of June 26, 2018.This information is current as Inflammation

Blockade Only Ameliorates Joint αCollagen-Induced Arthritis, Whereas TNF-

Bone Destruction in Murine Type II Blockade Prevents Cartilage andβαIL-1

A. J. van de Loo, Dick Heinegård and Wim B. van den BergLeo A. B. Joosten, Monique M. A. Helsen, Tore Saxne, Fons

http://www.jimmunol.org/content/163/9/50491999; 163:5049-5055; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/163/9/5049.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1999 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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IL-1 ab Blockade Prevents Cartilage and Bone Destruction inMurine Type II Collagen-Induced Arthritis, Whereas TNF- aBlockade Only Ameliorates Joint Inflammation1

Leo A. B. Joosten,2* Monique M. A. Helsen,* Tore Saxne,†‡ Fons A. J. van de Loo,*Dick Heinegard,‡ and Wim B. van den Berg*

Anti-TNF- a treatment of rheumatoid arthritis patients markedly suppresses inflammatory disease activity, but so far no tissue-protective effects have been reported. In contrast, blockade of IL-1 in rheumatoid arthritis patients, by an IL-1 receptor antag-onist, was only moderately effective in suppressing inflammatory symptoms but appeared to reduce the rate of progression of jointdestruction. We therefore used an established collagen II murine arthritis model (collagen-induced arthritis(CIA)) to study effectson joint structures of neutralization of either TNF- a or IL-1. Both soluble TNF binding protein and anti-IL-1 treatment ame-liorated disease activity when applied shortly after onset of CIA. Serum analysis revealed that early anti-TNF-a treatment of CIAdid not decrease the process in the cartilage, as indicated by the elevated COMP levels. In contrast, anti-IL-1 treatment ofestablished CIA normalized COMP levels, apparently alleviating the process in the tissue. Histology of knee and ankle jointscorroborated the finding and showed that cartilage and joint destruction was significantly decreased after anti-IL-1 treatment butwas hardly affected by anti-TNF-a treatment. Radiographic analysis of knee and ankle joints revealed that bone erosions wereprevented by anti-IL-1 treatment, whereas the anti-TNF-a-treated animals exhibited changes comparable to the controls. In linewith these findings, metalloproteinase activity, visualized by VDIPEN production, was almost absent throughout the cartilagelayers in anti-IL-1-treated animals, whereas massive VDIPEN appearance was found in control and sTNFbp-treated mice. Theseresults indicate that blocking of IL-1 is a cartilage- and bone-protective therapy in destructive arthritis, whereas the TNF-aantagonist has little effect on tissue destruction. The Journal of Immunology,1999, 163: 5049–5055.

T umor necrosis factor-a is currently being investigated asa target in the efforts to treat rheumatoid arthritis (RA)3.It has been postulated that TNF-a drives most of the IL-1

production by synovial tissue in RA. Therefore, blocking ofTNF-a is assumed to sufficiently down-regulate all facets of thearthritic process (1, 2). Several clinical trials using anti-TNF-aAbs or soluble TNF receptors in RA patients showed suppressionof clinical disease and inflammatory activity (3, 4). Although theobserved effects are indeed encouraging with regard to the acutefacets of the disease, there is as yet no information regarding pro-tective effects against longer term bone and cartilage destruction.

The first trials with IL-1 receptor antagonist (IL-1Ra) in RAshowed significant, but moderate, suppression of clinical diseaseactivity. However, a beneficial effect on the rate of joint erosionprogression was suggested in a 6-mo clinical trial (5, 6).

