Comparison of collagenase-cleaved articular cartilage collagen in ...

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Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, U.K.. †Joint Diseases Laboratory, Shriners Hospital for Children and Departments of ...
Osteoarthritis and Cartilage (2002) 10, 172–179 © 2002 OsteoArthritis Research Society International doi:10.1053/joca.2001.0500, available online at http://www.idealibrary.com on

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Comparison of collagenase-cleaved articular cartilage collagen in mice in the naturally occurring STR/ort model of osteoarthritis and in collagen-induced arthritis* J. S. Price*, M. G. Chambers*, A. R. Poole†, A. Fradin‡ and R. M. Mason* *Cell and Molecular Biology Section, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, U.K. †Joint Diseases Laboratory, Shriners Hospital for Children and Departments of Surgery and Medicine, McGill University, Montreal, Quebec, Canada ‡Department of Rheumatology, Institut de Recherches Servier, Suresnes, France Summary Objective: The STR/ort mouse develops a naturally occurring osteoarthritis of the femorotibial joint that provides a model with which to establish the time course of biochemical changes taking place in articular cartilage in the disease. Our objective was to define the onset, location and progression of type II collagen cleavage by collagenase in the tibial cartilage of the STR/ort mouse. For comparison, cartilage collagen cleavage was also studied in collagen-induced arthritis in DBA mice. Design: STR and control CBA mice aged 6–45 weeks were examined. DBA/1 mice were studied 2 and 3 weeks after initiating collagen-induced arthritis. Collagen cleavage was detected by immunolocalization using the antibody COL2-3/4Cshort which recognizes a carboxy terminal neoepitope created by collagenase cleavage of type I and II collagens. Results: No COL 2-3/4Cshort immunostaining was observed in the intact cartilage of healthy young or old mice. The earliest detectable collagen degradation occurred at the cartilage surface coincident with the appearance of surface roughening. As fibrillations developed, further collagen degradation was evident around the edge of the lesion and in adjacent extracellular matrix. In contrast, staining was observed throughout the cartilage matrix in type II collagen-induced arthritis prior to the development of histopathological lesions. Conclusion: No evidence was found for collagen cleavage in intact/pre-lesional cartilage from STR/ort mice. Local collagen cleavage was, however, clearly associated with very early histopathological lesions and immunostaining with COL 2-3/4Cshort increased with progression of the latter. In contrast, type II collagen cleavage occurs throughout the articular cartilage at an early stage in collagen-induced arthritis. © 2002 OsteoArthritis Research Society International Key words: Osteoarthritis, Collagenase, Animal model.

limited turnover of type II collagen in mature cartilage5 so cleavage is likely to be a critical step resulting in the irreversible loss of cartilage structural integrity. Cleavage of type II collagen during arthritis is thought to be carried out mainly by the matrix metalloproteinase (MMP) enzymes, particularly the collagenases; interstitial collagenase (MMP-1), neutrophil collagenase (MMP-8) and collagenase-3 (MMP-13) which can all be produced by chondrocytes6,7. The collagenases cleave within the triple helical region of fibrillar collagens resulting in characteristic one-quarter and three-quarter fragments4. Antineoepitope antibodies have been produced to recognize the cleaved ends of these collagen fragments, permitting their detection and measurement4. Type II collagen breakdown in OA appears to involve collagenase-38. Reports that gelatinase A (MMP-2) has collagenolytic activity (on type I collagen)9 have been contradicted by others10. Membrane type 1 metalloproteinase (MT1-MMP; MMP-14)11 and cathepsin K12 also have collagenolytic activity although their role, if any, in type II collagen breakdown in arthritis is unclear. Previous evidence has shown that there is both a loss of type II collagen and an increase in denatured type II collagen during late stage human OA2,13. Furthermore, Billinghurst et al.4 reported enhanced cleavage of type II

