Annals ofthe Rheumatic Diseases 1992; 51: 869-873

PathQgenic importance of fibronectin in thesuperficial region of articular cartilage as a localfactor for the induction of pannus extension on

rheumatoid articular cartilage

S Shiozawa, R Yoshihara, Y Kuroki, T Fujita, K Shiozawa, S Imura

AbstractTo identify the local factors in cartilage thatare responsible for the induction of pannusinvasion, a 14 day organ culture study inwhich rheumatoid synovium was grown incontact with cartilage pieces was carried out.Rheumatoid synovium preferentially extendedover hyaluronidase treated cartilage pieces,but detached from untreated pieces. Rheuma-toid synovium extended over hyaluronidasetreated cartilage surfaces containing fibro-nectin more extensively than over surfacestreated with hyaluronidase only. Extensionover hyaluronidase treated cartilage surfacescontaining immune complexes was small. Theadherence of synovial cells to hyaluronidasetreated cartilage slices in vitro was specificallyinhibited by the synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, which is the adhesiveportion of the fibronectin molecule. Further-more, synovial fibroblast-like cellular exten-sion, morphologically similar to rheumatoidpannus, was observed in the organ cultureexperiments in which rheumatoid synoviumgrew over hyaluronidase treated cartilagesurfaces containing fibronectin. Synovialtissue extension over fibronectin coatedsurfaces was inhibited when hyaluronic acidand chondroitin-4-sulphate, major com-ponents of cartilage proteoglycans, werepresent on the cartilage surface. Thesefindings suggest that fibronectin present inthe superficial region of cartilage potentiatesrheumatoid synovial extension and proteo-glycans and immune complexes inhibitrheumatoid synovial extension. It is likely thatfibronectin deposited on the eroded surface ofarticular cartilage induces pannus formationin rheumatoid arthritis.

(Ann Rheum Dis 1992; 51: 869-873)

Pannus invasion begins at the margin of articularcartilage that borders the junction betweensynovial tissue and cartilage. Pannus destroyscartilage either by growing over its surface' ordirectly penetrating the bare zone betweencartilage and synovial insertion, therebymigrating into the medullary space of bone.2On the articular surface of cartilage, pannusfirst extends as a morphologically quiescentlayer of fibroblast-like cells. This is followed bymatrix degradation by macrophage-like cellsunderneath this fibroblastic cell layer in theabsence of blood vessels. ' 3

Degradation of cartilage in the absence ofvascular supply suggests that local factors in thecartilage matrix might stimulate pannusformation. The most likely factors are immunecomplexes4 5 and fibronectin,6 detectable in thesuperficial region of rheumatoid articularcartilage in a disease specific manner.To identify the local factors in cartilage that

are responsible for the induction of pannusformation, an organ culture study allowing theinteraction of rheumatoid synovium withcartilage pieces was carried out. The resultsobtained indicate that fibronectin may be a localfactor responsible for rheumatoid pannusformation.

Patients and methodsSAMPLES AND REAGENTSSamples of rheumatoid synovium were obtainedduring joint operations on patients fulfilling thediagnostic criteria for classic rheumatoidarthritis.' Cartilage specimens obtained fromosteoarthritic knee joints were stored at -20°C.Cartilage specimens were trimmed to a flatshape, and freed of the original articular surfaceto remove any inequalities between the articularsurface and cut surface with regard to synovialderived tissue or deposited substances, or both.They were either left untreated, or treated with300 U/ml of bovine testicular hyaluronidase(Sigma Chemical, St Louis, MO, USA) at 37°Cfor three hours to remove proteoglycans fromthe cartilage according to previous results.6 Oneside of the cartilage specimens was treated witheither immune complexes, human fibronectin(Sigma), or proteoglycans (10 mg/ml hyaluronicacid and 3% chondroitin-4-sulphate) at roomtemperature for one hour (fig 1). After washingwith 0-1 M phosphate buffer, pH 7-2, smalllong cartilage pieces were cut out perpendicularlyto the flat cartilage surface. Horseradish perox-idase-antiperoxidase' (Dakopatts, Glostrup,Denmark) was used as the immune complex.Histochemical staining for the detection offibronectin on the cartilage surfaces was carriedout as described previously using antibodies tohuman fibronectin and the diaminobenzidinehydrogen peroxide reaction,6 and immunecomplexes were detected by directly applyingthe diaminobenzidine hydrogen peroxidereaction.The culture medium was Iscove's modified

Dulbecco's minimal essential medium (GibcoLaboratories, Grand Island, NY, USA) supple-mented with transferrin, soybean lecithin,