Inflammation or clinical disease activity appears not necessarilylinked to progression of joint destruction (7). Studies of the lattercomponent of the disease is difficult to document in patients, shortof using extremely long trials. Therefore we turned to animal mod-els to allow documentation of all parameters, including the processin the joint structures. Murine collagen-induced arthritis (CIA) is awidely used experimental model of arthritis and has histopatho-logical features in common with RA. It has been shown that neu-tralization of TNF-a ameliorates disease activity when adminis-tered before or shortly after onset of the disease (8–10). However,anti-TNF-a treatment did not decrease disease activity when givenduring established CIA. Blocking of IL-1, either after onset orduring established CIA, effectively suppresses the arthritic process,recently demonstrated in several studies (10, 11).

We now investigated whether neutralization of the monokinesTNF-a or IL-1 during CIA influences the development of cartilageand bone destruction. Treatment with either soluble TNF bindingprotein or anti-IL-1a1b was started shortly after onset of disease,and clinical as well as effects on the tissues were monitored. Serumcartilage oligomeric matrix protein levels were analyzed as a markerof cartilage pathological turnover, and radiography was used todetermine bone destruction. Expression of the aggrecan neoepitopeVDIPEN in the cartilage layers, as marker of metalloproteinaseactivity, potentially a component in the destructive process, wasdemonstrated by immunohistochemistry. Furthermore, histology ofknee and ankle joints was performed to investigate the effect ofanti-TNF-a or anti-IL-1a1b treatment on the joint tissues.

The findings in this study showed that both anti-TNF-a andanti-IL-1a1b treatment ameliorates the inflammatory componentof the disease but only neutralization of IL-1a1b preventscartilage and bone destruction in CIA.

*Department of Rheumatology, University Hospital Nijmegen, Nijmegen, The Nether-lands; and Departments of†Rheumatology and‡Cell and Molecular Biology, Sectionfor Connective Tissue Biology, Lund University, Lund, Sweden

Received for publication April 19, 1999. Accepted for publication August 17, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Catholic Rheumatism FoundationNijmegen, The Swedish Medical Research Council, The Crafoord, Kock, O¨ sterlundand Ax:son Johnson Foundations, The Swedish Rheumatism Foundation, and KingGustaf V 80-Year Foundation.2 Address correspondence and reprint requests to Dr. Leo A. B. Joosten, Departmentof Rheumatology, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, TheNetherlands. E-mail address: l.joosten@reuma.azn.nl3 Abbreviations used in this paper: RA, rheumatoid arthritis; sTNFbp, dimericallylinked PEGylated soluble p55 TNFRI receptor; COMP, cartilage oligomeric matrixprotein; CIA, collagen-induced arthritis; IL-lRa, IL-1R antagonist.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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Materials and MethodsAnimals

Male DBA-1 mice were obtained from Bomholdgård, Rye, Denmark. Micewere kept in filter top cages and were fed a standard diet and tap water adlibitum. Mice were used at 10 to 12 wk of age.

Collagen arthritis induction and anti-cytokine treatment

Mice were immunized with 100mg bovine type II collagen in CFA en-riched withMycobacterium tuberculosisH37Ra (4 mg/ml) at the base ofthe tail. Bovine collagen was isolated as described elsewhere (12). Themice were boostered i.p. with 100mg collagen dissolved in saline. Afterdisease onset at day 28, mice were selected and divided into separategroups of at least 10 mice. The mean arthritis score of the control andanti-cytokine groups was comparable at the start of treatment. To neutralizeTNF-a, mice were injected i.p. every other day with 3 mg/kg dimericallylinked PEGylated soluble p55 TNFRI receptor (Amgen, Boulder, CO).This so-called TNFbp showed efficacy in murine streptococcal cell wallarthritis (13). The 50% effective dose (ED50) of TNFbp to block the cy-totoxic effect of murine TNF-a (mTNF-a) in the L929 bioassay was 531029 M. To eliminate IL-1a1b, mice received one single injection ofpurified (IgG fraction) rabbit anti-murine IL-1a and anti-IL-1b (1 mg ofeach Ig). This dose revealed to be sufficient to suppress several murinearthritis models such as Ag-induced arthritis, immune complex-inducedarthritis, and CIA (10, 14–16). One microgram of these anti-IL-1 Absneutralized 50–100 pg IL-1 in the NOB-1 bioassay. As control, we usedeither BSA (3 mg/kg) or normal rabbit Igs (2 mg i.p.).