Introduction The breakdown of articular cartilage is an important feature of both osteoarthritis (OA) and rheumatoid arthritis (RA). Type II collagen is the characteristic fibrillar collagen of articular cartilage and is one of the main macromolecular components of its extracellular matrix. It forms a fibrillar network throughout the extracellular matrix of articular cartilage and provides tensile strength by resisting the swelling pressure resulting from the hydration of the highly negatively charged proteoglycan aggregates within the matrix1. The proteolytic cleavage of type II collagen is thought to be one of the key steps involved in cartilage breakdown during arthritis2–4. There is a very Received 27 March 2001; revision requested 5 June 2001; revision received 30 August 2001; accepted 10 September 2001. We gratefully acknowledge financial support from the Arthritis Research Campaign U.K. and from Institut de Recherches Servier, Suresnes, France. Address correspondence to: Roger M. Mason, Cell and Molecular Biology Section, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, Sir Alexander Fleming Building, Exhibition Road, London SW7 2AZ, U.K. Tel.: +44 20 7594 3019; Fax: +44 20 7594 3015; E-mail: [email protected]

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Osteoarthritis and Cartilage Vol. 10, No. 3 collagen by collagenases in human OA. Similarly, using in situ zymography, Freemont et al. showed that type II collagen degrading activity varies with cartilage lesions and co-distributes with MMP-13 in human OA14. Stoop and colleagues investigated collagen degradation in spontaneous OA in cartilages from the femorotibial joint of aged C57B1/6 and BALB/c mice and found that it was present at sites of degradation15. However, it remains unclear whether type II collagen degradation is an early or late event in the pathobiology of OA. Most studies have been carried out on human arthritic tissue from late stage disease (i.e. following joint replacement) or in explant culture systems that cannot relate type II collagen degradation with the clinical progression of OA. From a therapeutic point of view it is important to determine the time course and pattern of collagen degradation during the disease process and its relation to cartilage changes. There is some evidence to suggest that collagen degradation may occur at an early time-point in the OA disease process. Early events in OA include the loss of cartilage tensile strength and swelling from an increase in water in the matrix, indicative of damage to the collagen network16,17 One way in which early (i.e. pre-lesional) and late changes in cartilage can be monitored during arthritis is by the use of animal models. The STR/ort mouse is a naturally occurring model for OA of the human knee18. Male STR/ort mice develop histopathological lesions in the medial tibial plateau which are similar in severity and rate of progression to those in the human disease19. Around 85% of male mice develop such lesions by 35 weeks of age18. Type II collagen cleavage has not been studied previously in this model, although aggrecan cleavage by matrix metalloproteinases and aggrecanases has been described20. In this study we used the antibody COL 2-3/4Cshort which recognizes a carboxyterminal neoepitope on the threequarter fragment generated by collagenase cleavage of type I and II collagens4, to determine how collagen degradation in cartilage is related to the development of OA lesions in the STR/ort model. Young and mature mice were investigated at all stages of the disease. Collagen cleavage by collagenase in articular cartilage from mice with murine type II collagen-induced-arthritis (CIA), a severe inflammatory model with rapid cartilage damage similar to human RA, was examined for comparison.

173 with 100 l of a mixture consisting of 4 mg/ml of chicken type II collagen emulsified in an equal volume of complete Freund’s adjuvant (strain H37Ra; Difco, U.S.A.)21. Type II collagen (a generous gift of M. Huc, Coletica, France) had been purified from normal chicken sternal cartilage and was dissolved overnight with 0.1 M acetic acid to a concentration of 4 mg/ml at 4°C with mild agitation. Twenty-one days later, arthritis was induced by injecting mice intraperitoneally with 200 g of chicken type II collagen in phosphate buffered saline (PBS; 0.5 ml/mouse). In order to boost the arthritic process, mice received 40 g of bacterial lipopolysaccharide from Escherichia coli (strain B55:b5; Difco, U.S.A.)22. All CIA and control animals were examined after sacrifice by cardiac puncture under isoflurane anaesthesia ForeneR, Abbott, France) either 2 or 3 weeks after arthritis induction.

