Annals ofthe Rheumatic Diseases 1992; 51: 978-982

Detection of cytokine activated chondrocytesin arthritic joints from pigs infected withErysipelothrix rhusiopathiae

M Elisabeth Davies, Alan Horner, Burkart Franz, Hans-Joachim Schuberth

AbstractChronic polyarthritis was induced in pigs byinjection of Erysipelothrix rhusiopathiae andthe in vivo activation of chondrocytes bycytokines was then investigated in the affectedjoints by immunocytochemistry. A polyclonalantiserum which recognises surface markerson in vitro interleukin 1 activated porcinechondrocytes was used to detect activatedchondrocytes in all zones of the cartilage fromdiseased joints. In contrast, cartilage removedfrom an unaffected joint in the same animalshowed no chondrocyte activation. Inflam-matory synovial tissue removed from diseasedjoints and cocultured with cartilage from theunaffected joint induced activation of adjacentchondrocytes. The presence of interleukin 1in the inflammatory cells of the synovium wasconfed and major histocompatibilitycomplex (MHC) class II antigens weredetected as a marker of synovial activation.Chondrocytes were found not to express classII antigens in cartilage from either the diseasedor the unaffected joint. These observationsshow that the porcine erysipelas model ofarthritis will be useful in facilitating a novelapproach to monitoring the behaviour ofindividual chondrocytes under pathophysio-logical conditions.

(Ann Rheum Dis 1992; 51: 978-982)

Strangeways ResearchLaboratory,Worts Causeway,Cambridge CBI 4RN,United KingdomM-- E DaviesA HomerInstitute of Microbiologyand Infectious Diseases,The Veterinary School,Bischofsholer Damm 15,D-3000 Hannover,GermanyB FranzThe Immunology Unit,The Veterinary School,Bischofsholer Damm 15,D-3000 Hannover,GermanyH-J SchuberthCorrespondence to:Dr M Elisabeth Davies,Tissue PhysiologyDepartment,Strangeways ResearchLaboratory,Worts Causeway,Cambridge CBI 4RN,United Kingdom.Accepted for publication10 March 1992

Rheumatoid arthritis is a chronic inflammatoryjoint disease of unknown aetiology which ischaracterised by pannus formation resulting insevere degeneration of the articular cartilageand bone.

Articular cartilage consists of chondrocytesembedded in an extracellular matrix made up ofcollagen fibrils and large aggregating proteo-glycans. The dense fibrillar network providedby these matrix macromolecules is essential formaintenance of the structure and function of thecartilage and is controlled continuously by theactivity ofthe chondrocytes.' In healthy cartilagethe role of these cells is to sustain a dynamicequilibrium between synthesis and degradationof these components.Immune related disturbance of this balance in

matrix turnover occurs in inflammatory jointdiseases such as rheumatoid arthritis, eventuallyresulting in extensive depletion of the matrixconstituents. The mechanisms involved in thesedegradative processes are, however, stillunclear. Although it is generally accepted thatthe catabolic cytokines interleukin la, inter-leukin 1,B, and tumour necrosis factor a play amajor part in chondrocyte induced cartilage

destruction,2 3 the identity of the secondmessenger(s) remains to be determined. It istherefore of interest that high levels of tumournecrosis factor and interleukin 1 have beenobserved in the acute inflammatory responseduring the development of arthritis in Erysipelo-thrix rhusiopathiae infected rats.4

Alterations in the behaviour of chondrocytesin response to cytokine activation may wellprovide some useful clues to the processestaking part in joint damage in rheumatoidarthritis. Therefore, as a means of identifyingcytokine responsive chondrocytes we raised apolyclonal antiserum which we have shown invitro to be specific for interleukin 1 inducedsurface markers on pig chondrocytes.5 6 Wehave now been able to extend our previous invitro studies to investigate the behaviour ofchondrocytes in vivo in pigs with chronicpolyarthritis. The porcine erysipelas model hasbeen extensively studied (for a review, seeSchulz et al 7) and is considered to be asatisfactory animal model of human rheumatoidarthritis.8The aim of this study was to locate sites of in

vivo cellular activation induced by inflammatorycytokines produced during the disease processin this animal model.

