Immunolocalization of cathepsin B in equinedyschondroplastic articular cartilage

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T/.' I}'h'u..ry fl,m.al I!1%%156, 193-201

Immunolocalization of Cathepsin B in Equine Dyschondroplastic Articular Cartilage ( ;. I I ERN,\NI)I'~Z-\q

I)AI., 1,. Ik,IH:F( :( )'1+1 "* :rod M. E. I)AVIES

.S'lraH+~qTt,aJ~I~v,w'mch I.abmalm'L |'|'orl~'('aH.w'way, (:amln'id£rr (:BI -tlLV. I ~h'." *l :nit,rr~ily O/'('ambrid.ffe, l)e/mrlmeHI o/Climcal 'eh'6.mX A h'diHm', Madi.gV O' I¢oad, Cambrid,g~r ('B ~ OI'S, I :K

SUMMARY

A I)olychmal a u t i s c r u m raised in s h r r p a e,+tinst h u m a n c,tthrl)sin B was tcslcd for Sl)ccilicity and cross-,eactivitv w i t h t h e h ( ) r s c h(>m<>l<)gtlc I)y S D S - P A ( ; E n n d W e s t e r n 1)lollillg, p r i o r to l ) c i n g u s e d f o r i m m t m o h w a l i z a t i ( m of tht" e n z y m e in cquint" articular c.u'lilagc. In Western I)h)ts, the antis<+'runl rccognizc(I the 3()kl)a sin,gh." oh,tin and 25kl)a hcavv chai,1 (ff th<." m,mu+r enzyme iu l)uril]cd bovi,+w c,tlh,+:psirl B, and co,r,.'Sl)o,lding hands at ?,~ and 27kDa iu cqt,inc ch(m(l,'ocvtc aud l]l)rol)Insl Ivsalcs. This antist'rtH+n was lhcn usrd l~> cO,nl)m'c th,+' ('Xl)rcssion ,m(l dislrihulirm:tl in thr (Ircp zl th<." <_'n/vmc in ('hcvtcs isol:ttr(l lrom r.:,rm+d c:u'tihtg+¢` (.=6). l)ul increased prst<+'ochondrosis.

INTRODUCTION

(~:tthcl)sin B, ,t Ivsosom,tl ('vslcinc i)rolc.inasc, is Found in re,my tissues .tnd i)htys an i m p o , t a n l rolr in intracclhtlar p,(>lc(flysis, ,mtigcn proccssine, and ;.lUglllC'lllcd pr()lcin t t n n o v c r in a Val'it'lv o[" l)alho logical conditions (Komin,tmi rl a/.. 1991). Mt,ch inlcrcsl h,ts rcccnth' focused on the r()]c of calhcpsin B in cartilagc m a t r i x t u r n o v e r , lolh)win,g the disc()vcrv that this e n z y m e dircctlv mc(liatcs cartil a g e a t t l o d c , ~ r a d a l i o n a n d c o n t r i b u t c s to l h c d e s t r u c t i o n o[ the c,u+tilagc c x t r a c c l l u l a r m a l t i x (E(;M) iu inlla,nmat(wy arthritis (Van No()rflcn e/ a/., 1988; Buttlc el al., 1997,, 1995).

(hwrcsl)ondcncc Io: M. E. Davir~,. thfi','cr,dt', ol (:amlwi(IRr. Drl)m'inwnl ol (:linic'alVrlrrin,wv Xh'dichw..MadinRh'~ Road. (:amhridRc (:I~,3I)ES.U K. Fax: I)IL)'_)3337 (iII). I090-0~37, '.qM:0li0193+(}9SI~.(R).'I)