Department ofMedicine,Third Division,Kobe UniversitySchool of Medicine,7-5 Kusunokicho,Chuoku, Kobe 650,JapanS ShiozawaR YoshiharaY KurokiT FujitaDepartment ofMedicine andRheumatology,Kakogawa NationalHospital, Kannocho,Saijo, Kakogawa, JapanK ShiozawaS ImuraCorrespondence to:Dr Shiozawa.Accepted for publication4 November 1991

869

Shiozawa, Yoshihara, Kuroki, Fujita, Shiozawa, Imura

delipidated albumin, and 10% fetal calf serum(IMDM).9

ORGAN CULTURERheumatoid synovium, minced with scissors,was layered in the centre of a Millipore filter(HATF 02500, Nihon Millipore Kogyo KK,Yonezawa, Japan). Rheumatoid synovium wasattached to the ends of cartilage pieces. Theywere cultured in an organ culture dish (FalconNo 3037, Becton Dickinson, Cockeysville,MD, USA) in a humidified atmosphere of 5%CO2 at 37°C for 14 days (fig 1).

SYNOVIAL CELL CULTURE ON CARTILAGE SLICESCartilage specimens punched out in a cylindricalshape (4 mm diameter) were snap frozen.

Frozen cartilage 20 urm thick was cut within acryostat, attached to cover slips using 1%bovine serum albumin, and allowed to dry. Thecartilage slices were then treated with 300 U/mlof hyaluronidase at 37°C for three hours.Synovial tissue cells were dispersed by treatmentwith 2 mg/ml collagenase from Clostridiumhistolyticum (Wako Pure Chemicals, Osaka,Japan) at 37°C for 45 minutes and 0-05% trypsin(Flow Laboratories, McLean, VA, USA) at37°C for 30 minutes. After overnight culture,synovial adherent cells were harvested and laidon a cartilage slice (2 x 102/mm3 cartilage slice in10 1t IMDM). After incubation at 37°C for onehour they were cultured in a 24 well culture dish(No 25820, Becton Dickinson) by adding 1ml/well of IMDM in the presence of 0-6 mMfibronectin peptide (Gly-Arg-Gly-Asp-Ser-Pro)or its control peptide (Gly-Arg-Gly-Glu-Ser-Pro) (Iwaki Glass, Tokyo, Japan) at 37°C for sixdays.

Na Ic o n |

scove s MEMr1 FCS

Mi1 ip 1 it'

4

i..

Minced fhetirmatoi i-:v novitj .n

One side of the cartilage specimeis reacted vvith IC. Fn and!or Pfr

rhinr cartilage slice isOLU

L j Untreated cartilage

[ ]|Hyaluronidase treatedcartilage

MORPHOLOGICAL STUDYCulture was stopped by fixing specimens with0-5% glutaraldehyde at 4°C for 24 hours.Tissues were then observed with a Nikon SMZ10 stereomicroscope, or processed for de-hydration in graded alcohol and embedding inEpon 812. Sectiors 300 nm thick were cut andobserved under light microscopy with toluidineblue staining. The extent of synovial tissueextension over the cartilage surfaces wasmeasured in a stereomicroscope from thereverse side (the side attached to the Milliporefilter) of the tissue by placing a scale, accurate to0-1 mm, underneath the tissue (fig ID).

ResultsIn the organ culture experiments in whichrheumatoid synovium was reacted with cartilagepieces for 14 days (fig IA), rheumatoid synoviumpreferentially extended over hyaluronidasetreated cartilage pieces, but detached fromuntreated cartilage pieces (fig 2).To examine the part played by immune

complexes and fibronectin localised in thesuperficial region of cartilage matrix in pannusformation, the hyaluronidase treated cartilagesurfaces were covered with horseradish perox-idase-antihorseradish peroxidase immunecomplexes or with fibronectin (fig IB). Theywere then reacted with rheumatoid synovium.

Figure I Organ culture experiment. Rheumatoid synovium is grown in conitact withcartilage pieces on a Millipore filter at 37°C for 14 davs. One side of'the cartilage specimens is

treated with immune complexes (IC), fibronectin (Fn), andlor proteoglvcans (1;). T'hincartilage slices are cut out and positioned in contact with rheumatoid svnovium as shownin A-C. The distance ofrheumatoid synovial extension is measured as shown it 1).

Figure 2 Rheumatoid synovium extends on thehyaluronidase (HA) treated cartilage piece, but detachesfromuntreated cartilage after 14 days oforgan culture. Theoriginal portion of the ends ofthe cartilage piece is indicatedby dotted lines.

