Type IX collagen immunoreactive peptides in synovial fluids from arthritis patients.

OBJECTIVES: To determine whether type IX collagen-related peptides can be detected in the synovial fluids of arthritis patients and to assess their potential as molecular markers of arthritis. PATIENTS/METHODS: Synovial fluids from a set of carefully diagnosed arthritis patients and from healthy volunteers were used. Hydroxyproline assays were carried out to determine the content and concentration of collagen. Collagen cross-link determinations were conducted by reversed-phase HPLC. SDS PAGE and immunoblotting were used to identify the collagenous components, and N-terminal sequencing was performed to confirm these identities. RESULTS: All the synovial fluids were found to contain measurable amounts of collagen at similar concentrations. This appeared to be mainly high-molecular-weight material consisting of type I and type IX collagens, but not type II collagen. However, other smaller molecular weight type IX immunoreactive peptides were detected which were more apparent in the synovial fluids from arthritis patients. These peptides were also found to contain non-collagenous material. Collagen cross-links were also present in the arthritis synovial fluids. CONCLUSION: Collagenous material can be detected in all synovial fluids and the presence of pyridinoline cross-links indicates that at least some of this is derived from a mature collagen matrix. Type IX immunoreactive peptides were identified, but were found to contain significant amounts of non-collagenous material, and their presence, even at lower levels, in synovial fluids from normal subjects limits their potential for use as molecular markers of disease. Nevertheless, this is the first report of type IX collagen-related fragments in synovial fluids.

Type III collagen in normal human articular cartilage.

Type III collagen in normal human articular cartilage has been detected biochemically and its location in a diffuse area around the chondrocytes demonstrated by immunofluorescence. It can be found pericellularly throughout the depth of the cartilage and is evident in specimens ranging in age from 17 to 81 years.

Quantitative analysis of cyanogen bromide-cleaved peptides for the assessment of type I: type II collagen ratios in equine articular repair tissue.

Cyanogen bromide was used to solubilise and specifically fragment purified equine Type I and II collagen and equine articular surface repair tissue. The resultant peptides were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and quantified by densitometric scanning. Measurement of the relative amounts of the peptides alpha 2(I) CB3, 5 and alpha 1(II)CB10 provided an accurate method of establishing the ratio of Type I to Type II collagen in mixtures of purified equine collagens. The method was sensitive to 6% Type II collagen when the band areas were corrected for peptide molecular weight and the number of chains in the parent tropocollagen molecule which contain that particular peptide. Use of this technique showed that repair tissue in full thickness osteochondral defects in the dorso-distal margins of the intermediate carpal bones of ponies did not contain detectable amounts of Type II collagen 11 weeks after defect induction.

Effect of central or marginal location and post-operative exercise on the healing of osteochondral defects in the equine carpus.

The effects of osteochondral defect location and post-operative walking exercise on structural repair and recovery of joint function were examined in the midcarpal joints of ponies. Functional recovery was monitored by measuring ground reaction forces using a force plate. Structural repair was evaluated histologically and by measuring the total collagen and uronic acid content and relative proportions of Type I and II collagen in the repair tissue. Central defects tended to cause a more marked functional disturbance but were repaired with fibrocartilage rather than fibrous tissue in 3 out of 6 ponies while marginal defects were repaired almost exclusively with fibrous tissue. There was no significant difference between defect locations with regard to the biochemical measures evaluated. Exercise produced no beneficial effect on structural repair of the defects. Secondary 'kissing' lesions in the third carpal bones opposite the defects appeared grossly more severe in the exercised ponies which also showed a trend to more marked functional disturbance.

Influence of intra-articular sodium hyaluronate and polysulphated glycosaminoglycans on the biochemical composition of equine articular surface repair tissue.

The influence of repeated intra-articular injections of sodium hyaluronate and polysulphated glycosaminoglycan on the repair of full-thickness osteochondral defects was examined in the midcarpal joints of ponies. The study showed no significant difference between treated and control groups with regard to total collagen content, uronic acid content or the relative proportions of Type I and Type II collagen in the repair tissue, indicating that the drugs did not affect the biochemical composition of the repair tissue 11 weeks after defect induction.