Materials and methodsINFECTION OF ANIMALSFive pigs (four to five months old) wereexperimentally infected by intraperitonealinjection of 5 x 106 colony forming units ofErysipelothrix rhusiopathiae Serovar 2 strainT28. This strain had been isolated from pigswith natural chronic erysipelas infection andwas shown to be highly virulent.9 10 Theexperimentally infected pigs showed visibleclinical signs of developing polyarthritis (stiff-ness, mild to moderate swelling of knee andmetacarpophalangeal joints, and reluctance tomove). Eight weeks after infection histologicalexamination at necropsy showed typical arthriticinflammatory lesions in the affected joints. Atthis time synovium and cartilage explants wereremoved from the affected knee joints and wereeither used immediately for culture experimentsor were frozen in OCT embedding compound(Miles) in liquid nitrogen and stored at -70°Cuntil used for histology. In one animal cartilagefrom an unaffected metacarpophalangeal jointwas removed for use as a control cartilage.

CLINICAL MONITORINGThe clinical status was evaluated by recordingthe body temperature as a marker ofsepticaemia.

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Cytokine activation in porcine rheumatoid joints

To monitor the arthritis an arthritic index wasdevised by assigning a value to the visiblealterations of 1 (low degree of alterations such asredness, swelling, pain) or 2 (high degree ofalterations) of each of the following parameters:general locomotion disorders (stiffness,reluctance to move, kyphosis, limping), arthritisof carpometacarpal joints, arthritis of tarso-metatarsal joints, arthritis of elbow joint, arth-ritis of knee joint. Every animal could attain amaximum score of 5 x 2=10.

COCULTURE OF NORMAL PIG ARTICULARCARTILAGE WITH AUTOLOGOUS RHEUMATOIDSYNOVIUMCocultures were set up as described pre-viously6 11 except that rheumatoid-like synoviumtaken from the cartilage/pannus junction wasused instead of synovial mince and cultureswere not placed on grids and Millipore mem-branes. Cartilage explants were removed froman unaffected metacarpophalangeal joint of thesame pig and were cocultured for 1, 2, 4, and 7days and then frozen in liquid nitrogen asdescribed previously. Cultures were carried outin Dulbecco's minimal essential medium (Gibco)containing 580 ,ug/ml L-glutamine, 120 [ig/mlbenzyl penicillin, and 200 ,ug/ml streptomycin,supplemented with 5% heat inactivated (56°Cfor 30 minutes) fetal calf serum. The culturemedium was changed at two day intervals.

ANTISERAA polyclonal antiserum was raised in rabbitsand has previously been shown to be specific forinterleukin 1 activated porcine chondrocytes.5 6An antiserum raised against purified porcineinterleukin 113 was also used.'2 A polymorphicmouse monoclonal antibody (B0191) was raisedagainst bovine MHC class II antigen and shownto cross react with the porcine molecule. Apolyclonal sheep antihuman factor VIII antigenwas purchased from Serotec and screened forreactivity with porcine vascular tissue.

All fluorescein (FITC) labelled secondaryantisera, FITC swine antirabbit IgG, FITCrabbit antimouse IgG, and FITC rabbit anti-sheep IgG, were purchased from Dako(High Wycombe, Buckinghamshire, UnitedKingdom).

IMMUNOHISTOLOGICAL TECHNIQUESFrozen sections 4-6 ,um thick were cut at -25 to-35°C onto poly-L-lysine (Sigma) coated eightwell multitest slides (Flow Labs). Sections wereair dried for 5-15 minutes then either fixed with4% paraformaldehyde, pH 7-4, or stainedunfixed within four hours. Unfixed sectionswere used for interleukin 113 localisation; allother antisera were effective on fixed sections.

Sections were incubated with primary andsecondary antisera essentially as describedpreviously5 6 except that all antisera dilutionswere made with 1% bovine serum albumin inphosphate buffered saline and for MHC class IIlocalisation some sections were pretreated withhyaluronidase (1 mg/ml for 15 minutes tomaximise the exposure of antigen. Sectionswere viewed and photographed using the methoddescribed previously.6

Loss of proteoglycans from the cartilagematrix was assessed visually by toluidine bluestaining of adjacent unfixed frozen sections.'3

ResultsCLINICAL SYMPTOMSThe first symptoms were obvious two days afterinfection when changes in the behaviour of theanimal (malaise, general lethargy), an increasein body temperature, and signs of arthritiscould be observed. The fever disappeared afterone week but arthritic symptoms could beobserved until the animals were killed (fig 1).