HISTOLOGY AND GRADING OF CARTILAGE LESIONS

STR/ort mice were killed by cervical dislocation. For all mice, the skin was removed from the legs and the knee joints dissected out with the associated muscle. Knee joints were dipped briefly in a 5% aqueous solution of polyvinyl alcohol and then snap frozen at −70°C in n-hexane (BDH Laboratories, Poole, U.K.). Joints were stored at −70°C until sectioned. Serial sections of 10 m were cut in an anterior to posterior direction using a Bright’s bone cutting cryostat. The sections were mounted onto polylysine coated slides (BDH) and stored at −70°C until use. Unstained sections were graded for histological OA changes using the grading system reported previously23; 0=no change; 1=roughened articular surface and small fibrillations; 2=fibrillations down to zone 2 (chondrocytes just below the surface spindle cells) and some loss of surface lamina; 3=loss of surface lamina and fibrillations extending down to the calcified cartilage; 4=major fibrillations and cartilage erosion down to subchondral bone; 5=major fibrillations and erosion of up to 80% of cartilage; 6=greater than 80% loss of cartilage. Each section was graded and the overall grade of damage to the whole joint was defined as the most severe grade observed within all sections of that joint.

DETECTION OF TYPE II COLLAGEN DEGRADATION

Materials and methods ANIMALS

Twenty-three male STR/ort mice aged 6–45 weeks from the Imperial College inbred STR/ort colony were examined along with 12 age- and sex-matched CBA control mice (from Charles River, U.K.). Ten DBA/1 male mice (from Charles River, France) with CIA together with 10 age- and sex-matched control DBA/1 mice were examined for comparison. Animals had access to standard diet and water ad libitum. Animal husbandry and experiments were carried out under authority of national and local licences. INDUCTION OF COLLAGEN-INDUCED ARTHRITIS

Nine- to ten-week old DBA/1 mice were randomly assigned to four groups of five animals each (CIA 2 and 3 weeks post-induction, controls for 2 and 3 week CIA groups). CIA mice were sensitized at the base of the tail

Type II collagen degradation was visualized with the rabbit polyclonal antibody COL2-3/4Cshort, using a modification of the previous method4. Briefly, thawed sections were fixed with freshly prepared 4% paraformaldehyde (BDH) in PBS for 5 min at room temperature and then rinsed extensively with PBS. In order to enhance the permeability of the extracellular matrix, sections were treated with 0.5% testicular hyaluronidase (type I–IV, EC.3.2.1.35; Sigma) in PBS for 30 min at 37°C. Sections were washed and any endogenous peroxidase activity was blocked using 1% v/v hydrogen peroxide (BDH) in absolute ethanol for 30 min at room temperature. Sections were washed and non-specific staining was blocked by incubation with 10% normal swine serum (Dako, Denmark) in PBS with 1% bovine serum albumin (PBS-BSA; Sigma). Sections were incubated overnight with COL2-3/4Cshort (1:800 dilution) in PBS-BSA at 4°C in a humidified chamber. Negative control sections were incubated with COL2-3/ 4Cshort that had been preabsorbed with 500 g/ml of the immunizing peptide for 1 h at 37°C. In some cases APMA

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activated human recombinant proMMP-13 (20 g/ml; a kind gift from Professor G. Murphy, School of Biological Sciences, University of East Anglia, Norwich) was added to the sections (20 min at 37°C) prior to incubation with COL2-3/4Cshort to provide a positive control for the antibody. Unbound COL2-3/4Cshort antibody was removed by washing in PBS (3×5 min at room temperature) and bound antibody was detected using a biotin labelled swine antirabbit IgG secondary antibody for 1 h at room temperature (1:400 dilution; Dako). The bound secondary antibody was detected with a biotin-streptavidin detection system (Vector ABC reagent; Vector Laboratories, Peterborough, U.K.). The peroxidase was detected using a DAB detection kit (Vector Laboratories) according to the manufacturers instructions. After rinsing, sections were air dried and mounted with aquamount (BDH). Staining was recorded as either being present (+) or absent (−) at two locations, the articular surface and the extracellular matrix of the tibial cartilage. DETECTION OF PROTEOGLYCAN DEGRADATION

Sections were stained as described previously20 for the VDIPEN and NITEGE neoepitopes, indicative of cleavage of the core protein of aggrecan by MMPs and aggrecanases, respectively. Proteoglycan glycosaminoglycan side chains were stained using the Alcian Blue method24. DETECTION OF INFLAMMATORY MACROPHAGES IN COLLAGEN-INDUCED ARTHRITIS (CIA)

