Protection against cartilage and bone destruction by systemic interleukin-4 treatment in established murine type II collagen-induced arthritis

Abstract

Destruction of cartilage and bone are hallmarks of human rheumatoid arthritis (RA), and controlling these erosive processes is the most challenging objective in the treatment of RA. Systemic interleukin-4 treatment of established murine collagen-induced arthritis suppressed disease activity and protected against cartilage and bone destruction. Reduced cartilage pathology was confirmed by both decreased serum cartilage oligomeric matrix protein (COMP) and histological examination. In addition, radiological analysis revealed that bone destruction was also partially prevented. Improved suppression of joint swelling was achieved when interleukin-4 treatment was combined with low-dose prednisolone treatment. Interestingly, synergistic reduction of both serum COMP and inflammatory parameters was noted when low-dose interleukin-4 was combined with prednisolone. Systemic treatment with interleukin-4 appeared to be a protective therapy for cartilage and bone in arthritis, and in combination with prednisolone at low dosages may offer an alternative therapy in RA. . Of great importance is that IL-4 could not be detected in synovial fluid or in tissues. This absence of IL-4 in the joint probably contributes to the disturbance in the Th1/Th2 balance in chronic RA. Collagen-induced arthritis (CIA) is a widely used model of arthritis that displays several features of human RA. Recently it was demonstrated that the onset of CIA is under stringent control of IL-4 and IL-10. Furthermore, it was demonstrated that exposure to IL-4 during the immunization stage reduced onset and severity of CIA. However, after cessation of IL-4 treatment disease expression increased to control values. Because it was reported that IL-4 suppresses several proinflammatory cytokines and matrix degrading enzymes and upregulates inhibitors of both cytokines and catabolic enzymes, we investigated the tissue protective effect of systemic IL-4 treatment using established murine CIA as a model. Potential synergy of low dosages of anti-inflammatory glucocorticosteroids and IL-4 was also evaluated. DBA-1J/Bom mice were immunized with bovine type II collagen and boosted at day 21. Mice with established CIA were selected at day 28 after immunization and treated for days with IL-4, prednisolone, or combinations of prednisolone and IL-4. Arthritis score was monitored visually. Joint pathology was evaluated by histology, radiology and serum cartilage oligomeric matrix protein (COMP). In addition, serum levels of IL-1Ra and anticollagen antibodies were determined. ). Systemic IL-4 administration increased serum IL-1Ra levels and reduced anticollagen type II antibody levels. Treatment with low-dose IL-4 (0.1 μg/day) was ineffective in suppressing disease score, serum COMP or joint destruction. Synergistic suppression of both arthritis severity and COMP levels was noted when low-dose IL-4 was combined with prednisolone (0.05 mg/kg/day), however, which in itself was not effective. In the present study, we demonstrate that systemic IL-4 treatment ameliorates disease progression of established CIA. Although clinical disease progression was only arrested and not reversed, clear protection against cartilage and bone destruction was noted. This is in accord with findings in both human RA and animal models of RA that show that inflammation and tissue destruction sometimes are uncoupled processes. Of great importance is that, although inflammation was still present, strong reduction in serum COMP was found after exposure to IL-4. This indicated that serum COMP levels reflected cartilage damage, although a limited contribution of the inflamed synovium cannot be excluded. Increased serum IL-1Ra level (twofold) was found after systemic treatment with IL-4, but it is not likely that this could explain the suppression of CIA. We and others have reported that high dosages of IL-1Ra are needed for marked suppression of CIA. As reported previously, lower dosages of IL-4 did not reduce clinical disease severity of established CIA. Of importance is that combined treatment of low dosages of IL-4 and IL-10 appeared to have more potent anti-inflammatory effects, and markedly protected against cartilage destruction. Improved anti-inflammatory effect was achieved with IL-4/prednisolone treatment. In addition, synergistic effects were found for the reduction of cartilage and bone destruction. This indicates that systemic IL-4/prednisolone treatment may provide a cartilage and bone protective therapy for human RA. < 0.05, versus control, by Mann-Whitney U test.

Introduction:

. Of great importance is that IL-4 could not be detected in synovial fluid or in tissues. This absence of IL-4 in the joint probably contributes to the disturbance in the Th1/Th2 balance in chronic RA.