Biochemical changes in the collagen of human osteoporotic bone matrix.

Although it is known that collagen imparts mechanical strength to bone no detailed biochemical analysis has been made of osteoporotic bone collagen. We report for the first time significant changes in the properties of the collagen. Analysis of collagen types revealed little change in the proportion of Type III collagen, but in some cases there was a significant loss of the Type VI. However, the major differences were observed in the post-translational modifications, namely, in the stabilizing cross-links and the hydroxylation of the collagen. These changes indicated a higher turnover in the head region compared to the neck region of the femoral head and are consistent with the susceptibility of the neck region to fracture. Clearly, the collagen is altered in osteoporosis and these changes may play a role in the pathogenesis of the disease.

Post-translational modifications in the collagen of human osteoporotic femoral head.

No detailed biochemical analysis has been made of the possible compositional changes in the collagen relating to the fragility of osteoporotic bone. We report for the first time significant changes in the compositional properties of the collagen. The major differences were observed in the post-translational modifications, namely, in the hydroxylation of lysine residues and the nature of the stabilizing cross-links of the collagen fibre. The increase in hydroxylation was greater in the head region compared to the neck region of the femoral head, whilst the decrease in the intermediate cross-links was greater in the neck region. Clearly, the collagen is altered in osteoporosis and it is important that these changes are recognised in studies of bone metabolism in osteoporosis since they may play a role in the pathogenesis of the disease.

Type III collagen in the intervertebral disc.

Several collagen types have now been isolated from the intervertebral disc, although type III collagen has previously only been extracted from human pathological disc. In this study, type III collagen has been isolated from normal human and bovine intervertebral disc and immunolocalized in sections of rat, sheep, bovine and 'normal' human intervertebral disc of various ages. Staining with antisera to type III collagen is localized primarily around the cells. Results indicate that cells of the disc sit in 'chondrons', similar to those seen in the deep and mid zones of articular cartilage. We suggest that type III collagen is present in the intervertebral disc and hypothesize that it may be involved in the organization of the pericellular environment, perhaps linking the chondron capsule to the interterritorial matrix.

The application of scanning confocal microscopy in cartilage research.

Scanning confocal microscopy has been used in conjunction with immunofluorescent localization to address two areas of debate in cartilage research. With the enhanced resolution and optical sectioning capability of this new technique, we have demonstrated that type IX collagen is preferentially located in an area around the chondrocyte, even in young cartilage. We have also shown that cathepsin B production is not confined to de-differentiated chondrocytes. The advantages and versatility of scanning confocal microscopy have thus clearly been demonstrated.

Degradation of cartilage matrix components by the cysteine proteinases, cathepsins B and L.

Rat chondrosarcoma chondrocytes are able to secrete both the precursor and mature forms of cathepsins B and L. When these two cysteine proteinases were added to a native cartilaginous matrix near neutral pH they were found to release and degrade both the proteoglycan and collagen components. One cleavage site in the proteoglycan monomer was found to be near the amino terminal globular domain. Collagen types II, IX, and XI in the matrix were also released and partially degraded. Ultimately these proteinases act as matrix depolymerases as they attacked the regions which are involved in collagen crosslinking. We conclude that these enzymes may play a role in matrix destruction as seen in arthritis.

Susceptibility of the cartilage collagens types II, IX and XI to degradation by the cysteine proteinases, cathepsins B and L.

We have investigated the susceptibility of both the helical and non-helical regions of isolated rat chondrosarcoma collagens, types II, IX and XI, to degradation by the cysteine proteinases, cathepsins B and L. Both enzymes degrade these collagens at temperatures from 20 to 37 degrees C and pH values from 3.5 to 7.0. Cleavage occurs only within the non-helical domains unless the helix is destabilized. Cathepsin L is more effective than cathepsin B on a molar basis and they appear to cleave at different sites. Since these cathepsins can degrade cartilage collagens at pH values near neutrality, they may contribute to the destruction of cartilage observed in arthritis.

Degradation of cartilage collagens type II, IX, X and XI by enzymes derived from human articular chondrocytes.

