Neoepitopes as biomarkers of cartilage catabolism

Progressive degradation of articular cartilage is a central feature of arthritis and a major determinant of long term joint dysfunction. There are no treatments able to halt the progression of cartilage destruction presently available, and monitoring the benefit of potential therapies is hampered by our inability to measure the "health" of articular cartilage. Serial radiographic assessment of joint space narrowing, the current gold standard, requires measurements over a prolonged time (1-5 years) and is prone to technical difficulties. Other strategies for evaluating cartilage degradation are needed to enable both short and long term monitoring of disease progression and response to therapy. One avenue that holds promise is the use of biomarkers that accurately reflect the degradative state of the articular cartilage. Antibodies that recognise terminal amino acid sequences generated by proteolysis at specific sites in the core protein of both aggrecan and type II collagen (neoepitope antibodies) have become available in recent years. These antibodies have been invaluable for identifying the proteinases responsible for cartilage breakdown both in vitro and in vivo. The presence of neoepitope sequences generated by specific metalloenzyme cleavage of aggrecan and type II collagen correlates well with the progression of cartilage degeneration, both in vitro and in mouse models of arthritis. Preliminary results with quantitative assays of type II collagen neoepitopes suggest that they may be useful markers of joint disease in humans. Long term studies correlating neoepitope concentration with clinical and radiographic disease are now required to validate the utility of neoepitopes as surrogate markers of cartilage degeneration and joint disease.     

Anatomical localization of cartilage degradation markers in a surgically induced rat osteoarthritis model

Osteoarthritis (OA) is a degenerative disease characterized by an irreversible loss of articular cartilage. Although surgically induced animal OA models are commonly used in drug efficacy assessment, degradation of type II collagen, an important component of articular cartilage is not routinely evaluated. Here, the medial meniscectomy surgical model (MMT) in Lewis rats was evaluated for proteoglycan loss with toluidine blue staining and collagen degradation with immunohistochemical staining for a collagen cleavage C-neoepitope, using a novel anti-type II collagen neoepitope antigen (TIINE) antibody. Femorotibial joints were collected for histology at 0 (no surgery), 3, 7, 14, 21, 28, 35, and 42 days postsurgery. Following MMT surgery, the medial tibial articular cartilage had proteoglycan matrix loss by day 3 that reached subchondral bone by days 28-42. Femoral cartilage damage occurred by day 14. TIINE staining was present at basal levels in growth plates and articular cartilage of all joints while all MMT-treated animals had increased intensity and area of staining in erosions that colocalized with proteoglycan loss. The MMT model produces a progressive pattern of cartilage damage resembling human OA lesions, making it useful, when evaluated with cartilage biomarkers, for assessing changes in cartilage degradation.     

Analysis of collagenase-cleavage of type II collagen using a neoepitope ELISA

We have developed monoclonal antibody 5109 against a unique highly acidic sequence in type II collagen. When paired with previously reported monoclonal antibody 9A4, 5109 can be used as the capture antibody in an ELISA assay for the neoepitope generated by collagenase-cleavage of type II collagen. The assay detects the sequence ZGlyGluX(759)GlyAspAspGlyProSerGlyAlaGluGlyProX(771)GlyProGlnGly(775) where Z is a variable length polypeptide, X is proline or hydroxyproline, and Gly(775) corresponds to C-terminal amino acid of the 3/4 piece after collagenase cleavage. Antibody 5109 detects the first and 9A4 the second underlined sequence. Antibody 5109 recognizes its epitope with a K=1.2x10(-8) M independently of hydroxylation of X(759). When X(771) is proline, the sequence is 90x more sensitively detected by this ELISA than when it is hydroxyproline. Type II collagen of human articular cartilage was fragmented by cyanogen bromide (CNBr) and trypsin. The immunoreactive fragment was captured with 5109 and sequenced. Proline(771) averaged 81% hydroxylated. Other 3rd position prolines were >97% hydroxylated. In urine of control individuals of 50-70 years of age, we failed to detect the presence of the collagen fragment in a majority (8/10) of specimens. The two controls with measurable levels averaged 123 pM. In a similar age cohort of osteoarthritic patients, the majority (9/10) showed measurable values of urinary collagen fragments averaging 312 pM. This assay can be used for monitoring type II collagen metabolism in patients with osteoarthritis.     

NG2/HMPG modulation of human articular chondrocyte adhesion to type VI collagen is lost in osteoarthritis.