Collagen-induced arthritis (CIA) is a widely used model of arthritis that displays several features of human RA. Recently it was demonstrated that the onset of CIA is under stringent control of IL-4 and IL-10. Furthermore, it was demonstrated that exposure to IL-4 during the immunization stage reduced onset and severity of CIA. However, after cessation of IL-4 treatment disease expression increased to control values.

Aims:

Because it was reported that IL-4 suppresses several proinflammatory cytokines and matrix degrading enzymes and upregulates inhibitors of both cytokines and catabolic enzymes, we investigated the tissue protective effect of systemic IL-4 treatment using established murine CIA as a model. Potential synergy of low dosages of anti-inflammatory glucocorticosteroids and IL-4 was also evaluated.

Methods:

DBA-1J/Bom mice were immunized with bovine type II collagen and boosted at day 21. Mice with established CIA were selected at day 28 after immunization and treated for days with IL-4, prednisolone, or combinations of prednisolone and IL-4. Arthritis score was monitored visually. Joint pathology was evaluated by histology, radiology and serum cartilage oligomeric matrix protein (COMP). In addition, serum levels of IL-1Ra and anticollagen antibodies were determined.

Results:

). Systemic IL-4 administration increased serum IL-1Ra levels and reduced anticollagen type II antibody levels. Treatment with low-dose IL-4 (0.1 μg/day) was ineffective in suppressing disease score, serum COMP or joint destruction. Synergistic suppression of both arthritis severity and COMP levels was noted when low-dose IL-4 was combined with prednisolone (0.05 mg/kg/day), however, which in itself was not effective.

Discussion:

In the present study, we demonstrate that systemic IL-4 treatment ameliorates disease progression of established CIA. Although clinical disease progression was only arrested and not reversed, clear protection against cartilage and bone destruction was noted. This is in accord with findings in both human RA and animal models of RA that show that inflammation and tissue destruction sometimes are uncoupled processes. Of great importance is that, although inflammation was still present, strong reduction in serum COMP was found after exposure to IL-4. This indicated that serum COMP levels reflected cartilage damage, although a limited contribution of the inflamed synovium cannot be excluded.

Increased serum IL-1Ra level (twofold) was found after systemic treatment with IL-4, but it is not likely that this could explain the suppression of CIA. We and others have reported that high dosages of IL-1Ra are needed for marked suppression of CIA. As reported previously, lower dosages of IL-4 did not reduce clinical disease severity of established CIA. Of importance is that combined treatment of low dosages of IL-4 and IL-10 appeared to have more potent anti-inflammatory effects, and markedly protected against cartilage destruction. Improved anti-inflammatory effect was achieved with IL-4/prednisolone treatment. In addition, synergistic effects were found for the reduction of cartilage and bone destruction. This indicates that systemic IL-4/prednisolone treatment may provide a cartilage and bone protective therapy for human RA.

< 0.05, versus control, by Mann-Whitney U test.

Introduction

].

], and this lack of IL-4 is likely to contribute to the uneven Th1/Th2 balance in chronic RA.

] are downmodulation of histone acetyltransferase and upregulation of histone deacetyltransferase, which both affected messenger RNA transcription negatively.

].

In the present study the effects of systemic high dose IL-4 therapy in established CIA were investigated. Furthermore, the potential synergy of combined prednisolone and IL-4 treatment were examined. We investigated the protective effect of IL-4 alone or in combination with prednisolone on disease activity as well as cartilage and bone destruction as determined histologically, radiologically and by serum measurements of cartilage oligomeric matrix protein (COMP). Anticollagen type II specific antibodies and serum IL-1Ra levels were assessed, in order to obtain an insight into the mechanism of action. The findings suggest that IL-4 treatment protects against cartilage and bone destruction, and that combined IL-4/steroid treatment may provide a safe, anti-inflammatory and anti-destructive therapy in human RA.

Animals

Male DBA-1/Bom mice were purchased from Bomholdgård (Ry, Denmark). The mice were housed in filter top cages, and were given free access to water and food. The mice were immunized at the age of 10–12 weeks.

Materials

U/mg) was kindly provided by Dr S Smith (Schering-Plough, Kenilworth, NJ, USA).

Collagen preparation

]. It was dissolved in 0.05 mol/l acetic acid (5 mg/ml) and stored at -70ºC.