T h e p r e s e n c e ~I cathcpsin B has l)ccn d c u . m s m . c d by several g r o u p s (Bayliss & A I i , 1978: V , m Noordcn & Vogcls, 1986; Baici el al., lC)95a, l); Hc'l+nfindcz-Vidal e/al., 1996). T h e e n z y m e has l)c'cn

hw.dizcd within the cho,~d,+ocylcs, whcrc it is svuthcsizcd and slorcd l},w use iu i,ltraccllular protc.ill d c g r a d a l i o n associated with n o r m a l matrix turnover. A h h o u g h the m~!jor rolc of cathcpsin B is as am inlrac<.'llular lvsosomal digcslivc cnzymc, s('<:lt'lion <:h)cs o c c u r u n d c r p a l h o h ) g i c a l c o n d i l i o u s (Mort el al., 1984; (.al),!jcl("i(" el al., It.)90; Trabandt e/a/.. 1991 ). I . vi/r0 studies conli rm that cathc'pshl B can degrade both p,otcoglyca,~ subunits (aggrccau) ;rod link p,'otci,I (Nguycu el al., ]c)gl)). h also clcavc's collagcn typc's I (Bu,lcigh el al.. 1974), II. IX and X1 (Macicwicz el al., 1991) and X (Sires el al., 1995). (;athepsin B is also known to bc capable of trigg<.'rin,g a casc'ach.' of degradative pathways in cartilage, by a c t i v a t i n g proenzymcs s u c h as © 199~ Bailli&c Tindall

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THE VETERINARY JOURNAL, 156, 3

pro-stromelysin, pro-collagenase and pro-plasminog e n a c t i v a t o r (Peele, 1993; Buttle e[ al., l~-)q~ Thus, a p r o m i n e n t role has been p r o p o s e d for tile cysteine proteinases in ECM turnover, in collaboration with tile aspartic and matrix m e t a l l o p r o t e i n a s e s . Suggestive e v i d e n c e for tile involvement of cathepsin B in cartilage and b o n e p a t h o l o ~ ' has bccn provided in studies using the selective cysteinc proteinase inhil)itors Z-Phe-AIaCH,2F and peptidyl methyl t h m r o k e t o n e (Van Noon-den el al., I t388; ' Esser el al. 1994), w h e r e a s tile speci['ic i n h i b i t o r o f c a t h c p s i n B, CAO74Mc, prevented interlcukin-l-induced release o f proteoglycan fronl cartilagc c u h u r e d in vitro (Buttlc el al., 1993). T h e s e findings imply an i m p o r t a n t role of this enzyme both in hernial connective tissue ttlrllover and in tilt" tissue d a m a g e associated with joint disease. Catllepsin B has als<> been implicated in the clegradation
cathepsin B-rich cell, the sepioclast, has been identitled in growth plate cartilage (l+ee e/ a/., 1995). Although secretion of tile' +llZ'¢llle bv scptoclasts has not vet beell dt'mOllstr;_ited, its role in the resorption of tilt' trailsverse septa, which lie directly al the interl+ace I:>ctwecn the elfiphyseal growth plate and metal)hysis, is implied (Lee el a/., 1993). D)'schondropiasia, tile early stilge
attenlpt to establish a possible role fi>r this enzvnle in tile pathogenesis o f dyschondr
MATERIALS AND METHODS A n tisera T h e polyclonal anti-cathepsin B serum used in this study was kindly d o n a t e d by Dr D..]. Bttttle (University o f Sheffield Medical School). T h e a n t i s e r u m was i+aised in s h e e p against c a t h e p s i n B p u r i f i e d from httman liver and characterized its prcviously described (Bttttlc el al., 1988). Prior to time in this sltidx', the specificity and cross-rcactivitv of tile antis c r m n with e q u i n e c a t h c p s i n B wcrc c h e c k e d by SDS-PA(;E a n d W e s t e r n b l o t t i n g . F l u o r c s c c i n (FITC)-colljugatcd rabbit anti-sheep immtmogh)bulins were from Dako, UK. Calhepsin B Purified bovinc spleen cathcpsin B (E(: 3.4.22.1) was purchased from Sigma. Crude preparations of equine cathcpsin B were obtained using cell Ivsates p r e p a r c d from horse ch
(;ATIIEPSIN B IN DL'SCHONDROPLASTIC EQUINE ('ARTILA(;E mice, cartilage llaps) were e l i m i n a t e d f r o m the investigation. Cartilage samples fi'om live o f these horses were used for detailed immunocytoclaemical analysis, samples from the o t h e r two were ttsed for isolation of chondrocytes.

