Reduction of joint inflammation and bone erosion in rat adjuvant arthritis by treatment with interleukin-17 receptor IgG1 Fc fusion protein

Authors


Abstract

Objective

To investigate the role of interleukin-17 (IL-17) in inflammatory arthritis by blockade with an IL-17 receptor/human IgG1 Fc fusion protein (muIL-17R:Fc) in adjuvant-induced arthritis (AIA) in the rat.

Methods

AIA was induced in 39 DA rats with the use of Freund's complete adjuvant. Rats received either 7.3 or 20 mg/kg of muIL-17R:Fc or phosphate buffered saline intraperitoneally every other day from the time of arthritis induction for ∼17 days. Paw volume, arthritis severity, and weight were assessed every 3–4 days. Rats were killed between days 21 and 23 postinduction. Ankles were removed for quantitative radiology and histology and for immunohistochemistry for T cells.

Results

Treatment with muIL-17R:Fc attenuated paw volume in a dose-dependent manner. Both the 7.3 and 20 mg/kg doses of muIL-17R:Fc significantly reduced radiographic scores in the treated rats compared with the controls. The 20 mg/kg dose of muIL-17R:Fc significantly reduced histology scores compared with the controls. T cell numbers were unchanged in the muIL-17R:Fc–treated rats as a function of dose.

Conclusion

In vivo blockade of IL-17 by muIL-17R:Fc treatment attenuated AIA and reduced joint damage, suggesting that IL-17 plays an important role in the inflammation and joint destruction of AIA. IL-17 may be a potential therapeutic target for inflammatory diseases in humans, such as rheumatoid arthritis.

Interleukin-17 (IL-17) is a recently discovered cytokine that is secreted by CD4 T cells (1, 2) and is spontaneously produced by rheumatoid arthritis (RA) synovial membrane cultures (3). High levels of IL-17 have been detected in the synovial fluid of patients with RA (4). IL-17 has been shown to have a wide range of proinflammatory properties in vitro (1–3, 5); it shows synergy with tumor necrosis factor (TNF) for bone resorption (6) and stimulates osteoclast differentiation (7). These in vitro properties have been supported by in vivo studies which demonstrated that IL-17 increased inflammatory cell infiltration of the synovium and cartilage degradation when injected into the knee joint of mice (8, 9). Therefore, IL-17 has properties that could contribute to both proinflammatory immune mechanisms and cartilage and bone damage in inflammatory arthritis.

A murine recombinant soluble IL-17R:Fc fusion protein (muIL-17R:Fc) has previously been shown to inhibit T cell proliferation in vitro and prolong allograft survival time in mice (10). However, there have been no published studies investigating the effects of blocking IL-17 in animal models of inflammatory arthritis or in human RA.

We have previously detected IL-17 messenger RNA (mRNA) in the inguinal lymph nodes and synovium of rats with adjuvant-induced arthritis (AIA) (11). In the inguinal lymph nodes, IL-17 mRNA expression increased early in AIA, before the onset of disease, suggesting an important role in the initiation of AIA. In the synovium, there was an increase in IL-17 expression around the time of arthritis onset. This increase in IL-17 mRNA occurred at the same time as increases in interferon-γ and IL-2 mRNA expression, suggesting that it was part of a synovial T cell–activation process in the early phase of AIA.

The aim of the present study was to define the role of IL-17 in the inflammatory arthritis and joint damage of AIA by investigating the effect of treatment with muIL-17R:Fc on joint swelling, radiographic changes, changes in synovial histologic features, and T cell infiltration.

MATERIALS AND METHODS

Animals. Male dark agouti (DA) rats 4–6 weeks old and weighing ∼140 gm (Institute of Medical and Veterinary Science, Gilles Plains, South Australia, Australia) were housed in a temperature-controlled room (22°C; ±1°C) with a 12-hour alternating cycle of light and dark. Animals were given rat chow (Gordon's Specialty Stockfeeds, Yandera, New South Wales, Australia) and water ad libitum prior to and throughout the experiments. All experiments were approved by the Animal Care and Ethics Committee of the University of New South Wales.

Induction of adjuvant arthritis. Animals were handled 1–2 weeks prior to experiments and every 2–3 days throughout the study; food and fluid intake and body weight were monitored. To induce adjuvant arthritis, rats were anesthetized with ketamine (50 mg/kg intraperitoneally [IP]; Parnell Laboratories, Alexandria, New South Wales, Australia) and xylazine (5 mg/kg IP; Troy Laboratories, Smithfield, New South Wales, Australia) and injected with Freund's complete adjuvant (1 mg of heat-killed and dried Mycobacterium butyricum suspension in paraffin oil and mannide monoleate; Difco, Detroit, MI) intradermally into the base of the tail.