ACTIVATION OF CHONDROCYTES IN ARTHRITICJOINTSAnimals which developed polyarthritis werekilled eight weeks after infection with theorganism. Dissected joints showed characteristicmacroscopic lesions including swelling, in-creased amounts of viscous synovial fluid,extensive proliferation and vascularisation ofthe synovium, and pannus formation over alarge area of the articular surface. At this stagethe cartilage was not visibly eroded.

Immunohistological staining of the cartilageshowed the expression of interleukin 1 activationantigens by most ofthe chondrocytes throughoutthe cartilage, from the articular surface to thehypertrophic zone. Chondrocytes in the articularand subarticular cartilage underlying thepannus showed particularly intense fluorescence(fig 2A). Midzone and hypertrophic chondro-cytes showed fainter but more discrete membranestaining. Occasional non-activated chondrocytes

0 10 20 30 4t . Time (days)

InfectionFigure I Development ofbody temperature changes (0) and arthritis (@) in pigs aftierinfection with Erysipelothrix rhusiopathiae. For definition ofthe arthritic index see underMaterials and methods. All data are mean values (n=S).

were visualised by methyl green staining of thenuclei. The synovial inflammatory cells showedno immunoreactivity with the antiserum (fig2A).

Invasion of the cartilage by inflammatorysynovium was often observed. These infiltrating

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Davies, Homer, Franz, Schuberth

Figure 2 Immunolocalisation in frozen sections ofcartilage and pannus.from pigs infe'cted with Ervsipelothrtx rhusiopathiae.(A) Articular surface chondrocytes at cartilage (C)/pannus (P) junction. (B) Articular surfadie chondrocvtes from an unaffectedjoint. (C) Immunolocalisation ofporcine interleukin Ir) at cartilage (C)Ipannus (P)junction. (1)) Immunolocalisation ofmajorhistocompatibility complex (swine leucocyte antigen class II) antigen on the svnovial inflammatorv cells of'the pannus (P). Insections (A), (B), and (D) nuclei were counterstained (red) to aid visualisation of'nlo?l-reactive cells.

'fingers' consisted of closely packed inflam-matory cells which were sometimes vascularisedas confirmed by staining of the vessel endo-thelium with antiserum to factor VIII. Chondro-cytes in the cartilage immediately surroundingthese synovial infiltrates were also found to bestrongly activated.

In contrast the normal unaffected jointshowed no signs of swelling and dissectionshowed little synovial fluid, no synovial pro-liferation, and no pannus formation. Chondro-cytes in the cartilage from these joints had nosigns of activation and were detected only bytheir counterstained nuclei (fig 2B).

MATRIX PROTEOGLYCAN DEPLETIONToluidine blue staining of non-fixed frozensections of cartilage from the arthritic jointindicated trivial depletion of proteoglycans.Loss of metachromasia was seen only in areasimmediately adjacent to synovial tissue-that is,minimal depletion was observed at the articularsurface beneath the pannus and in a narrowzone surrounding the inflammatory infiltrates(fig 3). This restricted depletion ofproteoglycanspresumably resulted from localised secretion ofdegenerative enzymes by the synovial cells. Noloss of proteoglycan was seen surroundingindividual chondrocytes in any region of thecartilage.No loss of metachromasia was observed in

cartilage from the unaffected joint.

ACTIVATION OF CHONDROCYTES IN NORMALCARTILAGE BY AUTOLOGOUS INFLAMMATORYSYNOVIAL TISSUEAlthough the chondrocytes in cartilage removedfrom the unaffected joint were not expressing

activation markers, they were capable of beingactivated, as shown after coculture with auto-logous inflammatory synovium taken from anarthritic joint. Activation of the chondrocyteswas observed after 24 hours of coculture.Maximum expression of activation markers wasreached by 48 hours and was maintained duringcoculture for up to seven days. The presence ofinterleukin 1 in the inflammatory synoviumfrom the arthritic joint was confirmed using apolyclonal antibody to porcine interleukin 1p. 12The cytokine was localised in the matrix and ina high proportion of the cells of the synovialtissue (fig 2C). Some staining was also seen onthe immediately adjacent chondrocytes.