Assessment of CIA

Mice were carefully examined three times a week for the visual appearanceof arthritis in peripheral joints, and scores for disease activity were givenas previously described (10, 14). The clinical severity of arthritis (arthritisscore, Fig. 1) was graded on a scale of 0–2 for each paw, according tochanges in redness and swelling. At later time points, ankylosis was in-cluded in the macroscopic scoring.

IL-6 bioassay

IL-6 activity was determined by a proliferative assay using B9 cells.Briefly, 5 3 103 B9 cells in 200ml 5% FCS-RPMI 1640 medium per wellwere plated in a round-bottom microtiter plate and incubated for 3 daysusing human recombinant IL-6 (R&D systems, Minneapolis, MN) as stan-dards. At the end of the incubation, 0.5mCi of [3H]thymidine (NEN-Du-Pont, Boston, MA) was added per well. Three hours later, cells were har-vested, and thymidine incorporation was determined. Detection limit forthe IL-6 bioassay was 1 pg/ml.

Cartilage oligomeric matrix protein measurements

At the end of the experiments, serum samples were taken and murinecartilage oligomeric matrix protein (COMP) levels were determined byELISA using similar conditions as described for the assay for humanCOMP (17). The assay was modified by using rat COMP for coating themicrotiter plates and for the standard curve included in each plate as wellas by using a polyclonal antiserum raised against rat COMP (18). A highcross-reactivity to murine COMP was shown both by parallel dilutioncurves of murine sera to the standard curve as well as by experimentswhere a dilution of murine serum was added to the standard curve.

Radiology and histology

Knee and ankle joints were removed at the end of the experiments and werefixed and used for radiographic analysis as a marker for bone destruction.Radiographs were carefully examined by using a stereo microscope. Jointdestruction was scored on a scale from 0–5, where 0 denotes no damage;1, minor bone destruction observed in one enlightened spot; 2, moderatechanges, 2–4 spots in one area; 3, marked changes, 2–4 spots in moreareas; 4, severe erosions afflicting the joint; and 5, complete destruction ofthe joints. Radiographs were scored by two observers without knowledgeof the experimental group. For histology, joints were decalcified, dehy-drated, and embedded in paraffin (10). Standard sections of 7mm weremade and stained with either hematoxylin and eosin or safranin O. Serialsections were scored by two observers on decoded slides. Inflammationwas graded on a scale from 0 (no inflammation) to 3 (severe inflamed joint)as influx of inflammatory cells in synovium and joint cavity. Cartilagedestruction and matrix proteoglycan depletion were scored on a scale from0–3 ranging from no abnormalities to completely destroyed or destained(with safranin O) cartilage. Bone erosions were graded on a scale 0–3,

ranging from normal bone appearance to fully eroded cortical bone struc-ture in patella and femur condyle (10, 14).

Immunohistochemical VDIPEN staining

For immunostaining, sections of knee joints were deparaffinized, rehy-drated, and digested with proteinase-free chondroitinase ABC to removethe side chains of the proteoglycans. Subsequently, sections were treatedwith 1% hydrogen peroxide, 1.5% normal goat serum, and affinity-purifiedrabbit anti-VDIPEN IgG (kindly provided by Dr. I. I. Singer and Dr. E. K.Bayne, Merck, Rahway, NJ). This Ab has been characterized before (19,20). Thereafter, sections were incubated with biotinylated goat anti-rabbit,and avidin-streptavidin-peroxidase (Elite kit, Vector Labs, Burlingham,CA) staining was performed. Counterstaining was done with orange G.

Statistical methods

The significance of difference between group means was determined by aMann-WhitneyU test in the program SigmaStat (SPSS Software Products,Chicago, IL).