Conditioned culture medium derived from Interleukin-I alpha-activated human articular chondrocytes contained both collagen- and proteoglycan-degrading activities. Preparations of soluble type I collagen and the cartilage collagens type II, IX, X and XI were all degraded when incubated with the conditioned culture medium at 35 degrees C. Fractionation of the enzymic activities using column chromatography with Ultragel AcA 34 and Heparin-Sepharose allowed the separation and identification of neutral proteinase, collagenolytic and proteoglycan-degrading activities. Eluant fractions which contained type I collagenase activity effectively degraded collagen type II, but these fractions did not correspond precisely with those which degraded collagen types IX, X and XI. These observations indicate that chondrocytes have the potential to produce a conventional interstitial type II collagenase together with other enzymes having some specificity for the minor collagens. Thus IL-1-activated chondrocytes produce a range of collagenolytic and proteoglycan-degrading enzymes which can process most of the structural components of the cartilage matrix.

Localization of type IX collagen in chondrons isolated from porcine articular cartilage and rat chondrosarcoma.

Chondrocytes, each with their pericellular matrix bounded by a fibrous capsule, can be extracted singly or in groups from both mature pig articular cartilage and chondrosarcoma tissue. These structures, termed chondrons, are thought to anchor the chondrocytes in the matrix and protect them from the compressive forces experienced when articular cartilage is under load. The capsule of these chondrons contains both type II and type IX collagens and is composed of fine fibrillar material, unlike the large banded fibres of type II collagen found in the rest of the matrix. This suggests a role for type IX collagen in regulating the diameter of type II fibres to produce the fine fibrillar structure of the chondron capsules.

Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase.

The action of purified rheumatoid synovial collagenase and human neutrophil elastase on the cartilage collagen types II, IX, X and XI was examined. At 25 degrees C, collagenase attacked type II and type X (45-kDa pepsin-solubilized) collagens to produce specific products reflecting one and at least two cleavages respectively. At 35 degrees C, collagenase completely degraded the type II collagen molecule to small peptides whereas a large fragment of the type X molecule was resistant to further degradation. In contrast, collagen type IX (native, intact and pepsin-solubilized type M) and collagen type XI were resistant to collagenase attack at both 25 degrees C and 35 degrees C even in the presence of excess enzyme. Mixtures of type II collagen with equimolar amounts of either type IX or XI did not affect the rate at which the former was degraded by collagenase at 25 degrees C. Purified neutrophil elastase, shown to be functionally active against soluble type III collagen, had no effect on collagen type II at 25 degrees C or 35 degrees C. At 25 degrees C collagen types IX (pepsin-solubilized type M) and XI were also resistant to elastase, but at 35 degrees C both were susceptible to degradation with type IX being reduced to very small peptides. Collagen type X (45-kDa pepsin-solubilized) was susceptible to elastase attack at 25 degrees C and 35 degrees C as judged by the production of specific products that corresponded closely with those produced by collagenase. Although synovial collagenase failed to degrade collagen types IX and XI, all the cartilage collagen species examined were degraded at 35 degrees C by conditioned culture medium from IL1-activated human articular chondrocytes. Thus chondrocytes have the potential to catabolise each cartilage collagen species, but the specificity and number of the chondrocyte-derived collagenase(s) has yet to be resolved.

Type IX collagen: a possible function in articular cartilage.

The effect of type IX on in vitro fibrillogenesis of type II collagen indicated that, while not preventing fibrillogenesis, the presence of type IX collagen reduced the size of the type II fibre aggregates. This observation is consistent with the in vivo localisation studies of type IX collagen. Using the immunogold labelling technique, type IX collagen was shown to be located evenly on small fibrils which occur at higher concentration closer to the cell. Therefore type IX collagen may function as a regulator of fibre diameter in articular cartilage.

Isolation and characterization of the precursor of type M collagen.

A 225000-Mr peptide has been purified from rat chondrosarcoma which is immunologically and biochemically related to type M collagen. Rotary shadowing shows this molecule to be twice the length of the type M molecule and has a prominent kink close to one end. We believe this molecule represents parent type M, the form of the molecule in vivo.