Isolation and propagation of articular chondroo, tes C h o n d r o c y t e s were isolated u n d e r sterile conditions from normal cartilage and from three samples o f dyschondroplastic cartilage by enzymatic digestion, a n d c u l t u r e d in n a o n o l a y e r s as p r e v i o u s l y described (HernfindczA:idal el al., 1997). C h o n d r o cvie p h e n o t y p e was m o d u l a l e d to i n d u c e dedilterentiation by serial sul)cuhtwe of tD'psinized m o n o l a v e r s essentially as d e s c r i b e d by Baici el al. (1995b). T h e p r e s e n c e o f calhepsin B was monit o r e d in p r i m a r y c u l t u r e s a n d in s u b s e q u e n t passages by intmun()cvtochemistrv. ( ; h o n d r o c v t e Ivsates for use as a c r u d e c,tthepsin B preparatio,i were made by h o m o g e n i z a t i o n in Ivsis buffer containing a selected inhibitor cocktail (Hernfindez-Vidal el aL, 1997).

Isolation and pro[)agalion of skin fibroblasts For c o m p a r i s o n p u r p o s e s , fibroblasts (known to contain cathepsin B, Everts el al., 1994) were isol a t e d fr()m s h a v e d s k i n s l i c e s u n d e r s t e r i l e c<)ndilio,,s its prcviousl.v d e s c r i b e d ( H c r n f i n dez-Vidal el al., 1997). Fibr()blasts were sul)cuhured from the o r i g i n a l o r g a n c u l t u r e until suft]cient n u m b e , s were o b t a i n e d ti," cell lysate preparation and for intnnmocytochemistry.

SI)S-I'~AGI'~ and Western blotting S o d i u m dodecylsulplaate-i)olyacrylamide gel electropho,-esis (SDS-PA(;E) was p e r f o r m e d using the m e t h o d of Laemmli (1970). Puritied bovine cathepsin B and c r u d e enzynte p r e p a r a t i o n s in lysates ( ---4x 107 cells.gel lane -1 ) of chondrocytes and fibroblasts were s e p a r a t e d by e l e c t r o p h o r e s i s , and the p r o t e i n s were t h e n trallsferl'ed to n i t r o c e l l u l o s e m e m b r a n e s (BioRad) for Western blot analysis, as previously described (He,'nandez-Vidal el al., 1997). Standard ntolecular weight ntarkers (Sigma) were also rt|n.

hnmunoo, lo<'hemi.slly Sections o f cartilage were cut at a thickness o f 68Urn, mounted on poly-L-lysine (Sigma)-coated slides, and air dried for 5-]Omin at room temperature. T h e c h o n d r o c y t e and tibroblast m o n o l a y e r s were fixed i,t 4% p a r a t o r m a l d e h y d e , pH 7.4, for

195

301nin. C a t h e p s i n B was i m m u n o l o c a l i z e d with s h e e p a n t i - h u m a n cathepsin B (1:50) as p r i m m T antibody and tluo,'escein (FITC)-conjugated rabbit anti-sheep immunoglol)ulins (1:200) as secondary antibody, using the protocol previously described (Hernandez-Vidal el al., 1997). T h e inamunofluorescent staining was viewed with a Nikon Diaphot l i g h t m i c , ' o s c o p e f i t t e d with e p i f l u o r e s c e n c e (Hernfmdez-Vidal el aL, 1997). Positive staining of cells was recognized as bright apple-green fluorescence. Positive cells were quantified ,nicroscopically by c o u n t i n g , which was usually p e r f o r m e d by two i n d e p e n d e n t assessors. Negative cells were visualized by the red nuclear cotmter-stain and counted. Six fields per cell m o n o l a y e r or per cartilage zone were selected at r a n d o m and scored. T h e percentage p o s i t i v e cells was c a l c u l a t e d . S a n t p l e s o f cartilage trom five of the seven horses with dyschondroplasia were fully analysed.

Histolr~©, Paraffin sections (6--81.ma) were stained with toluidine blue and haematoxvlin and eosin according to established procedures.