DISTRIBUTION OF MHC CLASS II EXPRESSIONMajor histocompatibility complex class IIantigens, designated the swine leucocyteantigen complex II1,4 were detected using amouse polymorphic monoclonal antibody raisedagainst bovine MHC class II antigens which hasbeen shown to cross react with the porcinemolecule (Schuberth, unpublished obser-vations). In arthritic joints of five experimentallyinfected pigs we showed a strong expression ofswine leucocyte antigen class II on all inflam-matory synovial tissue, but were unable todetect the molecule on chondrocytes (fig 2D).The chondrocytes showed no green fluorescenceand were visualised only by the methyl greenstaining of their nuclei. Pretreatment of sectionswith hyaluronidase had no effect on thedistribution of swine leucocyte antigen class IIexpression.A similar localisation was observed when

normal pig cartilage was cocultured withinflammatory synovium from an arthritic joint.

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Cytokine activation in porcine rheumatoidjoints

Figure 3 Toluidine blue staining offrozen sections ofcartilage (C)from pigs infected with Erysipelothrix rhusiopathiaeshowing (arrows) restricted loss ofmetachromasia (proteoglycan depletion) surrounding inflammatory sVnovial infiltrate (si)in mid zone cartilage.

Chondrocytes were activated by synovialmediators as already described but were notstimulated to express swine leucocyte antigenclass II.

DiscussionWe have developed a polyclonal antiserumwhich recognises phenotypic changes inducedon pig chondrocytes in response to interleukin1. Studies in vitro have already shown theusefulness of this antiserum to immunolocaliseindividual cytokine 'activated' chondrocytes insitu in cultured cartilage explants.5 6 We alsohave available the porcine erysipelas polyarthritisanimal model which has allowed us to use thisantiserum to investigate activation of chondro-cytes in vivo by cytokines secreted underpathophysiological conditions. An additionaladvantage of this model is that the diseaseprogresses to a chronic self perpetuating auto-immune disease making it a suitable animalmodel for human rheumatoid arthritis.8

In this study we have identified activatedchondrocytes in all samples of cartilage removedfrom arthritic joints of five pigs infected withErysipelothrix rhusiopathiae. As activation wasobserved only in diseased cartilage and wasmost marked at the cartilage/pannus junctionand surrounding synovial infiltrates, and inview of the specificity of the antiserum (Dingleand coworkers5 6and unpublished observations)we assume that the chondrocytes in the diseasedjoints were activated in response to interleukin 1secretion by the inflammatory synovium. Releaseof increased levels of cytokines including inter-leukin 1 has been reported in human rheumatoidsynovium 1 '" and we now contirm the presenceof interleukin 1 in porcine inflammatorysynovium.From our observations we can conclude that

at the particular stage of the disease studied inthese experiments sufficient interleukin 1 hadbeen produced in the arthritic joints to inducephenotypic changes in the chondrocytes inlocalised areas within the cartilage. Such changesmay represent an early event, reflecting theresponse of the cells to the episodic or low levelrelease of cytokines before the onset of persistentcytokine production'6 in the chronic stages ofthe disease. Certainly the integrity of thecartilage matrix in diseased joints, as assessedby minimal loss of metachromasia, suggests thatthe chondrocytes, though activated, were notyet in the process ofdestroying their surroundingextracellular environment.We currently have little information on the

nature of the surface markers induced byinterleukin 1 and recognised by the polyclonalantiserum. The only well characterised cytokine'activation' marker on chondrocytes so farreported is the MHC class II antigens. Thesemolecules have been shown to be expressed inhuman rheumatoid arthritic cartilage'7 and inresponse to the cytokine interferon y.' Class IIantigens have also been reported on rabbit'9 andrat20 chondrocytes following isolation fromnormal articular cartilage. Although we showedexpression of swine leucocyte antigen II (MHCclass II) on the inflammatory synovial cells wewere unable to detect this marker on thechondrocytes in cartilage from arthritic pigs.This result again implies limited cytokineproduction in the joints at this stage of thedisease. Our findings so far, however, haveencouraged us to undertake further experimentsto study cytokine production and the activationof chondrocytes at different times during theprogress of the disease to the chronic stage. Inparallel we are raising monoclonal antibodies tomake available additional markers of activatedporcine chondrocytes.

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In conclusion our observations using thepolyclonal antiserum in the erysipelas animalmodel have shown a novel way to simul-taneously monitor cytokine production andchondrocyte behaviour during the developmentof arthritis. It is anticipated that this facility willprovide new information to aid our under-standing of the pathogenesis of inflammatoryjoint diseases such as rheumatoid arthritis.

The antiserum raised against purified porcine interleukin 1 wasa gift from Dr J Saklatvala (Strangeways Research Laboratory).MED and AH thank the British Council for financial support.

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