Experimental protocol. Arthritis was induced in a group of 39 male DA rats. Rats received either 7.3 mg/kg (n = 9) or 20 mg/kg (n = 10) of muIL-17R:Fc (kindly donated by Immunex, Seattle, WA) IP every other day from the day of induction of arthritis up to days 16–18. Control rats received an equal volume of sterile phosphate buffered saline (n = 20). Rats were killed on day 21 or on day 23 after adjuvant administration.

Assessment of arthritic damage. Disease progression was monitored from the induction of arthritis (day 0) until the day when the last group of rats was killed. The rats were euthanized with pentobarbital, 60 mg IP (Lethobarb; Virbac, Sydney, New South Wales, Australia). Three indices of arthritis activity were used: joint swelling assessed by paw volume, radiographic changes, and histologic changes. Joint swelling in both ankles was measured every 3–4 days by plethysmometry (Ugo Basile, Comerio, Italy). The animals were killed and the right ankle was removed for radiographic and histologic examination to assess joint damage, as previously described (see ref. 12). Briefly, radiographs were scored for soft tissue swelling, joint space narrowing, and joint damage, each of which was scored on a scale of 0–3 (range 0–9 per joint). Following radiography, paraffin sections were prepared for histologic analysis as described previously (12). Each section was evaluated for periarticular inflammation and pannus formation and scored on a scale of 0–8 (maximum score 16 per section).

Immunohistochemistry for T cells. Paraffin sections of rat ankle joints were stained for T cells as previously described (11). Briefly, sections were stained with polyclonal rabbit anti-human CD3 (1:200; Dako, Carpinteria, CA). Negative controls were incubated with rabbit serum (Gibco, Mulgrave, Victoria, Australia) at the same protein concentration. Stained cells were counted in high-power fields (400× magnification) using a 0.02-mm2 graticule. Ten fields were counted for all samples in a blinded manner. Results were expressed as the number of cells per square millimeter of joint tissue.

Statistical analysis. Data are presented as mean ± SEM. Raw scores for both the left and the right paw volumes were normalized as the percentage of scores from day 0. Differences between means were calculated using repeated-measures analysis of variance. For radiology, histology, and T cell data, differences between means were calculated using one-way analysis of variance. If a significant difference was found (P < 0.05), post hoc analyses were performed on planned comparisons using Fisher's least significant difference multiple comparison tests (NCSS, Kaysville, UT).

RESULTS

Time course of AIA. Paw volume progressively increased in untreated control rats from day 0 (P < 0.05) (Figure 1). All arthritic animals groomed themselves well and maintained their original body weight throughout the course of the disease.

Figure 1.

Percentage change in paw volume from day 0 in rats with adjuvant-induced arthritis (AIA) treated with phosphate buffered saline (control; n = 20) or with 7.3 mg/kg (n = 9) or 20 mg/kg (n = 10) of muIL-17R:Fc fusion protein. * = significantly different from the controls.

Effect of muIL-17R:Fc treatment on joint swelling. Joint swelling in the hind paws was reduced in a dose-dependent manner by IL-17 blockade. The 7.3-mg/kg dosage of muIL-17R:Fc significantly reduced paw volume by 31% in treated rats compared with controls on days 21–23. The 20-mg/kg dosage significantly reduced paw volume on days 13, 17, and 21–23 by 31%, 38%, and 44%, respectively, in treated rats compared with controls (P < 0.05) (Figure 1).

Effect of muIL-17R:Fc treatment on joint damage assessed by radiology. There was a dose-responsive reduction in the radiographic scores of joint damage in treated rats compared with untreated controls. Radiographic scores were significantly reduced in both the 7.3- and the 20-mg/kg muIL-17R:Fc groups compared with controls, by 36% and 44%, respectively (P < 0.05) (Figure 2).

Figure 2.

Radiology scores on the day of killing in rats with adjuvant-induced arthritis treated with phosphate buffered saline (control; n = 20) or with 7.3 mg/kg (n = 9) or 20 mg/kg (n = 10) of muIL-17R:Fc fusion protein. * = significantly different from the controls.