RESULTS

Specifici O, and cross-reacltvi(~, of anliser~tnl with equine cathepsin B Purified bovine cathepsin B ran characteristically (Buttle et al., 1988) on 15% reducing SDS-PA(;E as two bands r e p r e s e n t i n g the single chain (30kDa) and the hea D, chain (25kDa) of the two-chain form of the mature enzyme [Fig. 1 (a) ]. Equivalent bands could not be distinguished in the crude extracts of the e q u i n e honaologue of the e n z y m e from chondrocyte and fibroblast lysates, which both exhibited p r o m i n e n t multiple protein banding. W e s t e r n blots [Fig. 1 (b)] d e m o n s t r a t e d two intensely stained bands, confirming that the a n t i - h u m a n c a t h e p s i n B seruna r e c o g n i s e d b o t h chains of bovine cathepsin B. T h e antiserunl also ,'eacted with slightly higher molecular weight bands o f approximately 32 and 27kDa, correspo,lding to the horse hom,)h,gue in the c h o n d r o c y t e and t]broblast lysates. T h e weak staining reaction seen with these two bands presumal)ly rellects the low conc e n t r a t i o n o f cathepsin B in the u n e n r i c h e d cell lysates. Since no o t h e r protein bands in these crude prepa,'ations were recognised, the a n t i s e r u m was c o n s i d e r e d to be s p e c i f i c f o r c a t h e p s i n B, to

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THE VETERINARY JOURNAL, 156, 3 (a)

Cath B Bovine

Mr

BSA

(b)

Horse Chond Fibro

Cath B Bovine

Horse Chond Fibro

I

W

30-30--

25--

25-~llll~

°

Fig. 1. (a) SDS-PA(;E of purified bovine cathepsin B and crude enzyme 19reparations ti'om horse chondr(wyte and Iibroblast lysates. Bovine seruna albumin (BSA) and stanclatxl m()lecular weight markers are indicated. (b) Western blot of purified bovine cathepsin B and crude enzy,ne li'om horse claonch'ocyte aqcl fil)roblast lysates. Standard molecular weight markers are indicated (Mr). cross-react with the e q u i n e e n z y m e a n d t h e r e l b r e to be a suitable a n t i s e r u m t:or i m m u n o c y t o c h e m i c a l studies.

itive cells c o u l d he d e t e c t e d . M o r e s t a i n i n g was p r e s e n t at the a r t i c u l a r s u r f a c e , w h e r e a p p r o x i mately 20% of the chondrocytes were weakly positive. Since the distribution of cathepsin B i m m u n o s t a i n i n g was similar in cartilage fi'om all six n o r m a l horses e x a m i n e d , c o n f i r m i n g data f r o m a previous preliminary study (Hernfindez-Vidal el al., 1996), the o b s e r v a t i o n s have b e e n c o m b i n e d a n d p r e s e n t e d as a typical n o r m a l in T a b l e I. Elevated levels o f c a t h e p s i n B i m m u n o r e a c t i v i t y were d e t e c t e d in all cartilage samples e x a m i n e d in detail fi'om the five horses with d y s c h o n d r o p l a s i a c o m p a r e d to n o r m a l cartilage (Table I). T h e p r o m -

Cathepsin B distribulion in equine articular cartilage In the s a m p l e s o f cartilage o b t a i n e d fi'om n o r m a l joints, a distinct zonal distribution o f c a t h e p s i n B was o b s e r v e d . S i g n i f i c a n t i m m t t n o r e a c t i v i t y was p r e s e n t only in the d e e p zone, w h e r e strong intracellular staining was seen in a p p r o x i m a t e l y 60% of chondrocytes. In the mid-zone, c h o n d r o c y t e s were essentially negative, a l t h o u g h r a n d o m i m m u n o p o s -

Table I Zonal distribution of cathepsin B in equine growth cartilage

Ca/hepsin B Staining Staining Staining Staining

in in in in

articular zone mid zone deep zone clusters

N

63

68

56

99

64

+

_+

_+

+

+

+

+

+

_+

+

+

+

+++

++++

++++

++++

++++

++++

+++++

+++++

+++++

No clusters

No clusters

+++++

+=<10%; +=10-30%; ++=40-50%; +++=50-70%; ++++=70-90%; +++++=>90% cells staining positive. N, combined data f'ronl six normal horses; 63, 68, 56, 99, 64-dyschondroplastic horses.