K

"/ 4.l

L77 i_,,

/// r/

.i r ~~Fr

F n Fn PG

.4

1-

A-

870

Fibronectinforpannus extension

Atter 14 days of culture, rheumatoid synoviumextended poorly over the immune complexcoated cartilage surfaces (table), but extendedover fibronectin coated cartilage surfaces moreextensively than over hyaluronidase treated cutsurfaces of cartilage (table). On the cut surfaceof cartilage, fibronectin endogenously presentin the matrix was exposed by the hyaluronidasetreatment, and the amount of fibronectin on thecartilage was additionally increased by treat-ment with fibronectin (fig 3; see also table 1 inShiozawa et al6).

A B C D E

Surface - -

Cartilagematrix

DAB DAB +antibody tofibronectin

Figure 3 Cut surfaces ofcartilage treated with: (A) 300U/ml hyaluronidase at 37°Cfor three hours, andsubsequently with horseradish peroxidase-antiperoxidaseimmune complexes; (B) and (C) with hyaluronidasealone;(D) with hyaluronidase and fibronectin; or (E) withhyaluronidase, fibronectin, and subsequently coated withhyaluronic acid and chondroitin4-sulphate. Immunecomplexes are visualised by directly applying thediaminobenzidine hydrogen peroxide (DAB) reaction inAand B. Fibronectin is visualised bv antibodies to humanfibronectin and theDAB reaction in C-E.

Rheumatoid synovial extension on hyaluronidase treatedcartilage in organ culture of rheumatoid synovium witha carttlage piece

Experiment I Experinient 2 Experiment 3

Fibronectin/nil' 1-60t 1-50 1-38Immune complex/nil 0 40 0 21 0-12Fibronectin and

proteoglycans/nil 0-42 0 01 0 51

''Hyaluronidase treated cartilage surfaces are either left untreated(nil), coated with fibronectin (fibronectin), coated with horse-radish peroxidase-antiperoxidase immune complexes (immunecomplex), or coated with fibronectin and then with hyaluronicacid and chondroitin-4 sulphate (fibronectin and proteoglycans).tRatio of synovial extension on one side of the cartilage (a) to theother (b) in fig ID.

To examine further the part played byfibronectin, a dispersed adherent p,pulation ofsynovial cells (2x 102/mm3) was cultured on ahyaluronidase treated cartilage slice in thepresence of the synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, which is the adhesive portion ofthe fibronectin molecule that specificallyinhibits binding of fibronectin to cells. ° Aftersix days of culture, synovial cell adherence wassignificantly inhibited by Gly-Arg-Gly-Asp-Ser-Pro, but not by the control peptide, Gly-Arg-Gly-Glu-Ser-Pro (fig 4). In the culture containingthe control peptides, synovial cells adhered toand extended with long dendritic processes onthe cartilage slice. In the culture containing thefibronectin peptides, the number of cellsattached to the cartilage slice was less than1/mm2 in three experiments.

Synovial fibroblast-like cellular extension,morphologically similar to rheumatoid pannus,was observed in the organ culture specimens ofrheumatoid synovium with the hyaluronidasetreated cartilage surfaces containing fibronectin(fig SA-C). Some cells beginning to invade thecartilage underneath the tissue were noted(arrow). This type of mesenchymal cellular

* -C~~~~~~~~~~~~~~~~~~.-t.-.....~~~~~~~~~~~~~~~~~~~~~.. I f

f..

:. ........i:

:: .:

B

..R-XgSi--:~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.....SI .~~~~~.jint~~~~~~ib'*~.C~~~~~~~~~~~~~~~~~- _

*:tjj ^ E~ -A_

Figure 5 Rheumatoid synovium extends on thehyaluronidase treated cartilage surface containingfibronectin. A continuous tissue section is shownfrom A to C.Cartilage matrix isfocally degraded underneath the tissue(arrow). Bar=50 ,um.

Figure 4 Synovial adherent cells (2 x 102-mm2) Cultured on a cartilage slice for six davs in the presence of0-6 mM ofeitherGly-Arg-Gvy-Glu-Ser-Pro, a control peptide (A) or (Glv-Arg-(ilv-Asp-Ser-Piro, a fibronectin peptide (B). Svnovial cellsextend with long dendritic processes and a cell cluster is formed on the cartilage slice in A, whereas onlv one cell is detectable inB. Bar=-50 im.