NG2/human melanoma proteoglycan (HMPG) is a chondroitin sulphate proteoglycan (CSPG), expressed by chondrocytes in fetal and in normal and osteoarthritic (OA) adult articular cartilage. NG2/HMPG is a receptor for extracellular matrix proteins, including type VI collagen, and regulates beta1 integrin binding to fibronectin. This study was undertaken to identify whether NG2/HMPG had similar activities in human articular chondrocytes (HACs). Normal and OA adult HAC adhesion to fibronectin, type II or type VI collagen was assessed using a methylene blue assay. The requirement for integrins, NG2/HMPG, and integrin-associated signalling molecules was investigated using anti-beta1 integrin and anti-HMPG antibodies and pharmacological inhibitors of signalling molecules. The adhesion of normal and OA HACs to fibronectin, type II and type VI collagen was beta1 integrin-dependent. Normal HAC adhesion to type VI collagen was stimulated by anti-HMPG antibodies. This effect was inhibited by pertussis toxin. Anti-HMPG antibodies had no effect on OA chondrocyte adhesion to type VI collagen, or on normal and OA cell adhesion to fibronectin and type II collagen. The results show that NG2/HMPG modulates integrin-mediated interactions of normal HACs with type VI collagen. Loss of this activity may be of importance in the progression of osteoarthritis.     

Gene expression and matrix proteinase activity in osteochondrosis dessicans

Introduction Osteochondrosis dessicans (OCD) is a disorder of unknown aetiology where often a fragment of cartilage and subchondral bone separates from the articular surface. Previous studies have shown histological changes in glycosaminoglycan content in OCD cartilage compared to normal cartilage ( Koch et al. 1997 ). It has also been shown in equine OCD cartilage that there is excessive degradation of type‐II collagen compared to normal cartilage ( Laverty et al. 2002 ). The present study was undertaken to examine the gene expression in human OCD cartilage compared to its normal autologous articular cartilage and human osteoarthritic (OA) cartilage. Methods Cartilage from five OCD patients (18–34 years) was obtained at the time of surgery. Pieces of cartilage were either snap‐frozen (in preparation for RNA isolation) or the proteoglycans extracted with 4 m GuHCl. Total RNA was isolated from the cartilage using RNeasy minicolumns and reagents (Qiagen) according to the manufacturer's protocol. RT‐PCR was performed using an RNA PCR kit (Perkin‐Elmer) using a number of oligonucleotide primers. GuHCl‐extracted proteoglycan fragments were analysed using Western blotting with a number of antibodies to aggrecan metabolites, collagen metabolites and the small leucine‐rich proteoglycans. Results and discussion When OCD cartilage was compared to normal and human OA cartilage, there was an increase in aggrecan, collagen type‐II and collagen type‐X RNA expression. There was no change in RNA expression of link protein or type‐I collagen. The RNA expression of the aggrecanases (ADAMTS enzymes) was also different in the three different cartilage samples. Neither ADAMTS‐1, ‐4 or ‐5 was present in the normal cartilage. In contrast, in the OCD cartilage, there was expression of both ADAMTS‐1 and ‐4, whereas in the OA cartilage, there was expression of ADAMTS‐4 and ‐5. In the case of MMP RNA expression, MMP‐3 was decreased and MMP‐13 increased in OCD cartilage compared to both normal and OA samples. In addition, the expression of all three TIMP isoforms was increased in the OCD cartilage. Although inflammatory components are not expected in OCD pathology, expressions of inflammatory mediators such as COX‐2, IL‐1‐α and TNF‐α were all increased in the OCD cartilage when compared to normal, but expression of these mRNAs in the OA cartilage was higher. Analysis of proteoglycan fragments in the OCD cartilage by Western blotting showed the presence of aggrecan fragments containing the G1 domain, interglobular domain and the C‐terminal neoepitope generated by aggrecanase cleavage. There was also immunoreactivity for biglycan and link protein. Conclusion These results suggest that the phenotypic expression of chondrocytes at the site of the OCD lesion are markedly different from ‘normal’ articular cartilage and also pathological OA cartilage. Interestingly, the expression patterns of matrix proteinases and their natural inhibitors were also markedly different in OCD cartilage, again suggesting that there are specific biochemical expression patterns in OCD pathology, which may potentially be biomarkers of the disease process. Further studies are necessary to elucidate how the differences in gene expression and matrix protease activity may be involved in the aetiology of OCD.     