Immunization

Bovine type II collagen was diluted with 0.05 mol/l acetic acid to a concentration of 2 mg/ml and was emulsified in an equal volume of complete Freund's adjuvant (2 mg/ml MT H37Ra). The mice were immunized intradermally at the base of the tail with 100 μl emulsion (100 μg collagen). At day 21 the animals were boosted with an intra-peritoneal injection of 100 μg collagen type II, diluted in phosphate-buffered saline (pH 7.4).

Assessment of arthritis

]. Mice were considered arthritic when significant changes in redness and/or swelling were noted in digits or in other parts of the paws. At later time points ankylosis was also included in the arthritis score. Clinical severity of arthritis was graded on a scale of 0–2 for each paw, according to changes in redness and swelling: 0, no changes; 0.5, significant; 1.0, moderate; 1.5, marked; and 2.0, maximal swelling and redness, and later on ankylosis. Arthritis score (mean± stan-dard deviation) was expressed as cumulative value for all paws, with a maximum of eight and expressed as percentage of the initial score at the beginning of treatment.

Treatment of collagen-induced arthritis with interleukin-4,

			prednisolone or interleukin-4/prednisolone

To evaluate the effect of IL-4, prednisolone or the combination IL-4/prednisolone on established CIA, mice with CIA were selected at day 28 and divided into groups of at least 10 mice with similar arthritis scores. Thereafter, mice were treated twice a day intraperitoneally with IL-4 (0.1 or 1μg/day), prednisolone (0.05 mg/kg/day), or with IL-4 and prednisolone (at the same doses for the noncombined regimens) for each of several days as indicated in the results.

Determination of interleukin-1 receptor antagonist levels

IL-1Ra was measured using enzyme-linked immunosorbent assay (ELISA). Briefly, Nunc Maxisorb ELISA plates (Nunc, Rostilde, Denmark) were coated with capture antibodies (5 μg/ml, carbonate buffer, pH 9.6, 24 h at 4°C), and thereafter nonspecific binding sites were blocked with 1% bovine serum albumin/phosphate-buffered saline-Tween. Standards and unknown samples were diluted in normal DBA-1 serum and incubated for 3 h at room temperature. Biotinylated detection antibodies were added at concentrations of 0.2–0.4 μg/ml in 0.5% bovine serum albumin in phosphate-buffered slaine-Tween for 1.5 h at room temperature. Thereafter plates were incubated with PolyHRP (0.1 μg/ml in 1% caseine colloid buffer) for 45 min and orthophenylenediamine (0.8 mg/ml) was used as substrate. Plates were read at 495 nm.

Measurement of cartilage oligomeric matrix protein

]. This was shown by parallel dilution curves of murine sera to the standard curve prepared with rat COMP, as well as in experiments in which a dilution of murine serum was added to the standard curve.

Determination of anticollagen antibodies

were measured. Briefly, plates were coated with 10 μg bovine type II, and thereafter nonspecific bindings sites were blocked with 0.1 mol/l ethanolamin (Sigma Chemicals). Serial 1 : 2 dilutions of the sera were added, followed by incubation with isotype-specific goat antimouse peroxidase (Southern Biotechnology Associates, Birmingham, AL, USA) and substrate (5-aminosalicyclic acid; Sigma Chemicals). Plates were read at 492 nm. Titres were expressed as means ± standard deviation dilution, which gives the half maximal value.

Radiological analysis of bone destruction

].

Histology

]. Tissue sections (7 μm thick) were stained with haematoxylin and eosin, or safranin O. Histopathological changes were scored using the following parameters.

].

Statistical analysis

Differences between experimental groups were tested using the Mann-Whitney U test, unless otherwise stated.

treatment of interleukin-4

).

Interleukin-4 protects against cartilage destruction

shows that elevated COMP in CIA were not reduced by treatment with low-dose IL-4. It is of particular interest, that treatment with high-dose IL-4 (1 μg/day) significantly reduced serum COMP levels to values found in nonarthritic control animals.

Interleukin-4 protects against bone destruction

).

Combined interleukin-4/prednisolone treatment

). Treatment of CIA with 1 μg/day IL-4 alone and in combination with prednisolone (0.05 mg/kg/day) for 7 days caused similar reduction in osteoclast numbers (data not shown).