('ATHEPSIN B IN DYSCHONDROPLASTIC EQUINE CARTILAGE

(a)

197

(b)

~ Y,

•

I

i

7~ N\

~

t

e

I

#

(d)

(f)

Fig. 2. hmnunolocalization and hislochenfical staining ill sections of (a-d) dyschondmplastic ca,-tilage and (e. t) isolated dyschondroplastic chondrocytes. (a) Fluo,-escent immu,lostaining of cathepsin B in chondrocyte clttsters in tht4 deep zone. (b) Haematoxylin and eosin staining of chondrocyte clusters in the deep zone. (c) Tohfidine blue staining of chondrocyte clusters at the articular surlhce. (d) Fluorescent immunostaining ofcathepsin B in normal early deep zone. (e) Fluorescent immunostaining of cathepsin B in primary culttu-es of dyschondroplastic chondrocytes. (.tO Control, showing flt,orescent immtmostaining of isolated chondrocytes with normal sheep serum.

inent feature of dyschondroplastic cartilage was the presence (in 4 out of the 5 horses) of chondrocyte clusters which stained intensely for cathepsin B [Table I, Fig. 2(a)]. It was estimated that >90% of

the chondrocytes in these clusters were immunopositive. Histological examination showed that the clusters consisted of tightly packed groups of cells of varying size enclosed within one lacuna. Metach-

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romatic staining of the matrix in the areas of cluster formation was noticeably reduced. Although usually found associated with foci of abnormal cartilage in the deep zone [Fig. 2(b)], randomized areas of clusters, extending through the mid-zone to the articular surface, were also seen [Fig. 2(c)]. The rather scattered arrangement of the clusters contributed to the general lack of zonal organization seen in tile cartilage, and gave rise to variahility in the distribution of cathepsin B between the different dyschondroplastic cartilage samples. This was in s h a r p c o n t r a s t to tile m o r e t ~ n i f o r m z o n ally-restricted disn'ibution in normal cartilage. Moving away fi'om the lesions, areas of apparently normal cartilage devoid of chondrocyte clusters were seen. Here, the zoning of the cartilage was more distinct and the distribution of cathepsin B immunoreactivity resembled that in normal cartilage. The staining patterns in the three zones of these normal areas of cartilage are given in Table I for five different dyschond,-oplastic horses (numbers 56, 63, 64, 68, 99). It can be seen that tile number of cells staining positively for cathepsin B was increased fi'om <10% in normal cartilage to a p p r o x i m a t e l y 20% ( r a n g e 1 0 - 3 0 % ) in t h e mid-zone in three of the dyschondroplastic cartilage samples. In the deep zone, the n u m b e r of positive cells was increased to approximately 80% (range 70-90%) compared to normal (60%) in all samples of dyschondroplastic cartilage.

Cathepsin B in chondrocyte monolayer cultures PrimmT cuhures of chondrocytes isolated fi'om normal cartilage were found to contain little cathepsin B (Fig. 3). In five s e p a r a t e e x p e r i m e n t s , only approximately 10% of chondrocytes in primary monolayers stained positive. In these cells, staining was conspicuously granular, with a cytoplasmic distribution similar to that seen in subcuhured skin fibroblasts. The rather weak and diffuse intracellular staining seen in some cells was non-specific, as d e m o n s t r a t e d in c o n t r o l s using n o r m a l sheep serum [Fig. 2(f)]. During subcuhure of the chondrocytes, the p r o p o r t i o n of cells with positively stained cytoplasmic granules gradually increased, with staining reaching a maximum at >80% positive at the 5th-6th passage (Fig. 3). Dyschondroplastic cartilage was obtained in sufficient amount from three horses (12 months old) to allow isolation of enough chondrocytes for monolayer c u h u r e . In all the samples e x a m i n e d , the majority of chondrocytes in primary cuhures con-

100

,n ¢a

.~ 80 ..= ._~ 60

& 40 o

~ 20

0

0

1

2

3 4 Passage no.

5

6

Fig. 3. Perccnlagc of chondrocytes staining positively for cathcpsin B during serial monolayer subcuhtwe. Passage 0=prilnary cuhurt'. Values are the inean_+sl) (~1=5).

rained large numhers of positively stained cytoplasmic granules [Fig. 2(e)] comparable to those seen in normal cells after several subcuhures.