Inflammation in the CIA joints was assessed using a murine macrophage marker (rat anti-mouse F4/80 antigen; Serotec, Oxford, U.K.). Briefly, sections were fixed in 4% paraformaldehyde in PBS, washed in PBS then blocked using 5% normal rabbit serum for 30 min at 37°C. Sections were then incubated with the primary antibody, rat antimouse F4/80 antigen for 1 h at 37°C (1:50 dilution). Anti-F4/80 antibody binding was detected using a biotinylated rabbit anti-rat IgG (H+L) mouse absorbed secondary antibody (1:100 dilution; Vector) for 1 h at 37°C. The antibody was detected using a streptavidin-FITC antibody (1:200; Vector) for 30 min at 37°C. After rinsing, sections were air dried and mounted with fluorescent mounting medium containing propridium iodide to distinguish the cell nuclei (Vector).

Results TYPE II COLLAGEN DEGRADATION IN THE STR/ORT MODEL OF OA

STR/ort mice with no articular cartilage damage and with osteoarthritic changes ranging from grade 1 (surface roughening and small fibrillations) to grade 5 (up to 80% loss of cartilage) were examined (Table I). Type II collagen degradation was observed in all mice which had histological lesions, including those with grade 1. A very narrow zone of immunostaining was present on the surface of articular cartilages from the medial tibial plateau in regions where surface roughening occurred [Fig. 1(a)]. This was a consistent finding but was not evident in cartilages with a smooth surface (see below). In cartilage with the next stage of histopathological damage [grade 2; Fig. 1(b)], immunostaining was present along the fibrillated edge of the lesion, and along the surface of the articular

Table I Histological grading and presence of collagen degradation in the STR/ort model of OA Histology grade

0 0 0 0 0 0 0 0 1 1 1 1 2 2 2 3 3 3 4 4 4 4 5

Age (weeks)

6 12 13 13 20 22 24 30 14 20 22 33 18 20 24 24 30 33 12 22 24 45 22

COL2-3/4Cshort staining Articular surface

Extracellular matrix

− − − − − − − − + + + − − + + − − − − − − − −

− − − − − − − − − − − + + + + + + + + + + + +

The medial tibial cartilage of STR/ort mice of different ages was graded for osteoarthritic lesions using the scale described in the Methods section. Type II collagen degradation was visualized by immunostaining using antibody COL2-3/4Cshort and was seen at the articular surface and/or in the extracellular matrix adjacent to lesions. (+), Positive immunostaining for collagen degradation; (−), no staining.

cartilage adjacent to the lesion. With progression to more severe grades of arthritis [e.g. grade 4; Fig. 1(c)] marked type II collagen degradation was evident at the site of the lesion itself and also less strongly in the surrounding extracellular matrix. We investigated the specificity of COL2-3/4Cshort immunostaining by pre-absorbing the antibody with its immunizing peptide before adding it to the sections. This resulted in little or no immunostaining, even in severely damaged cartilage [e.g. grade 4 lesion; Fig. 1(d)], indicating specificity of the COL2-3/4Cshort antibody for collagenase-generated collagen fragments. STR/ort mice usually develop lesions on the medial side of the tibial plateau18 and the results shown in Table I and Fig. 1 are for the medial side of the joint. The lateral compartment of the joint develops lesions much less frequently and often at a later stage of the disease18. However, when lesions were present in the lateral cartilage they also showed evidence of type II collagen degradation (results not shown). There was no consistent evidence of type II collagen degradation in histologically normal cartilage of either young or old STR/ort mice [Table I, Fig. 2(a),(b)), although occasional faint staining was observed in a pericellular location around chondrocytes in young intact cartilage which was not present in antigen peptide absorbed controls (not shown). Moreover, control age-matched CBA mice, all of which had histologically normal cartilage, showed no evidence of type II collagen degradation in either young or old animals [Fig. 2(c)]. This was not the case for cartilage

