthritis was dissected away, fixed, and processed as described in Methods. (a–c) Safranin O staining. (d–f) Hematoxylin and eosin staining. (a and d) Normal mouse joint. Joint from control CIA mouse treated with 2 mg control IgG (b) and with saline (e) showing severe arthritis with cartilage destruction and proteoglycan depletion, with numerous infiltrating cells in the inflamed synovium and synovial space. (c and f) Joints from mouse treated with 2 mg of anti–mIL-18 IgG (c) or with 3 mg/kg rhIL-18BP (f). The cartilage appears well protected despite the presence of inflammatory cells. Original magnification: ×100 (a–c) and ×200 (d–f).

Figure 3

Neutralization of endogenous IL-18 decreases cartilage destruction in CIA mice. (a) Erosion scores of arthritic joints after treatment with 2 mg/mouse of control IgG (squares), anti–IL-18 IgG (triangles), and 0 mg/kg (inverted triangles), 0.25 mg/kg (diamonds), 0.5 mg/kg (circles), 1 mg/kg (open squares), and 3 mg/kg (triangles)of rhIL-18BP, as indicated. (b and c) Quantification of serum levels of COMP, a marker of cartilage turnover, after treatment with 2 mg of normal rabbit IgG (squares) or anti–mIL-18 IgG (triangles) (b), and with saline (0 rhIL-18BP)(squares) or with 1 mg/kg (triangles) and 3 mg/kg (inverted triangles) rhIL-18BP (c). *P < 0.05, **P = 0.0023, ***P = 0.0006, treated versus control groups.

Figure 4

Neutralization of IL-18 decreases paw swelling. Progression of swelling was followed on the first paw that showed clinical signs of disease by measuring paw swelling using precision calipers. Results are expressed as AUC ± SEM, after treatment with control IgG (n = 9) or anti–IL-18 IgG (n = 9) and saline (n = 11), or rhIL-18BP at 0.25 mg/kg (n = 7), 0.5 mg/kg (n = 7), 1 mg/kg (n = 12), and 3 mg/kg (n = 12). *P ≤ 0.05, ***P < 0.0001, treated versus control groups.

Figure 5

Neutralization of endogenous IL-18 decreases circulating levels of IL-6. (a) IL-6 bioactivity present in serum of arthritic mice treated with either control IgG or anti–IL-18 IgG (n = 9). (b) IL-6 levels measured by ELISA in the serum of arthritic mice treated with either saline or rhIL-18BP (n = 10). *P < 0.05, **P < 0.01, treated versus control groups.

Figure 6

rhIL-18BP regulates IL-18–induced TNF-α, IL-6, and IFN-γ production by peritoneal macrophages. Macrophages from DBA/1 mice were enriched by plastic adherence and stimulated with 200 ng/ml IL-18 (open bars), a combination of IL-12 (100 ng/ml) and IL-18 (200 ng/ml), or cultured with medium alone (black bars), in the absence or presence of rhIL-18BP (1 μg/ml). Cytokine levels in 24-hour culture supernatants were measured by ELISA. Data are expressed as mean ± SEM of triplicate cultures.