The assessment of cartilage degradation in vivo: development of an immunoassay for the measurement in body fluids of type II collagen cleaved by collagenases

A monoclonal antibody has been developed which recognizes a neoepitope in type II collagen which is generated by the intrahelical cleavage of collagenases. Antibody reactivity is directed at the carboxyl-terminus of the TCA or 3/4 piece of the degraded alpha1(II) chain. Reactivity is dependent upon hydroxylation of proline. Evidence is provided suggesting that epitope binding involves the recognition of a conformational neoepitope. Using an ELISA, we show that this neoepitope can be detected in the urines and sera of nonarthritic persons and patients with rheumatoid arthritis (RA). An increased content is observed in the sera and urines of patients. The assay may be of value in studying cartilage type II degradation both in vitro and in vivo such as in those with arthritis.     

Cathepsin K Is a Critical Protease in Synovial Fibroblast-Mediated Collagen Degradation

Synovial fibroblasts (SFs) play a critical role in the pathogenesis of rheumatoid arthritis (RA) and are directly involved in joint destruction. Both SF-resident matrix metalloproteases and cathepsins have been implicated in cartilage degradation although their identities and individual contributions remain unclear. The aims of this study were to investigate the expression of cathepsin K in SFs, the correlation between cathepsin K expression and disease severity, and the contribution of cathepsin K to fibroblast-mediated collagen degradation. Immunostaining of joint specimens of 21 patients revealed high expression of cathepsin K in SFs in the synovial lining and the stroma of synovial villi, and to a lesser extent in CD68-positive cells of the synovial lining. Cathepsin K-positive SFs were consistently observed at sites of cartilage and bone degradation. Expression levels of cathepsin K in the sublining and vascularized areas of inflamed synovia showed a highly significant negative correlation with results derived from the Hannover Functional Capacity Questionnaire (r = 0.78, P = 0.003; and r = 0.70, P = 0.012, respectively) as a measure of the severity of RA in individual patients. For comparison, there was no correlation between Hannover Functional Capacity Questionnaire and cathepsin S whose expression is limited to CD-68-positive macrophage-like synoviocytes. The expression of cathepsin K was also demonstrated in primary cell cultures of RA-SFs. Co-cultures of SFs on cartilage disks revealed the ability of fibroblast-like cells to phagocytose collagen fibrils whose intralysosomal hydrolysis was prevented in the presence of a potent cathepsin K inhibitor but not by an inhibitor effective against cathepsins L, B, and S. The selective and critical role of cathepsin K in articular cartilage and subchondral bone erosion was further corroborated by the finding that cathepsin K has a potent aggrecan-degrading activity and that cathepsin K-generated aggrecan cleavage products specifically potentiate the collagenolytic activity of cathepsin K toward type I and II collagens. This study demonstrates for the first time a critical role of cathepsin K in cartilage degradation by SFs in RA that is comparable to its well-known activity in osteoclasts.     

Behavior of human articular chondrocytes derived from nonarthritic and osteoarthritic cartilage in a collagen matrix

The purpose of this study was to evaluate the morphologic and biochemical behavior and activity of human chondrocytes taken from nonarthritic and osteoarthritic cartilage and seeded on a three-dimensional matrix consisting of collagen types I, II, and III. Human articular chondrocytes were isolated from either nonarthritic or osteoarthritic cartilage of elderly subjects, and from nonarthritic cartilage of an adolescent subject, seeded on collagen matrices, and cultured for 12 h, 7 days, and 14 days. Histological analysis, immunohistochemistry, and biochemical assays for glycosaminoglycans (GAGs) and DNA content were performed for cell-seeded and unseeded matrices. Chondrocytes of nonarthritic cartilage revealed a larger number of spherical cells, consistent with a chondrocytic phenotype. The biochemical assay showed a net increase in GAG content in nonarthritic chondrocytes, whereas almost no GAGs were seen in osteoarthritic cells. The DNA results suggest that more osteoarthritic cells than chondrocytes from nonarthritic cartilage attached to the matrix within the first week. Human articular chondrocytes isolated from osteoarthritic cartilage seem to have less bioactivity after expansion and culture in a sponge consisting of type I, II, and III collagen compared with chondrocytes from nonarthritic cartilage.     