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Fig. 1. Type II collagen degradation in STR/ort tibial cartilage is associated with cartilage damage. Antibody COL2-3/4Cshort was used to detect the collagenase-generated fragments of type II collagen in medial tibial cartilage with histological lesions of OA of varying severity. Lesions were graded as described in the Methods section. Type II collagen degradation is present: (a), along the surface of roughened cartilage (grade 1); (b), in the lesional area of a mild lesion (grade 2); (c), in the lesional area of severe damage (grade 4). (d) Pre-absorption of COL2-3/4Cshort with its immunizing peptide resulted in complete loss of staining even in severely damaged regions (grade 4). T—tibia; AC—tibial articular cartilage. Original magnification, ×50.

sections incubated with active MMP13, in which type II collagen degradation could be detected in intact normal articular cartilage of either CBA or STR/ort mice [Fig. 2(d)]. Immunostaining occurred throughout the cartilage matrix in these sections, indicating that collagen cleavage can be detected readily with the antibody, where present. COMPARISON WITH TYPE II COLLAGEN DEGRADATION DURING COLLAGEN-INDUCED-ARTHRITIS (CIA)

CIA is an inflammatory arthritis similar to RA and is associated with rapid onset of damage to articular cartilage25, including marked collagen degradation15. It was of interest therefore to compare the pattern of collagen cleavage in this disease with that in spontaneous OA of STR/ort mice. The results of the CIA experiments are summarized in Table II. Only one-third of the mice induced developed the characteristic synovial inflammatory changes associated with CIA, as detected with the antibody to the macrophage marker F4/80. This low incidence of inflammatory arthritis could have been due to the use of chicken type II collagen for the induction. The use of bovine collagen was banned at the time of the experiment because of the bovine spongiform encephalitis crisis in the U.K. Collagen degradation was not observed in age-matched non-induced DBA/1 control joints (Table II). Similarly, it did not occur frequently in joints of induced mice lacking signs of inflammation [Table II; Fig. 3(a)]. In two joints that showed weak immunostaining for collagen degradation there was no inflammation in the knee joint studied, as determined by F4/80 antigen staining, but there was evidence for inflammation in the contralateral knee joint (not shown). However, when joints showed clear histological signs of inflammation, there was marked type II collagen degradation (Table II). Moreover, the extent of degradation was

more generalized than in the STR/ort joints and occurred throughout the cartilage extracellular matrix [e.g. Fig. 3(b)]. Strong immunostaining was observed in these inflamed joints even when there was little evidence of histological damage to the cartilage (Table II). Furthermore, in contrast to the focal areas of collagen cleavage associated with STR/ort osteoarthritic lesions, CIA joints with histological damage showed COL2-3/4Cshort staining in areas of the joint distant from the lesion with an intact articular cartilage surface. The immunostaining was markedly reduced in sections from CIA mice by preabsorption of the antibody with its peptide, demonstrating its specificity [Fig. 3(c)]. CIA joints with severe inflammation showed a generalized loss of Alcian Blue staining throughout the cartilage matrix accompanied by a large increase in the aggrecan cleavage neoepitopes VDIPEN and NITEGE26,27 (not shown). Thus widespread enhanced proteoglycan cleavage occurred simultaneously with collagen cleavage in articular cartilage in this model from an early stage of the disease process. This contrasts with the pattern of increased loss of proteoglycan in developing OA in the tibial cartilage of STR/ort mice which occurs in more focal areas associated with lesion development18,20.

Discussion Our findings indicate that the level of type II collagen degradation in vivo in normal articular cartilage in mice aged 6 weeks or older is either non-existent, or so low as to be undetectable by immunohistochemistry with the COL23/4Cshort antibody. Since the immunohistochemical method was able to detect very discrete changes in the appearance of the COL2-3/4Cshort neoepitope at the surface of STR/ort cartilage with minimal histopathological change [e.g. Fig.