ResultsAmelioration of clinical disease activity by both sTNFbp andanti-IL-1 treatment

Anti-TNF-a treatment by injection of sTNFbp ameliorated clinicalexpression of CIA when started shortly after disease onset (Fig.1A). Significant reduction of joint swelling and redness was notedafter 4 days of treatment. Blockade of IL-1 during established CIAresulted in marked suppression of clinical disease activity, as canbe seen in Fig. 1B. Actually, one single injection of anti-IL-1a1b

FIGURE 1. Suppression of disease activity of CIA by either sTNFbp oranti-IL-1a1b treatment. Mice expressing CIA were selected at day 28after immunization of CIA and were divided into separate groups of at least10 mice. Mice were injected with 3 mg/kg sTNFbp (A) every other day orwith one single injection of 1 mg purified rabbit anti-IL-1a and anti-IL-1b(B). As control for sTNFbp, BSA was used. Normal rabbit Ig was used ascontrol for anti-IL-1a1b. Disease activity was visually scored as describedin Materials and Methods. Data are expressed as mean6 SD.p, p , 0.01,Mann-WhitneyU test, compared with control.

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Abs was sufficient to reduce clinical signs. The antiinflammatoryeffect of both sTNFbp and anti-IL-1a1b was further demonstratedby the reduced serum IL-6 levels determined at the end of treat-ment (Fig. 2.). IL-6 can be viewed as an acute phase protein asdescribed previously (21). The strong reduction of serum IL-6 in-dicated that TNF-a was sufficiently neutralized by sTNFbptreatment.

COMP, a circulating marker of cartilage turnover is decreasedby anti-IL-1 treatment

To obtain further insight into the protection against cartilage de-struction, we determined serum COMP levels in the variousgroups. COMP is released from cartilage as a result of increasedturnover in human and experimental arthritis (17, 18, 22). Fig. 3Ashows a strong correlation in disease with no intervention betweenclinical arthritis score and serum COMP levels (r 5 0.94). This isin line with previous findings in collagen-induced and pristane-induced arthritis in rats (18, 23). Although treatment with sTNFbpsuppressed clinical disease activity of the CIA, no reduction wasfound in serum COMP levels, indicating little effect on the processin the cartilage (Fig. 3B). In contrast, strong reduction of serumCOMP levels was seen in the anti-IL-1a1b-treated animals (6.56 0.9 mg/ml vs 3.46 0.5 mg/ml).

Prevention of bone erosions by anti-IL-1 but not by sTNFbptreatment

Radiological analysis, as an indicator of bone erosions of knee andankle joints, revealed that elimination of TNF-a in established CIAdid not retard the radiological progression of bone destruction (Fig.4). The lack of an effect on bone destruction was further illustratedin Fig. 4, which shows erosive processes in knee joints on femurand tibia. In contrast, anti-IL-1a1b treatment abolished bone ero-sions, as demonstrated in Fig. 4. Fig. 5 further emphasizes theprotective effect of the anti IL-1 treatment.

Effect of neutralization of TNF-a or IL-1 on joint pathology asdemonstrated by histological examination

To confirm that IL-1 has a key role in cartilage and bone destruc-tion in joint disease, we graded pathology on sections of wholeknee joints. Table I shows that sTNFbp treatment reduced the in-flammatory process, determined as the number of cells in the sy-

novium and joint cavity, but had only marginal effect on cartilagedamage, matrix proteoglycan depletion, and bone erosions. This isfurther illustrated in Fig. 6. Almost complete prevention of

FIGURE 2. Serum IL-6 levels after blockade of either TNF-a or IL-1.IL-6 levels were determined at day 36 after treatment with either sTNFbpor anti-IL-1 by using B9-cell bioassay. For treatment protocol see Fig. 1.Data are expressed as mean6 SD of at least seven mice per group.p, p ,0.01, Mann-WhitneyU test, compared with arthritic vehicle-treatedanimals.