Type IIA procollagen: expression in developing chicken limb cartilage and human osteoarthritic articular cartilage.

Type IIA procollagen is an alternatively spliced product of the type II collagen gene and uniquely contains the cysteine (cys)-rich globular domain in its amino (N)-propeptide. To understand the function of type IIA procollagen in cartilage development under normal and pathologic conditions, the detailed expression pattern of type IIA procollagen was determined in progressive stages of development in embryonic chicken limb cartilages (days 5-19) and in human adult articular cartilage. Utilizing the antibodies specific for the cys-rich domain of the type IIA procollagen N-propeptide, we localized type IIA procollagen in the pericellular and interterritorial matrix of condensing pre-chondrogenic mesenchyme (day 5) and early cartilage (days 7-9). The intensity of immunostaining was gradually lost with cartilage development, and staining became restricted to the inner layer of perichondrium and the articular cap (day 12). Later in development, type IIA procollagen was re-expressed at the onset of cartilage hypertrophy (day 19). Different from type X collagen, which is expressed throughout hypertrophic cartilage, type IIA procollagen expression was transient and restricted to the zone of early hypertrophy. Immunoelectron microscopic and immunoblot analyses showed that a significant amount of the type IIA procollagen N-propeptide, but not the carboxyl (C)-propeptide, was retained in matrix collagen fibrils of embryonic limb cartilage. This suggests that the type IIA procollagen N-propeptide plays previously unrecognized roles in fibrillogenesis and chondrogenesis. We did not detect type IIA procollagen in healthy human adult articular cartilage. Expression of type IIA procollagen, together with that of type X collagen, was activated by articular chondrocytes in the upper zone of moderately and severely affected human osteoarthritic cartilage, suggesting that articular chondrocytes, which normally maintain a stable phenotype, undergo hypertrophic changes in osteoarthritic cartilage. Based on our data, we propose that type IIA procollagen plays a significant role in chondrocyte differentiation and hypertrophy during normal cartilage development as well as in the pathogenesis of osteoarthritis.     

Akt与ERK1/2在人骨关节炎软骨细胞中的表达

目的: 观察蛋白激酶B(Akt)与细胞外信号调节激酶1/2(ERK1/2)在正常和骨关节炎(OA)软骨细胞中的表达,探讨Akt与ERK1/2在OA病程中的意义。方法: 手术中取5例正常和18例OA人膝关节软骨组织,包埋制备切片,免疫组织化学技术观察 p-Akt及p-ERK1/2在正常和OA 软骨组织中的表达;培养人软骨细胞,甲苯胺蓝染色、免疫组化鉴定并观察聚集蛋白聚糖及Ⅱ型胶原在正常和OA软骨细胞中的表达; Western blotting技术检测Akt、p-Akt、ERK1/2、p-ERK1/2、磷酸化70 kD核糖体蛋白S6激酶(p-p70S6K)及增殖细胞核抗原(PCNA)蛋白在正常和OA软骨细胞中表达水平; 实时荧光定量PCR技术检测聚集蛋白聚糖及Ⅱ型胶原在正常和OA软骨细胞中mRNA表达水平。结果: 与正常软骨细胞比较,OA软骨细胞内p-Akt和p-p70S6K蛋白表达明显降低(P<0.05),且聚集蛋白聚糖和Ⅱ型胶原mRNA和蛋白在OA软骨细胞中的表达水平降低(P<0.05),而 p-ERK1/2和PCNA蛋白表达明显提高(P<0.05)。结论: Akt可能通过p-p70S6K来调控OA软骨细胞外基质聚集蛋白聚糖及II型胶原的合成,ERK1/2可能通过PCNA来调控OA软骨细胞增殖;Akt与ERK1/2可能参与了OA的病理过程。     

Relationship among biomechanical, biochemical, and cellular changes associated with osteoarthritis

Articular cartilage that lines the surface of long bones is a multilayered material. The superficial layer consists of collagen fibrils and chondrocytes that run parallel to the joint surface. In the deeper layers, the collagen fibrils are more randomly arranged and support vertical units termed chondrons containing rows of chondrocytes. In the deepest layers, the collagen fibrils run almost vertically and ultimately insert into the underlying subchondral bone. Osteoarthritis (OA) is a disease that affects articular cartilage and is characterized by enzymatic and mechanical breakdown of the extracellular matrix, leading to cartilage degeneration, exposure of subchondral bone, pain, and limited joint motion. Changes in mechanical properties of articular cartilage associated with OA include decreases in modulus and ultimate tensile strength. These changes parallel the changes observed after enzymatic degradation of either collagen or proteoglycans in cartilage. Results of recent viscoelastic studies on articular cartilage suggest that the elastic modulus of collagen and fibril lengths decrease in OA and are associated with a loss of the superficial zone and a decreased ability of articular cartilage to store elastic energy during locomotion. It is suggested that osteoarthritic changes to cartilage involve enzymatic degradation of matrix components and fibril fragmentation that is promoted by subsequent mechanical loading.     