Effect of muIL-17R:Fc treatment on synovial histology. The 20-mg/kg muIL-17R:Fc dosage significantly reduced histology scores by 35% in treated rats compared with controls (P < 0.05) (Figure 3). Although histology scores were lower in the rats treated with 7.3 mg/kg of muIL-17R:Fc compared with controls, this change was not statistically significant (P > 0.05).

Figure 3.

Histology scores on the day of killing in rats with adjuvant-induced arthritis treated with phosphate buffered saline (control; n = 20) or with 7.3 mg/kg (n = 9) or 20 mg/kg (n = 10) of muIL-17R:Fc fusion protein. * = significantly different from the controls.

Effect of muIL-17R:Fc treatment on synovial T cells. Synovial T cell numbers in rats with AIA were not significantly changed by treatment with either 7.3 or 20 mg/kg of muIL-17R:Fc. The mean ± SEM number of cells/mm2 in the controls was 980 ± 110. The value in the 7.3-mg/kg dosage group was 936 ± 181, and the value in the 20-mg/kg dosage group was 1,020 ± 199.

DISCUSSION

In this study, we have shown that muIL-17R:Fc treatment attenuates rat AIA, demonstrating in vivo that IL-17 plays an important role in both the joint inflammation and the joint destruction of inflammatory arthritis. The attenuation of AIA with muIL-17R:Fc treatment, as shown by a reduction in paw volume and radiographic and histologic scores, is consistent with blocking the proinflammatory actions of IL-17. Potential mechanisms of this antiinflammatory activity include suppressed IL-17 induction of a wide array of proinflammatory mediators demonstrated by in vitro experimentation. These functions include stimulation of IL-1β, TNF, IL-6, IL-8, granulocyte colony-stimulating factor, and prostaglandin E2 production by synovial fibroblasts and macrophages (1–3, 5). Our study supports previous in vivo studies of IL-17 function that showed that the exogenous introduction of IL-17 into the knee joint of mice produced inflammatory infiltration of the synovium and cartilage degradation (8, 9). Further studies are required to elucidate the critical time of action of IL-17 blockade, whether during the early prearthritic phase or during the established arthritis phase.

The reduction in joint swelling with muIL-17R:Fc treatment in this study is not as great as with other cytokine-blocking therapies, such as anti-TNF therapy. In our experience as well as in the experience of other investigators, specific blockade of TNF results in a more marked reduction of joint swelling in AIA, suggesting that TNF plays a greater role than IL-17 in controlling the signs of inflammation (13, 14).

The reduction in radiologic scores observed with muIL-17R:Fc treatment in this study is consistent with previous in vitro studies that demonstrated that IL-17 plays a role in bone damage and cartilage degradation, showing synergy with TNF for bone resorption (6) and for stimulating osteoclast differentiation (7). Comparing these effects with other treatments in this model, in our laboratory, specific blockade of TNF with a PEGylated soluble tumor necrosis factor receptor I produced changes in radiologic scores of joint damage that were similar to those produced by treatment with 20 mg/kg of muIL-17R:Fc, despite a greater improvement in joint swelling (13). Further studies are needed to determine whether higher doses of muIL-17R:Fc may further reduce the joint inflammation and damage. In this study, only partial inhibition of these scores was achieved, and this may reflect the actions of other cytokines, such as TNF and IL-1.

IL-17 has previously been shown to enhance the surface expression of the intracellular adhesion molecule 1 in human fibroblasts (1, 2). We found that at higher doses, there was a general reduction in synovial cellularity, although there were no differences in T lymphocyte numbers in the treated rats. These results suggest that IL-17 may play a role in cell trafficking to sites of inflammation, but it is unclear if this is a direct effect or one that is mediated via other mechanisms of inflammation.

The results of this study suggest that IL-17 may be a potential therapeutic target in inflammatory arthritis. In addition to investigating the effects of IL-17–blocking treatments alone, IL-17 may be a candidate for combination therapy with other cytokine-blocking treatments. TNF, IL-1β, and IL-17 have potent synergistic actions in vitro (7). In support of this suggestion, it has been demonstrated that the combination of TNF, IL-1, and IL-17 soluble receptors is more effective in controlling ex vivo synovial inflammation and bone resorption in RA than each of the receptors alone (15).

In summary, we have shown that blocking IL-17 in vivo by treatment with muIL-17R:Fc attenuates rat AIA. This demonstrates that IL-17 plays an important role in both the inflammation and joint destruction of inflammatory arthritis.

Acknowledgements

The authors thank Angelina Enno for her help with the immunohistochemistry, and Immunex Corporation for the kind donation of muIL-17R:Fc.

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