FIGURE 3. IL-1 neutralization reduced serum COMP levels. SerumCOMP were determined in sera of arthritic mice expressing different dis-ease activity at day 36 after immunization (A). Strong correlation wasfound between disease activity and circulating COMP levels,r 5 0.94.B,Serum COMP levels, determined at day 36 after treatment with eithersTNFbp or anti-IL-1a1b. For details see Fig. 1 andMaterials and Meth-ods. Dotted line indicates serum COMP level in normal DBA-1 mice (4.26 0.6). p, p , 0.001, Mann-WhitneyU test, compared with control.

FIGURE 4. Effect of either anti-TNF-a or anti-IL-1a1b treatment on jointdestruction. Joints were x-rayed on day 36 after induction of CIA. Destructionwas graded from x-ray photography by scoring bone erosions on a scale from0 (no alterations) to 5 (completely destroyed joints). For treatment protocol seeMaterials and Methods. Data represent the mean6 SD x-ray score of at least20 joints.p, p , 0.001, Mann WhitneyU test compared with control.

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cartilage and bone damage was achieved by anti IL-1a1b treat-ment during established CIA (Table I, Fig. 6).

Blocking of IL-1 prevents VDIPEN neoepitope expression

VDIPEN neoepitope is a marker of metalloproteinase (MMP)-me-diated cleavage of aggrecan, the major proteoglycan of articularcartilage. Previous studies have revealed that VDIPEN neoepitopewas more abundant at sites and stages of advanced damage (19).To further demonstrate the protective effects on cartilage by anti-IL-1a1b treatment, sections were stained for this neoepitope. Asa typical example, VDIPEN was highly expressed throughout thecartilage layers of the patella and femur in sections of vehicle-treated animals (Fig. 7A). Elimination of TNF-a for 8 days, initi-ated shortly after onset of CIA, did not reduce VDIPEN neoepitopeexpression in cartilage, as can be seen in Fig. 7B. In contrast,VDIPEN expression was almost absent in cartilage of anti-IL-1a1b-treated animals (Fig. 7C).

DiscussionThe onset of clinical symptoms and inflammation in collagen typeII arthritis is TNF-a dependent, which is in line with a role of thiscytokine also in human RA (24). Studies with neutralizing anti-TNF-a Abs or soluble TNF receptors have revealed a major sup-pressive effect of the clinical disease activity, when treatment wasstarted directly after onset of CIA (8, 9). Recently, we showed that,when arthritis is fully expressed, subsequent blocking of TNF-a

appeared only marginally effective, implying that TNF-a is crucialin onset but less important in propagation of arthritis (10). In con-trast, neutralization of IL-1a1b in early and established stages ofCIA markedly suppressed disease activity (10, 14). It has beenshown that IL-1b is the pivotal cytokine in collagen type II arthri-tis regarding disease expression by using anti-IL-1b Abs in DBA-1mice, by studies of CIA in IL-1b-deficient mice, and by adminis-tration of IL-1b-converting enzyme inhibitors (10, 14, 25, 26).Furthermore, it was showed that local inflammation was impairedin IL-1b-converting enzyme-deficient mice (27). In the presentstudy, we investigated whether blocking of TNF-a or IL-1 duringestablished CIA would, in addition to suppressing inflammatorydisease activity, prevent tissue destruction. As demonstrated pre-viously, anti-TNF-a treatment ameliorated CIA when started afteronset of disease. Despite the fact that suppression of clinical ap-pearance of CIA and reduced serum IL-6 levels were found byearly anti-TNF treatment, serum COMP levels, cartilage damage,and bone destruction were not affected, showing that joint destruc-tion is progressing. COMP is a major component of articular car-tilage, and serum levels are considered as a marker of generalizedcartilage turnover (17, 22). In joint disease, the contribution fromother tissues, e.g. synovia, which has been shown to be capable ofCOMP production (28, 29), appears insignificant (T. Saxne and D.Heinegård, unpublished observations). Thus, we have found thatserum COMP levels were increased after the occurrence of carti-lage destruction in CIA in both rats and mice, while, in early stages