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Fig. 2. Lack of type II collagen degradation in undamaged murine tibial cartilage. Antibody COL2-3/4Cshort was used to detect the collagenase-generated fragments of type II collagen in undamaged medial tibial cartilage of mice of different ages. (a), 6 week STR/ort; (b), 33-week STR/ort; (c), 35-week CBA control. No staining was observed in undamaged cartilage from either old or young CBA or STR/ort mice. Type II collagen degradation could be induced throughout the matrix of normal cartilage of either STR/ort or CBA mice by prior digestion of the section with MMP-13. (d) 12-week STR/ort digested with MMP-13. T—tibia; AC—tibial articular cartilage. Original magnification, ×50. Table II Histologic grading, inflammatory changes and the presence of collagen degradation in DBA/1 mice with CIA Histological grade

0 0 0 0 0 1 1 1 2 3

Weeks post-CIA

Joint type

COL2-3/4Cshort staining in matrix

Inflammation

2 3 2 3 2 2 3 3 3 2

Control Control CIA CIA CIA CIA CIA CIA CIA CIA

− − − − + − − ++ ++ ++

− − − − − − − ++ ++ ++

Arthritic lesions in the medial tibial cartilage were graded histologically according to the scale described in the Methods section. Type II collagen degradation was visualized by immunostaining using antibody COL2-3/4Cshort and, where present, was seen throughout the articular cartilage matrix. Inflammatory changes were assessed by positive staining for a macrophage marker (murine F4/80 antigen): (−), no staining; (+), weak staining; (+ +), strong staining. Control, non-induced DBA/1 mice; CIA, mice with collagen-induced arthritis.

1(a)], it appears to be a sensitive method for detecting collagen cleavage in vivo. Secondly, the study shows that collagenases degrade type II collagen during the development of spontaneous OA in STR/ort mice, but with a quite different pattern of activity to that occurring in murine CIA-arthritis. In the STR/ort model type II collagen cleavage is restricted to cartilage in the area of histopathological lesions. Even aged STR/ort mice with type II collagen cleavage in damaged areas of cartilage show no evidence of such degradation in regions

of non-lesional cartilage. Thus, collagen degradation is a very localized event in spontaneous OA in STR/ort mice and is specifically associated with histopathological lesions. In contrast, type II collagen cleavage in the inflammatory CIA model is a much more generalized process, with rapid onset of collagen degradation occurring throughout the cartilage even before histopathological lesions appear, and in intact regions of the cartilage distant from the lesions. Our results indicate that collagen degradation begins at the surface of the cartilage in the superficial zone in spontaneous OA in STR/ort mice and is restricted to focal sites where the cartilage is damaged. This is a very similar pattern of type II collagen degradation to that reported recently in spontaneous OA in very old C57Bl/6 and BALB/c mice15. In human OA, although the initial damage to type II collagen occurs in the superficial and upper mid zones, it later extends to the deeper zones of the cartilage and is more pronounced3. Nevertheless, recent studies on human OA also revealed evidence for very focal damage to type II collagen that is also associated with lesions (Webb et al., manuscript in preparation). An obvious candidate enzyme for collagen breakdown in the STR/ort model is MMP-13 as, so far, this is the only collagenase shown to be present in mouse tissue28. In situ hybridization experiments indicate an abundance of mRNA for MMP-13 in STR/ort mouse tibial cartilage compared to levels in CBA control mice. However, these transcripts occur predominantly in the chondrocytes of the mid and deep zones and are present before any sign of cartilage damage is evident. Moreover, there is a marked decrease in MMP-13 transcripts in chondrocytes in the vicinity of lesions and we found no immunohistochemical evidence for MMP-13 protein at these sites in STR/ort cartilage at any stage29. Thus the observed expression pattern of MMP-13 does not fit well with the mapping of the type II collagen cleavage sites in STR/ort tibial cartilage.

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Fig. 3. Type II collagen degradation in murine collagen-induced arthritis. Antibody COL2-3/4Cshort was used to detect the collagenase-generated fragments of type II collagen in the cartilage of the medial tibial plateau of DBA mice with collageninduced-arthritis. (a) No type II collagen degradation was observed in collagen-induced mice free of inflammation. (b) Type II collagen degradation was observed throughout the matrix of the inflamed joints, both when lesions were present and when the cartilage appeared intact. (c) Pre-absorption of COL2-3/4Cshort with its immunizing peptide resulted in a marked reduction of this staining. T—tibia; AC—tibial articular cartilage. Original magnification, ×50.