Effect of interleukin-4, or interleukin-4/prednisolone treatment

			on interleukin-1 receptor antagonist and anticollagen antibody levels

shows a twofold increase after IL-4 treatment (1μg/day dose). Treatment with 0.1μg/day IL-4 showed no significant effects on serum IL-1Ra levels. Prednisolone reduced IL-1Ra levels when compared with vehicle-treated animals. In accord with these findings, combined IL-4/prednisolone (1 μg per day/ 0.05 mg per kg per day) treatment resulted in lower IL-1Ra levels than found with IL-4 alone.

levels showed the most prominent reduction, indicating an effect on the Th1 rather than on the Th2 immune response. No decreased anticollagen type II antibody levels were found after treatment with low-dose IL-4. The high-dose IL-4/prednisolone regimen reduced anticollagen type II antibodies to levels similar to those found after treatment with 1μg/day IL-4.

< 0.05, versus vehicle, by Mann-Whitney U test.

< 0.05, versus vehicle, by Mann-Whitney U test.

Knee joint of a mouse treated with IL-4/prednisolone (1 μg per day/0.05 mg per kg). Note the marked reduction of cell influx. All specimens were sampled at day 35. P, patella; F, femur; JS, joint space; C, cartilage; S, synovium. Haematoxylin and eosin staining was used. Original magnifications: × 200 (a, b) and × 100 (c, d).

. Safranin O staining, original magnification × 100.

< 0.01, versus vehicle, by Mann-Whitney U test.

< 0.01, versus vehicle, by Mann-Whitney U test.

. S, synovium; B, bone; BM, bone marrow. Original magnifications × 200 (a, b), × 400 (c, d).

levels were reduced. Similar effects were found after treatment with IL-4/prednisolone (1 μg per day/0.05 mg per kg). Anticollagen type II levels were determined in at least six mice per group. Data are expressed as means ± standard deviation dilution, which gives the half maximal value.

Effect of prednisolone, interleukin (IL)-4 or IL-4/prednisolone treatment on the joint pathology of collagen-induced arthritis in Mice

<0.05, versus vehicle, by Mann-Whitney U test.

Serum interleukin-1 receptor antagonist (IL-1Ra levels) after treatment with either interleukin (IL)-4, prednisolone, or IL-4/prednisolone

< 0.05, versus vehicle, by Mann-Whitney U test.

Discussion

The present study demonstrates clear tissue-protective effects of IL-4, although IL-4 did not prove to be a very potent anti-inflammatory cytokine. Both cartilage and bone erosion were prevented by IL-4 treatment of established CIA. Combination with low-dose prednisolone enhanced the anti-inflammatory capacity of IL-4. This might offer an attractive alternative to the use of high-dose prednisolone, because it can circumvent the unwanted side effects of the drug, including steroid-induced osteoporosis.

].

]. Whether IL-4 acts locally or systemically is at present unknown. Further experiments on biodistribution of IL-4 are needed to resolve this issue.

balance.

]. Thus, evidence so far indicates that changes in serum COMP relate to changes in the cartilage turnover. In accord with these findings, low-dose IL-4/prednisolone treatment did not suppress disease activity, largely reflecting synovitis, but clearly reduced serum COMP levels. Histology interestingly revealed that serum COMP levels correlated more with cartilage erosions than with loss of matrix proteoglycans, which is a reversible process.

] we showed that early proteoglycan loss is mediated by aggrecanase, whereas erosive, late destruction is linked to stromelysin.

production in several cell types that play a role in the resorption process of the bone. Interestingly, blocking studies with neutralizing antibodies directed against IL-4 in CIA indicated that the endogenous cytokine inhibited bone destruction. In animals treated with anti-IL-4, bone destruction determined by radiographic analysis was aggravated compared with that in vehicle-treated animals (data not shown).

].

]. However, when combined with prednisolone the progression of CIA was completely arrested. Furthermore, synergistic suppression of cartilage destruction was demonstrated by lowered serum COMP levels, which was also reflected by histology. Only combined therapy with high-dose IL-4 and prednisolone was able to suppress the influx of inflammatory cells in joint tissues and reduce the loss of matrix proteoglycans.

], it must be considered that a cocktail of IL-4, IL-10 and low-dose glucocorticosteroids or glucocorticoids might be an even more efficacious therapy for human RA.

Keywords

• bone destruction
• cartilage oligomeric matrix protein levels
• collagen-induced arthritis
• interleukin-4
• prednisolone