Increased urinary concentration of collagen type II C-telopeptide fragments in patients with osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is characterized by a progressive degeneration of articular cartilage and loss of joint function. We hypothesized that degradation of articular cartilage results in increased fragmentation of collagen type II. Thus, the concentrations of degradation products of this major cartilage matrix protein may increase in body fluids of patients with OA. METHODS: Monoclonal antibodies specific for a human collagen type II C-telopeptide (CTx-II) fragment were used in an ELISA for quantification of collagen type II fragments in urine. Clinical assessment of 88 patients with advanced OA of either hip or knee and 48 age-matched controls was performed with the Harris hip score, the Merle d'Aubigné score and a knee score. Joint space narrowing and the Kellgren and Lawrence score were assessed as radiological signs of OA. RESULTS: The concentration of CTx-II was significantly higher in OA patients compared with controls (527 vs. 190 ng/mmol, p < 0.001) whether the patients were diagnosed with hip OA (n = 51) or knee OA (n = 37). Mean CTx-II levels were higher in hip OA than in knee OA and a slight increase in levels with age was observed in the controls, but not in OA subjects. CONCLUSION: Elevation of CTx-II in urine of patients with severe OA compared with a control group suggests that collagen type II derived fragments may serve as markers for OA.     

Fourier transform infrared imaging spectroscopy analysis of collagenase-induced cartilage degradation

Collagenase treatment of cartilage serves as an in vitro model of the pathological collagen degradation that occurs in the disease osteoarthritis (OA). Fourier transform infrared imaging spectroscopic (FT-IRIS) analysis of collagenase-treated cartilage is performed to elucidate the molecular origin of the spectral changes previously found at the articular surface of human OA cartilage. Bovine cartilage explants are treated with 0.1% collagenase for 0, 15, or 30 min. In situ collagen cleavage is assessed using immunofluorescent staining with an antibody specific for broken type II collagen. The FT-IRIS analysis of the control and treated specimens mirrors the differences previously found between normal and OA cartilage using an infrared fiber optic probe (IFOP). With collagenase treatment, the amide II/1338 cm(-1) area ratio increases while the 1238 cm(-1)/1227 cm(-1) peak ratio decreases. In addition, polarized FT-IRIS demonstrates a more random orientation of the collagen fibrils that correlate spatially with the immunofluorescent-determined regions of broken type II collagen. We can therefore conclude that the spectral changes observed in the collagenase-treated cartilage, and similarly in OA cartilage, arise from changes in collagen structure. These findings support the use of mid-infrared spectral analysis, in particular the minimally invasive IFOP, as potential techniques for the diagnosis and management of degenerative joint diseases such as osteoarthritis.     

Immunohistochemical characterization of reparative tissue present in human osteoarthritic tissue

Studies involving disease progression in osteoarthritis (OA) have typically focused on the deterioration of native articular cartilage (AC) rather than the de novo cartilage which is frequently present. In general, there are two categories of de novo tissue observed in OA: (1) a pannus-like fibrocartilage that overlays native AC and (2) osteophytes. In this study, 30 AC samples representing a range of disease stages consistent with early to intermediate OA were examined for the occurrence of pannus-like tissue. All AC samples were examined immunohistochemically and compared with cartilage from three mature-looking osteophytes. To accomplish this, serial cartilage sections, derived from total knee arthroplasty specimens, were stained with hematoxylin and eosin and probed with antibodies raised against collagen type I, collagen type II, and aggrecan. Pannus-like tissue ranging from fibrous tissue to fibrocartilage was observed in 3 out of 30 AC samples. The appearance of this tissue was restricted to cartilage displaying signs of intermediate deterioration consistent with Outerbridge grade 2. Collagen type I, collagen type II, and aggrecan were abundant in both pannus-like tissue and osteophyte cartilage. In OA, the intrinsic repair process can yield a range of tissue types between fibrous tissue and fibrocartilage that is well integrated with the underlying, eroded AC. The absence of repair tissue from osteoarthritic samples representing the early stages of AC deterioration indicated that a relationship exists between macroscopic damage and a localized cellular repair response. Several histological and immunohistochemical similarities were also observed between the pannus-like tissue and osteophyte-derived cartilage, suggesting a common developmental process.     