More recently other enzymes have been described as having collagenolytic activity such as gelatinase A9, MT1MMP11 and cathepsin K12 and are thus candidates for type II collagen cleavage in STR/ort cartilage. Gelatinase A (MMP-2) is reported to cleave type I collagen at the same site as interstitial collagenases9, although other groups disagree with this finding (Mort et al., unpublished observations). We found that MMP-2 mRNA and MT1-MMP mRNA and protein is expressed throughout the STR/ort tibial cartilage. However, the MT1-MMP is expressed at similar levels in CBA cartilage and expression decreases in chondrocytes near lesions in STR/ort cartilage, which does not support a role for this enzyme in type II collagen degradation at such sites29. The cysteine protease Cathepsin K is also capable of cleaving type II collagen. However, this cleavage is thought to occur in the N terminal

177 helical domain at a site distinct from the collagenase cleavage site12. In this study we looked specifically at the role of collagenases in the development of OA in STR/ort mice, albeit indirectly through the products of collagenase activity in the tibial cartilage. Although the COL2-3/4Cshort antibody we used specifically recognizes the C-terminus of a collagen fragment generated by collagenase cleavage at a point three-quarters of the way along the collagen molecule, we cannot rule out the possibility that other enzymes are able to produce this fragment. Similarly, it is possible that the amount of type II collagen degradation is being underestimated due to enzymes acting at a different site on type II collagen that this antibody cannot recognize. Furthermore, enzymatic processing of the fragments produced by collagenases can result in the loss of the neoepitope following its cleavage by gelatinase A,B and stromelysin (Poole et al., unpublished observations) and removal by secondary cleavage of the -chain releasing the epitope from the cartilage8,30. There is evidence from previous studies in the STR/ort murine model that biochemical changes occur in the STR/ ort cartilage before any lesions have developed. The expression of cytokines such as IL-1/, IL-6, TGF and IGF is up-regulated in STR/ort cartilage before lesions develop, whereas transcripts for these factors were not detected in control CBA cartilage23. Total proteoglycan levels in the STR/ort tibial cartilage also increase at an early stage in disease and then decrease at a later stage31. This initial increase followed by proteoglycan loss is a characteristic feature of some other animal models of OA and is thought to be due to an attempted repair response32,33. Levels of the MMP and aggrecanase generated aggrecan cleavage neoepitopes26,27, VDIPEN and NITEGE respectively, have been studied in the STR/ort model20. Each neoepitope mapped to a separate location in the prelesional stages of OA in the STR/ort cartilage. These locations were the deep zone for VDIPEN and the upper zone for NITEGE, the same as found in normal CBA cartilage. However, with progression to OA lesions both neoepitopes appear prominently at the site of the lesion. Thus, as with the specific enzymes involved in collagen degradation in the STR/ort model, proteases involved in proteoglycan degradation appear to be produced and activated in focal areas associated with lesion development. Interestingly, in antigen-induced arthritis, a less severe inflammatory arthritis than CIA, it has been shown that VDIPEN and COL2-3/4C staining co-localize, suggesting a coupling of both cleavage activities34. However, in collagen-induced arthritis, collagenase and proteoglycan degrading enzymes appear to be induced throughout articular cartilage. In two CIA mice there was evidence of cartilage collagen cleavage before any histopathological lesion was evident, suggesting that collagenases are active in the tissue at a very early stage of the disease. In general, cleavage of cartilage collagen in CIA mice coincides with the appearance of inflammatory cells in the synovium, suggesting that signals originating at this site induce chondrocytic collagenase activity. We have rarely detected inflammation in the knee joints of STR/ort mice in our colony and when present it occurs in late stage disease and is minimal. These differences in the mechanism of matrix breakdown and disease progression in the two models must be taken into account when using either to test new therapies.

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The data in the present report consolidate other reports about the biochemical changes underlying the development of OA in the tibial cartilage of STR/ort mice18,20,23,29,31,35. Overall these provide the most comprehensive account to date of such changes in naturally occurring OA in a small animal model.

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15.

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