871

Shiozawa, Yoshihara, Kuroki, Fujita, Shiozawa, Imura

extension was not observed in the hyaluronidasetreated cartilage surfaces containing immunecomplexes (not shown).When the hyaluronidase treated cartilage

surfaces containing fibronectin were additionallycoated with hyaluronic acid and chondroitin-4-sulphate (major components of proteoglycans),artificially reproducing the normal cartilagesurface (fig IC), synovial tissue extension wassignificantly inhibited (table). Adherence wasgreater in the presence of fibronectin, except inthe presence of proteoglycans (fig 3E).

DiscussionPrevious studies have shown that rheumatoidcartilage surfaces are already eroded at the timeof pannus invasion." 12 It remains unclearwhether pannus invades the intact cartilage toerode its surface, or whether pannus extendsover an eroded surface. We have shown here, byusing hyaluronidase treated cartilage pieces inwhich proteoglycans had been partially removedfrom the cartilage surface,6 that rheumatoidsynovium preferentially extends on the hyal-uronidase treated cartilage surfaces. Further-more, when the hyaluronidase treated cartilagesurfaces were covered by the components ofproteoglycans, hyaluronic acid, and chondroitin-4-sulphate, the extension of the rheumatoidsynovium was inhibited. These results suggestthat proteoglycans in the cartilage matrix inhibitrheumatoid synovial extension. Thus, inconjunction with the previous observation thateven the normal appearing joint surfaces ofrheumatoid cartilage are already eroded whenexamined under an electron microscope, 12-14 itis likely that pannus extends only on an erodedarticular cartilage.

It has been suggested that immune complexespresent in the superficial region of rheumatoidarticular cartilage might be an important factorin the invasion of the cartilage by pannus.4 5

The area covered by pannus failed to showimmune complexes, however, even in the areasimmediately adjacent to the advancing edge ofthe pannus which, presumably, had only recentlybeen invaded by the granulation tissue.'4 Thissuggests that immune complexes, even thoughthey may be conveying an initial signal topannus formation, cannot be the stimulus forthe advance of the pannus.3 The present obser-vations showing that immune complexes arepoor inducers of rheumatoid synovial extensionmay be compatible with this. In addition, thefinding is consistent with the previous obser-vation that patients with hypogammaglobulin-aemia presented with destructive arthritis farmore often than expected from their smallamounts of immune complexes.' 5 It thusappears that immune complexes sequestered inthe superficial region of rheumatoid articularcartilage do not play a major part in theinduction of pannus extension onto cartilagesurface.Rheumatoid synovium extended on the

hyaluronidase treated cartilage surfaces contain-ing fibronectin more extensively than on thesurfaces with hyaluronidase treatment alone.The attachment of synovial cells on the cartilage

matrix in vitro was inhibited by the fibronectinpeptide, Gly-Arg-Gly-Asp-Ser-Pro, whichspecifically inhibits binding of fibronectin tocells,'° indicating that fibronectin in the cartilagematrix is required for the adherence andextension of synovial cells. Furthermore,synovial fibroblast-like cellular extensionsimilar to rheumatoid pannus was experimentallyreproduced in vitro by allowing rheumatoidsynovium to grow over hyaluronidase treatedcartilage surfaces containing fibronectin. Thesefindings suggest that fibronectin, present in thesuperficial region of cartilage, potentiatesrheumatoid synovial extension. Fibronectin isendogenously present in the cartilage matrix,'6and can, therefore, be a source of continuousstimuli for pannus formation. Studies haveshown that fibronectin is present in largeamounts in the rheumatoid joint,17-20 and that itis detectable in the superficial region of therheumatoid articular cartilage.6 Fibronectin inthe cartilage matrix appeared to be revealed bypartial degradation of proteoglycan by proteo-lytic enzymes in the synovial fluid, as well as bythe deposition of fibronectin on the cartilagesurface.6 It thus appears that fibronectindeposited on the eroded surface of rheumatoidarticular cartilage potentiates pannus invasionin vivo.

Studies using in vitro culture of synovial cellswith collagenous matrix have shown that col-lagenase plays a central part in the promotion ofjoint destruction, in which the interactionbetween plasmin, an enhancing factor, and thetissue inhibitor of metalloproteinases, aninhibitory factor, regulates its final outcome.2'Fibronectin present in the collagenous matrixappears to promote matrix degradation bycollagenase by enhancing the attachment of

3 22pannus cells to the matrix.

This work was supported by the Japan Ministry of Education,Science and Culture grant (S.S). We thank our teacher, ProfessorMorris Ziff, for useful advice.