FIGURE 5. IL-1 blockade prevents bone erosions. X-ray photographs taken at day 36 after treatment with either sTNFbp or anti-IL-1a1b. A, Knee jointof control, rabbit Ig-treated group. Marked zones of bone erosions were found in the femur condyles and in the patella (arrows).B, Clear protection againstjoint destruction after blocking IL-1a1b during established CIA.C, No improvement of radiological joint destruction after sTNFbp treatment, comparedwith control. No differences in bone erosions were noted between the two control groups.

Table I. Effect of sTNFbp or anti-IL-1 treatment on cartilage pathologya

TreatmentInfiltration of

CellsCartilageDamage

ProteoglycanDepletion

BoneErosions

Control (BSA) 1.56 0.7 1.86 0.6 2.36 0.9 2.26 0.7sTNFbp 0.76 0.6 1.56 0.7 2.06 0.8 1.86 0.5Control (Ig) 1.86 0.6 1.66 0.5 2.16 0.5 2.56 0.9Anti-IL-1a 1 b 0.46 0.3* 0.46 0.3* 0.56 0.4* 0.66 0.3*

a Joint pathology was examined after treatment either with 3 mg/kg sTNFbp every other day or with one single injection ofrabbit anti-IL-1a 1 b (1 mg each). Histology was performed on whole knee joints as described inMaterials and Methods.Scoring was performed by two independent observers on decoded slides.p, p , 0.001, Mann-WhitneyU test, compared withcontrols.

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of CIA with marked inflammation, no elevation of serum COMPwas found (T. Saxne and L. A. B. Joosten, unpublished observa-tions; E. Larsson and T. Saxne, unpublished observations). Cor-

roborating the strong suppression of CIA and a cartilage protectiveeffect by anti-IL-1a1b treatment, no elevated serum COMP levelswere found in anti-IL-1-treated animals. This was further

FIGURE 6. Histopathology at day 36 of knee joints after treatment with either sTNFbp or anti-IL-1a1b. A, Severe inflammation and cartilagedestruction (arrows) in control, BSA-treated animal.B, Complete loss of matrix proteoglycans, indicated by destained cartilage layers, in control group.C andD, Knee joint of an animal treated with sTNFbp. Although reduced infiltrate, no ameliorated cartilage pathology when compared with control.E andF, Almost homogeneous safranin O staining indicated a cartilage-protective therapy by anti-IL-1 treatment. Furthermore, markedly reduced inflammationby the latter treatment. No difference was seen between the two control groups.A, C, andE, Hematoxilin and eosin staining.B, D, andF, Safranin Ostaining. P5 patella, F5 femur, C5 cartilage, JS5 joint space. Original magnification3100.

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supported by VDIPEN neoepitope appearance in the cartilage lay-ers. Marked presence of this neoepitope was found throughout thecartilage in control and sTNFbp-treated animals, whereas animals

treated with anti-IL-1a1b had almost no VDIPEN neoepitope.This neoepitope is formed by proteolytic cleavage of aggrecan bymetalloproteinases. The fragment remains attached to hyaluronanin the cartilage, as an indicator of the proteolytic activity (30). Inthe model of Ag-induced arthritis, we noted that VDIPEN expres-sion depended primarily on effects of IL-1, and the location cor-related with severe cartilage damage (19). More recently, it wasdemonstrated that VDIPEN expression reflects stromelysin(MMP-3) activity, since expression was absent in stromelysin-de-ficient mice (31).