DISCUSSION

Tile presence of the cysteine proteinase, cathepsin B, ill the deep zone of normal equine articular cartilage was d e m o n s t r a t e d in a preliminary study (Hern~ndez-Vidal el al., 1996). We now report that levels of cathepsin B are elevated in articular cartilage fl'om joints of horses with dyschondroplasia compared with normal heahhy equine articular/ epiphyseal cartilage. Levels of cathepsin B were increased in the deep zone in areas of apparently normal cartilage, but tile enzyme was particularly abundant in chondrocyte clusters, which were evident in four of tile five dyschondroplastic lesions e x a m i n e d in detail. A l t h o u g h these cathepsin B-containing clusters were predominantly associated with areas of abnormal cartilage in tile deep zone, their presence also in the mid-zone and at the articular surface was remarkable. This present investigation is tile first to report on tile distribution of this enzyme in pathological cartilage from dyschondroplastic lesions in the horse. At present, the importance of tile high level of c a t h e p s i n B in t h e c h o n d r o c y t e c l u s t e r s is unknown. Since cluster Ik)rmation is one of tile characteristic features of equine dyschondroplastic lesions (Savage el al., 1993), it is not unreasonable

CATHEPSIN B IN DYSCHONDROPLASTIC EQUINE CARTILAGE to suggest that the presence of cathepsin B in these structures may have some p a t h o g e n e t i c significance. C h o n d r o c y t e clusters, which d i f f e r in morphology and distribution from hypertrophic c h o n d r o c y t e s (Poole el al., 1991; Henson el aL, 1997) are present in other pathologies, particularly osteoarthritis (OA) (Pool & Meagher, 1990; Poole et aL, 1991; Baici et aL, 1995a, b). However, despite many clescriptions of their occurrence in both natural and experimental OA cartilage, little is known of the mechanism of their lormation or of their 11mction. As early as 1970, Mankin a n d L i p p i e l l o suggested that the reduced levels of proteoglycan observed in the cellular microenvironment trigger chondrocyte metabolism and proliferation, resulting in c l u s t e r f o r m a t i o n . Poole el al. (1991) proposed a role for these hyperplastic chondrocytes in progressive r e m o d e l l i n g resulting in gradual depletion of pericellular proteoglycan. Alternatively, the suggestion that the clusters are inw)lved in local repair processes at sites of OA cartriage damage was made by Baici et aL (1995a, h). It is certain that enzymes are involved in these ECM remodelling events in OA, but it has proved difficult to identify the enzyme(s) and define the initial steps. Attempts have been made to demonstrate enzyme-induced degradation of type I1 collagen in the cellular micro-environment (Dodge & Poole, 1989), and changes in the organization of pericellular collagens VI and IX have also been reported (McDevitt & Miller, 1989). Based on these observations, we suggest that the c a t h e p s i n B, p r e s e n t in increased a m o t l n t s in equine dyschondroplastic cartilage clusters, could play some as yet unknown part in ECM turnover in this disease. It is of interest that Baici et al. ( 1995a, b) also found high levels of cathepsin B in human OA clusters and proposed a role for the enzyme in cartilage r e g e n e r a t i o n . The m e c h a n i s m s by which cathepsin B exerts an enzymatic action in cartilage are not understood. From in vitro experiments, it is k n o w n t h a t c a t h e p s i n B has the p o t e n t i a l to degrade most of the major cartilage ECM components, including proteoglycan (Nyugen et al., 1990) and collagen types II, IX, X and XI (Maciewicz et al., 1991; Sires et al., 1995). Following discovelT of this e n z y m e in cartilage (Ali, 1967), studies have focused primarily on its potential to degrade the ECM in rheumatoid and osteoarthritis in humans (Ali & Bayliss, 1975; Martel-Pelletier et al., 1990; Trabandt et al., 1991; Gabrijeif:ie et al., 1990). Nothing