大骨节病关节软骨胶原表型表达和软骨细胞异常分化的研究

目的探讨大骨节病关节软骨胶原表型表达的变化特点和软骨细胞异常分化在发病中的意义。方法用单克隆免疫组化法测定５例大骨节病关节软骨Ⅰ、Ⅱ、Ⅲ、Ⅵ、Ⅹ型胶原表型的表达。结果（１）关节软骨表层的Ⅱ型胶原表型表达减少；（２）Ⅰ、Ⅲ和Ⅵ型胶原表型表达见于关节软骨全层，而Ⅹ型胶原仅限于关节软骨钙化层和深层软骨细胞团周围；（３）软骨细胞团有Ⅰ、Ⅱ、Ⅲ、Ⅵ型胶原表型表达，而软骨细胞坏死区内无任何胶原表型表达。结论大骨节病关节软骨胶原表型表达类似于原发性骨关节病的变化，但在关节软骨表层Ⅰ型胶原表型表达增强以及软骨坏死区内无任何胶原表型表达不同于原发性骨关节病。     

Viscoelastic behavior of osteoarthritic cartilage

We have studied the incremental stress-strain behavior of human articular cartilage in tension in an attempt to understand the molecular basis for fibrillation and fissure formation in osteoarthritis. Our results indicate that the elastic spring constant for collagen in the direction per pendicular to the cleavage line pattern is about 1.6 GPa (2.3 GPa after correction for the collagen content) and the collagen fibril length is between 0.558 pm at low strains and 1.24 pm at high strains for normal cartilage. Values for the elastic spring constant and collagen fibril length were both found to decrease in OA. The value of the elastic spring constant for collagen perpendicular to the cleavage line pattern is similar to that calculated based on stress-strain curves reported by Kempson. Our results indicate that the elastic spring constant for collagen and the collagen fibril length decrease as the extent of fibrillation and fissure formation increase. Decreases in the elastic spring constant of collagen are consistent with loss of the superficial layer, degradation of proteoglycans and collagen, and subsequent mechanical fatigue. However, changes in the polymer volume fraction are consistent with enzymatic degradation preceding mechanical disruption. It is concluded that osteoarthritic changes to cartilage involve enzymatic degradation of matrix components and fibril fragmentation that is promoted by subsequent mechanical loading.     

The Effects of Collagen Fragments on the Extracellular Matrix Metabolism of Bovine and Human Chondrocytes

Cartilage matrix degradation generates collagen type II fragments. The objective of this study is to explore the possibility that these collagen fragments may be part of an endogenous metabolic feedback. Initially, collagen fragments were extracted from normal or osteoarthritic cartilage, as part of a matrix fragment preparation. Later, collagen fragments were generated by digestion of bovine collagen type II with bacterial collagenase (col2f). These fragments were added to cultures of isolated chondrocytes (bovine and human) and cartilage explants (human). In a dose-dependent manner, col2f caused inhibition of cell attachment to collagen, inhibition of collagen synthesis, and induction of matrix degradation. In addition, when col2f were added to human cartilage explants, an induction of gelatinase activity was detected in the media. These data sets present first evidence that degradation products of collagen may be directly involved in the regulation of cartilage homeostasis.     

Absence of immunity to type II collagen and proteoglycan in osteoarthritic C57BL mice

We measured immunity to type II collagen and proteoglycans in osteoarthritic C57BL mice to determine whether it is related to osteoarthritis pathogenesis. Histological examination revealed articular cartilage lesions in all mice and synovitis in only a few mice. Immunological responses to type II collagen were found in collagen arthritic mice, but not in C57BL mice. Furthermore, immunological responses to proteoglycans were observed in proteoglycan-immunized mice, but not in C57BL mice. Therefore, articular cartilage degeneration may not result in autoimmunity to type II collagen and proteoglycans in osteoarthritis of C57BL mice, and immune responses to these components may not be a primary etiology of osteoarthritis in this model.