Amelioration of collagen-induced arthritis by thrombin inhibition

Abstract

The deleterious role of fibrin deposition in arthritic joints prompted us to explore the effect of the thrombin inhibition on the course of collagen-induced arthritis (CIA) in the mouse. CIA was induced in male DBA/1J mice using native chicken type II collagen. The thrombin inhibitor polyethyleneglycol-hirudin (PEG-hirudin) was given for 16 days, starting 20 days after the first immunization (preventive treatment) or at the onset of clinical signs of arthritis (curative treatment). All the mice treated with PEG-hirudin had a significantly prolonged clotting time compared with control mice. PEG-hirudin, administered in a preventive way, led to significantly reduced incidence and severity of CIA during most of the treatment period, as assessed by clinical scoring. Accordingly, histological features showed a significant diminution of synovial hyperplasia in PEG-hirudin-treated mice compared with untreated mice. There was also a significant downmodulation of the synovial proinflammatory IL-1beta and IL-12p35 cytokine mRNAs in treated mice. Intra-articular fibrin, evaluated by immunohistochemistry, was significantly reduced in treated mice compared with control mice and correlated with both clinical and histological scorings. Most importantly, once arthritis was established, PEG-hirudin also showed a curative effect. In conclusion, PEG-hirudin can both prevent the onset of CIA in a dose-dependent manner and ameliorate established arthritis, suggesting that thrombin inhibition may offer a new therapeutic approach in arthritis.

Figure 1

Histologies and immunohistologies of joints from control and PEG-hirudin–treated mice with CIA. (a and c) Safranin-O–stained sections of control and PEG-hirudin–treated knee joints at day 36 after immunization. Note the size difference of synovial membrane (Sy), which is thicker in the control arthritic mice (a) than in the PEG-hirudin–treated mice (c). Red proteoglycan staining of the articular cartilage matrix (Ca) is decreased in the control group. (b and d) Fibrin immunostaining of adjacent sections using a rabbit anti-murine fibrin(ogen) Ab. Brown color indicates positivity. (e and g) Safranin-O–stained sections of control and PEG-hirudin–treated paw joints at day 36 after arthritis induction. Note the difference in thickness of the synovial membrane, which is thicker in the control arthritic mice (e) than in the PEG-hirudin–treated one. (f and h) Fibrin immunostaining of adjacent sections.

Figure 2

Development of CIA in PEG-hirudin–treated mice. (a–c) From day 20 after type II collagen immunization, test mice were treated daily with PEG-hirudin (subcutaneous injection at 1 mg/kg/day, n = 13). Placebo mice received PBS (n = 13). Mice were observed daily for development of arthritis. (a) Percentage of mice that developed arthritis. (b) Arthritis severity. Mean arthritic index + SEM is shown. (c) Number of arthritic paws (mean + SEM). Dose-response effect of PEG-hirudin on incidence (d) and severity (e). From day 20 after immunization, test mice were treated daily subcutaneously with PEG-hirudin (0.1 mg/kg or 1 mg/kg; n = 13 in each group). Placebo mice received PBS only (n = 13). Mice groups were compared by statistical analysis using the nonparametric Wilcoxon/Kruskal-Wallis tests (rank sums). (a) P < 0.05 from day 24 to day 36. (b) P < 0.01 from day 24–27 and P < 0.05 until day 36. (c) P < 0.05 from day 24 to day 36. (d) P < 0.02 from day 21 until day 27 for the dose of 0.1 mg/kg. P < 0.02 from day 21 until day 28 and P < 0.05 from day 28–31 for the dose of 1 mg/kg. (e) P < 0.02 from day 21 until day 27 for the dose of 0.1 mg/kg and until day 32 for the dose of 1 mg/kg.

Figure 3

PEG-hirudin treatment of established arthritis. Test mice (n = 26) were treated subcutaneously daily with 1 mg/kg of PEG-hirudin from the first day of clinical arthritis for 16 days. Placebo mice (n = 28) received PBS. Groups of animals were compared by statistical analysis using the nonparametric Wilcoxon/Kruskal-Wallis tests (rank sums). (a) ^AP < 0.05 at day 2, 3, 4, 15, and 16. (b) ^AP < 0.05 at day 15 and 16.

Figure 4

Histological features of arthritic knee joints. Knee histologies from control (n = 13) and PEG-hirudin–treated (n = 13) mice were scored for cartilage damage (a), inflammatory cells (b), and synovial thickness (c) using an arbitrary four-point (0–3) scale. Fibrin deposition (d), as evidenced by fibrin immunohistochemistry, was scored in the synovial membrane using an arbitrary seven-point (0–6) scale. The results are expressed as mean + SEM. Statistical significance was tested by Wilcoxon/Kruskal-Wallis tests (rank sums). ^AP < 0.05 was considered significant.

Figure 5

Correlations between fibrin staining and clinical and histological scoring. Fibrin deposition, as scored in Figure 4 was correlated to clinical score of paws (a), plasma TAT levels (b), inflammatory cells (c), synovial hyperplasia (d), and cartilage damage (e) at day 36 after immunization. P < 0.05 was considered significant.

Figure 6

Synovial cytokine mRNA levels in CIA knee joints from untreated or PEG-hirudin–treated mice. (a) Total RNA was prepared from knee joints of nonimmunized, nonarthritic mice (normal knee), or from arthritic CIA knee joints of untreated or PEG-hirudin–treated mice (1 mg/kg/day for 16 days). Expression of different cytokine mRNA was analyzed using RNase protection assay (IL-1Ra, IL-1–receptor antagonist; MIF, macrophage migration inhibitory factor). (b) PhosphorImager analysis was performed to quantify mRNA levels of each of the studied genes. Comparison of mRNA levels is done after standardization with GAPDH mRNA and expressed in arbitrary units. The results are expressed as mean + SEM (three mice per group). ^AP < 0.05; ^BP < 0.01.

Figure 7

Synovial PAR-1 mRNA levels in CIA knee joints from untreated or PEG-hirudin–treated mice. Total RNA was prepared from dissected knee joints of nonimmunized, nonarthritic mice (naive knee) or from arthritic CIA knee joints of untreated (control CIA) or PEG-hirudin–treated mice (1 mg/kg/day for 16 days). PAR-1 mRNA was studied using RNase protection assay and PhosphorImager analysis. Comparison of mRNA levels is done after standardization with GAPDH mRNA. The results are expressed as percentage of control CIA mice (mean + SEM, three to four mice per group). ^AP < 0.05.