1 Shiozawa S, Shiozawa K, Fujita T. Morphological observationsin the early phase of the cartilage-pannus junction, Lightand electron microscopic studies of active cellular pannus.Arthnitis Rheum 1983; 26: 472-8.

2 Fassbender H G. Joint destruction in various arthriticdiseases. In: Kuettner K, ed. Articular cartilage biochemistrv.New York: Raven Press, 1986: 371-89.

3 Shiozawa S, Shiozawa K. A review of the histopathological.evidence on the pathogenesis of cartilage destruction inrheumatoid arthritis. Scand.j Rheumatol Suppl 1988; 74:65-72.

4 Cooke T D, Hurd E R, Ziff M. Identification of immuno-globulins and complement in rheumatoid articular col-lagenous tissues. Arthritis Rheum 1975; 18: 541-51.

5 Ishikawa H, Smiley J D, Jasin H E, Bienenstock J, Ziff M.Electron microscopic demonstration of immunoglobulindeposition in rheumatoid cartilage. Arthn'tis Rheum 1975;18: 563-76.

6 Shiozawa K, Shiozawa S, Shimizu S, Fujita T. Fibronectinon the surface of articular cartilage in rheumatoid arthritis.Arthritis Rheum 1984; 27: 615-22.

7 Arnett F C, Edworthy S M, Bloch D A, et al. The AmericanRheumatism Association 1987 revised criteria for theclassification of rheumatoid arthritis. Arthritis Rheum 1988;31: 315-24.

8 Sternberger L A, Hardy P H Jr, Cuculis J J, Meyer H G. Theunlabeled antibody enzyme method of immunohisto-chemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradishperoxidase) and its use in identification of spirochetes..7 Histochem (jvtochem 1970; 18: 315-33.

9 Shiozawa K, Shiozawa S, Shimizu S, Fujita r. Ilt,25-dihydroxyvitamin D3 inhibits pokeweed mitogen-stimulatedhuman B-cell activation: an analysis using serum-freeculture conditions. Immunologv 1985; 56: 161-7.

10 Pierschbacher M D, Ruoslahti E. Cell attachment activity offibronectin can be duplicated by small synthetic fragmentsof the molecule. Nature 1984; 309: 30-3.

11 Chaplin D M. The pattern of bone and cartilage damage in

872

Fibronectinforpannus extension

the rheumatoid knee. j Bone joInt Surg lgr/ 1971; 53:711-7.

12 Kimura H, Tateishi H, Ziff M. Surface ultrastructure ofrheumatoid articular cartilage. Arthritis Rheum 1977; 20:1085-98.

13 Mitcheli N, Shepard N. The ultrastructure of articularcartilage in rheumatoid arthritis. A preliminary report.J Bone J7oint Surg [AmI 1970; 52: 1405-23.

14 Shiozawa S, Jasin H E, Ziff M. Absence of immunoglobulinsin rheumatoid cartilage-pannus junctions. Arthritis Rheum1980; 23: 816-21.

15 Good R A, Rotstein J. Rheumatoid arthritis and agamma-

globulinemia. Bull Rheum Dis 1960; 10: 203-6.16 Weiss R E, Reddi A H. Appearance of fibronectin during the

differentiation of cartilage, bone, and bone marrow. CellBiol 1981; 88: 630-6.

17 Carson S, Mosesson M W, Diamond H S. Detection andquantitation of fibronectin in synovial fluid from patientswith rheumatic disease. Arthritis Rheum 1981; 24: 1261-7.

18 Vartio T- Vaheri A, von Essen R, Isomaki H, Stenman S.Fibronectin in synovial fluid and tissue in rheumatoidarthritis. EurJ Clin Invest 1981; 11: 207-12.

19 Scott D L, Delamere J P, Walton K W. The distribution offibronectin in the pannus in rheumatoid arthritis. BrJ ExpPathol 1981; 62: 362-7.

20 Shiozawa S, Ziff M. Immunoelectron microscopic demon-stration of fibronectin in rheumatoid pannus and at thecartilage-pannus junction. Ann Rheum Dis 1983; 42:254-63.

21 Reynolds J J, Lawrence C E, Gavrilovic J. Model systems forstudying the destruction of the extracellular matrix andtheir usefulness in testing inhibitors. In: Glauert A M, ed.The control of tissue damage. Amsterdam: Elsevier, 1988:281-%.

22 Shiozawa S, Tokuhisa T. Contribution of synovial mesen-

chymal cells to the pathogenesis of rheumatoid arthritis.Semin Arthritis Rheum 1992; 21: 267-73.

873