199

is k n o w n o f its p o t e n t i a l r o l e in e q u i n e dyschondroplasia. A l t h o u g h able to d e g r a d e proteoglycan, it is unlikely that cathepsin B is directly responsible for the low levels of this matrix component, as reflected by the reduced metachromatic staining often seen in the ECM adjacent to dyschondroplastic clusters. In the many cartilage samples e x a m i n e d in this study, no evidence was found of release of cathepsin B into the matrix surrounding the clusters. Depletion of proteoglycan by other degradative enzymes such as serine and metalloproteinases and 'aggrecanase' (Henderson & Blake, 1994; Buttle et al., 1995) c a n n o t be e x c l u d e d ; alternatively, the loss of m e t a c h r o m a s i a may indicate sites of localized reduction in proteoglycan synthesis. In dyschondroplastic cartilage, cathepsin B was detected intracellularly. It is much more likely, therefore, that it performs a fimction within the cell, presumably in the lysosomal granules (Kominami et aL, 1991). A strong possibility is that it is responsible for the final intra-lysosomal stages of proteolysis of extracellularly cleaved ECM components, fragments of which are internalized by the c h o n d r o c y t e s . Such a role for cathepsin B has already been proposed in the degradation of collagen type VI fi'agments internalized by fibroblasts (Everts el al., 1994) and tor type X collagen fi'agments in osteoclasts (Sires et al., 1995). It would be of great interest to know w h e t h e r cathepsi,a B degrades types VI and X in chondrocytes, since both of these collagens are relevant to the pathology of d y s c h o n d r o p l a s i a . H e n s o n et al. (1997) previously reported that type VI collagen is abnormally distributed and upregulated in d y s c h o n d r o p l a s t i c clusters, a f i n d i n g in broad agreement with observations in OA clusters (McDevitt et aL, 1988; Kuettner, 1992). Type VI collagen is t h o u g h t to have a s t r u c t u r a l role in the ECM (Bonaldo et al., 1990; Stallcup et al., 1990), and may be upregulated in dyschondroplasia at sites of ECM weakness within the lesions (Henson et aL, 1997). Others have shown that type X collagen, as well as being transien'tly expressed by hypertrophic chondrocytes during endochondral ossification, is also overexpressed in OA clusters (yon der Mark et aL, 1995). This distribution pattern has led to the infere n c e t h a t t y p e X has a d i r e c t f u n c t i o n in calcification of the cartilage (Iyama et aL, 1991; Kitsch &von der Mark, 1991), in addition to providing exu'a mechanical reinforcement in the growth plate d u r i n g r e s o r p t i o n of the m i n e r a l i z i n g m a t r i x

200

TIlE VETERINARY.]()LIRNAL,

( A s p d e n , 1994; R u c k l i d g c el aL, 1 9 6 6 ) . W i t h r e s p o n sibility for e i t h e r o f t h e s e functiol~S, t y p e X c o l l a g e n clearly has all i m p o r t a n t r o l c in the process o f c n d o c [ l o n d r a ] ossi|ic,ltion. Aiiolll~,l]OtlS ttll-ilOVtW o f thcsc two collagens, p a r t i t ' u l a r l y o f iypc X, as a rcsull pcrhaps o f a l ) n o r i n a l calhel)sin B activity, n l i g h i lca(I to a [,lihu'e o f c'n(tocholldt+al (>ssi|]t'ation ; . l l l d / o l H) weakness in the g r o w t h plate, two ch,u'actt'ristics which define dyschondrop]asia. The suggeslion that cathepsin B activity might somehow bc i l n p a i r e d in dyschon(h-ol)lastic lesions has llOi ycl b e e n s u b s t a n t i a l c d by t'xpeu'inltqlt,II t ' v i d e n c t ' , ht si/u a c t M t y studies | o r cathcpsin B in p a t h o h l g i c a l e;.utilage will foi-in i h c b;.isis o f | u l u r c invt, stigalions.

ACKNOWLEDGEMENTS

(;. H c r n f i n d e z - V i d a l is s u p l ) o r t e d by t h c (:()llSt~i() Nacional dc (:iencia v Tccnologia ((:ONAC~T), U n i v e r s i d a d A u l o n o n l a dc NtLcVO L e o n , M t ' x i c o ;.iild a ( ] a i n b r i d g e ()VelSt'as Rcscal-ch ~ l u d e n i s Award. M. E. D a v i t s is supl:~orted by the Isaac Ncwtoll T r t l s t ;.llld t h e Sybil E , i s t w o o d Tl'tlSl. ~'~t" ;.ire g r a t c t h l to Dr N. H o l d s t o c k a n d c o l l e a g u e s l o t st\pp l y i n g so111¢" o[" t h e m a t e r i a l a n d to Ms I+. Butler, Dr F. H e n s o n , Dr W. S h i n g l c l o n a n d Mr L. V o u t e for h e l p with tissue c o l l e c t i o n .

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(Acceptedfro'publication 14 April 1998)