Histological analysis of knee and ankle joints revealed that elim-ination of IL-1a1b, starting after onset of disease, reduced jointinflammation, cartilage damage, loss of matrix proteoglycan andbone erosions. The primary effect of treatment by sTNFbp injec-tions was a decreased influx of inflammatory cells. Previous stud-ies with anti-TNF-a Abs or sTNF receptor protein in CIA reportedsignificant reduction of clinical disease activity, but hardly anymeasurable effect on cartilage or bone destruction (8, 9, 10). Al-though anti-TNF-a treatment was started directly after onset ofdisease, 75% of the animals developed moderate or severe carti-lage erosions (8).

IL-1 appears to be an efficient mediator, since, in the zymosan-induced experimental arthritis, it is clearly demonstrated that IL-1is responsible for the inhibition of chondrocyte proteoglycan syn-thesis. This suppressive effect appeared mediated by NO, sinceNOS2 gene knockout mice, while having inflammation, showed noinhibition of chondrocyte metabolism (32). Furthermore, it hasbeen demonstrated that blocking of IL-1 activity prevented carti-lage proteoglycan depletion in murine Ag-induced arthritis, indi-cating protection against cartilage damage (15). Interestingly, inthis latter experimental arthritis, anti-IL-1 treatment did not ablatejoint inflammation.

The first clinical trials with IL-1Ra in human RA demonstratedthat IL-1Ra has a beneficial effect on the rate of progression ofjoint erosion as well as suppressing inflammatory disease activity(5, 6). Anti-TNF-a treatment on the other hand seems to be some-what more effective in suppressing clinical disease activity, i.e.,primarily inflammation. However, there are as yet no publisheddata on effects of anti-TNF-a treatment on the progression of jointerosions (3, 4). We have preliminary data showing that, in RApatients treated with human anti-TNF-a (D2E7), serum COMPlevels were not reduced, although impressive reduction of diseaseactivity was seen. Furthermore, IL-1b expression in synovial bi-opsies was not changed by anti-TNF treatment. This argues againstTNF-a-dependent IL-1 production in RA synovium (P. Barrera,L. A. B. Joosten, A. A. den Broeder, L. B. A. van de Putte,P. L. C. M. van Riel, and W. B. van den Berg, manuscript inpreparation).

The present study indicates that blocking of IL-1 during arthritisrepresents therapy that protects the cartilage and bone structures.This is demonstrated by reduced serum COMP levels, reducedappearance of the VDIPEN neoepitope in cartilage, and joint his-topathology with abolished erosions of the articular cartilage, aswell as absence of radiographically detectable bone erosions.

The effects of IL-1 inhibition, at the same time as TNF-a inhi-bition has little effect on tissue destruction, may appear puzzling.However, it is known that chondrocytes and osteoblasts may pro-duce IL-1 (33). It is thus possible that the initial events includesetting up a cycle with self-activated destruction in cartilage andbone. In view of these findings, although TNF-a inhibitors mayrelieve the actual clinical picture, the long-term outcome with re-gard to joint destruction is likely to have the same bad prognosis.Therefore, to suppress the inflammation and offer protection

FIGURE 7. Expression of cartilage proteoglycan breakdown neo-epitope VDIPEN. Fully affected cartilage in control rabbit Ig (A) andsTNFbp (B) treated animals. Note the marginal expression of VDIPEN incartilage of anti-IL-1a1b-exposed mice.C, VDIPEN expression was de-termined at day 36. No difference was found in VDIPEN expression be-tween the two control groups, BSA and rabbit Ig. P5 patella, F5 femur.Original magnification3100.

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against tissue destruction, combinations of TNF-a and IL-1 inhi-bition should be tested.

AcknowledgmentsChristina J. J. Coenen (Organon, Oss, The Netherlands) is acknowledgedfor the radiographic analysis of knee and ankle joints. We are indebted toMette Lindell for skillful technical assistance. The Central Animal Labo-ratory (CDL), University of Nijmegen, is acknowledged for taking care ofthe animals.

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