Articular osteochondrosis (OCD) occurs in both man and animals. The etiology remains to be determined. Studies of OCD lesions in animals may provide clues as to its pathogenesis. The aim of our study was to determine whether there was evidence for increased degradation namely proteoglycan (PG) release and type II collagen cleavage in articular cartilage harvested from OCD lesions. We examined ex vivo explants at post-mortem from equine OCD lesions and macroscopically normal site and age matched cartilage. These were cultured over a 10 day period in serum-free medium. Type II collagen cleavage was measured in articular cartilage and media using an Elisa assay to detect the COL2-3/4C(short) epitope, which is generated on cleavage of the triple helix of type II collagen by collagenases. PG release was measured by a dye-binding assay. Cumulative release of PG and COL2-3/4C(short) and their contents in cartilage at the end of the culture period were determined. In OCD lesions there was a significant increase in type II collagen cleavage by collagenase but no evidence for increase of PG degradation. These findings point to a selective increase in type II collagen cleavage by collagenases, in OCD lesions of the kind observed in osteoarthritis. Further work is needed to determine whether changes represent primary or secondary events in the pathogenesis of OCD.

The assembly and resorption of the extracellular matrix in the physis of the growth plate are poorly understood. By examining isolated fetal growth plate chondrocytes in culture and using immunochemical methods we show that type II collagen, proteoglycan aggrecan, and type IX collagen are assembled into a matrix that is initially enriched in type II collagen over proteoglycan and type IX collagen. When compared to the content of the COL2 domain in the alpha(1)(IX) chain it is evident that the majority ( 90%) of type IX molecules lack the NC4 domain unlike in articular cartilage. During matrix assembly the molar ratio of type II/COL2 of alpha(1)(IX) varied from 25:1 to 2.5:1. Following expression of the hypertrophic phenotype (initiation of type X collagen synthesis) there are parallel changes in both collagen and proteoglycan contents (inversely related to collagenase cleavage of type II collagen). The NC4 domain is then selectively, rapidly and irreversibly removed as mineralization is initiated, leaving the alpha(1)(IX) chain COL2 domain. Subsequently as mineralization progresses type II and type IX collagen (COL2 domain), but not the proteoglycan aggrecan, are resorbed coincident with a markedly increased cleavage of type II collagen by collagenase as mineral is deposited in the matrix. This study, therefore reveals a carefully orchestrated series of events in matrix assembly and resorption that prepares the extracellular matrix for mineralization.

OBJECTIVE:To determine whether patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS) express cellular immunity to cartilage link protein (LP).METHODS:LP was purified from human fetal epiphyseal and bovine adult nasal cartilage. It was used in proliferation assays with the peripheral blood lymphocytes (PBL) isolated from 83 patients with RA, 21 patients with AS, and 30 healthy controls.RESULTS:Patients with RA (34%) and AS (71%) expressed a significantly higher prevalence of cellular immune responses to human LP compared with the healthy control group (13%). Such significant differences were not observed for bovine LP. Half the patients with RA responding to LP exhibited cellular immunity to both human and bovine protein. In the AS group, PBL from a majority of responders to LP recognized only human LP.CONCLUSION:These data suggest that LP is a potential autoantigen in the development of RA and AS and that cellular immune reactivity to common and distinct LP epitopes in patients with RA and AS may play a role in the pathogenesis of these diseases.

Our previous work showed that the proteoglycan aggrecan can induce erosive polyarthritis and spondylitis in BALB/c mice, and that the G1 domain of the proteoglycan aggrecan (G1) is the arthritogenic region. In this study, two T cell epitopes residing on G1 within residues 70-84 (peptide G5) and 150-169 (peptide G9) were identified using synthetic peptides and aggrecan-specific T cell lines. Two G1-specific T cell hybridomas exclusively responded to peptide G5. When the G5-specific T cell line was injected intraperitoneally into BALB/c mice, it induced acute inflammatory arthritis in joints, but only in those that had been injected with the epitope recognized by these T cells. Furthermore, we also demonstrate that the keratan sulfate chain(s) (KS) on G1 possess immunosuppressive properties with respect to T and B cell epitope recognition. T cell lines that recognize both G1 and peptide G5 show an increased response to G1 after KS is removed. Antibodies in hyperimmune sera of mice immunized with G1 show increased epitope recognition (quantitative and qualitative) after KS removal before immunization. These studies reveal that a T cell line specific to an epitope on the G1 domain of aggrecan, also recognizing a corresponding mouse G1 epitope, can induce arthritis by adoptive transfer and homing to the intraarticular epitope, thereby implicating T cells in arthritis development caused by immunity to the G1 domain of aggrecan. Moreover, the presence of KS on G1 can inhibit arthritis development by suppressing T and B cell epitope recognition.

Both type II collagen and the proteoglycan aggrecan are capable of inducing an erosive inflammatory polyarthritis in mice. In this study we provide the first demonstration that link protein (LP), purified from bovine cartilage, can produce a persistent, erosive, inflammatory polyarthritis when injected repeatedly intraperitoneally into BALB/c mice. We discovered a single T-cell epitope, located within residues 266 to 290 of bovine LP (NDGAQIAKVGQIFAAWKLLGYDRCD), which is recognized by bovine LP-specific T lymphocytes. We also identified three immunogenic regions in bovine LP that contain epitopes recognized by antibodies in hyperimmunized sera. One of these B-cell regions is found in the most species-variable domain of LP (residues 1 to 36), whereas the other epitopes are located in the most conserved regions (residues 186 to 230 and 286 to 310). The latter two regions contain an AGWLSDGSVQYP motif shared by the G1 globulin domain of aggrecan core protein, versican, neurocan, glial hyaluronan-binding protein, and the hyaluronan receptor CD44. Our data reveal that the induction of arthritis is associated with antibody reactivities to B-cell epitopes located at residues 1 to 19. Together, these observations show that another cartilage protein, LP, like type II collagen and the proteoglycan aggrecan, is capable of inducing an erosive inflammatory arthritis in mice and that the immunity to LP involves recognition of both T- and B-cell epitopes. This immunity may be of importance in the pathogenesis of inflammatory joint diseases, such as juvenile rheumatoid arthritis, in which cellular immunity to LP has been demonstrated.

OBJECTIVE:To determine whether children with juvenile rheumatoid arthritis (JRA) express cellular immunity to cartilage link protein.METHODS:Link protein was purified from human fetal epiphyseal and bovine adult nasal cartilage. It was used in proliferation assays with the peripheral blood lymphocytes isolated from 54 children with JRA and 22 nonarthritic controls.RESULTS:Patients with JRA expressed a significantly higher prevalence of cellular proliferation to human link protein compared with the control group, independent of whether they had pauciarticular, polyarticular, or systemic disease. In the case of bovine link protein, significant differences were only noted for the polyarticular group. There was significant correlation between the immunity to bovine and to human link proteins. Furthermore, immune responses to both molecules correlated with measures of joint disease activity.CONCLUSION:These results suggest that immunity to link protein may play a role in the pathogenesis of JRA.

Earlier work from this laboratory showed that the human proteoglycan aggrecan from fetal cartilages can induce a CD4+ T cell-dependent inflammatory polyarthritis in BALB/c mice when injected after removal of chondroitin sulfate chains. Adult keratan sulfate (KS)-rich aggrecan does not possess this property. We found that two CD4+ T cell hybridomas (TH5 and TH14) isolated from arthritic mice recognize bovine calf aggrecan and the purified G1 domain of this molecule, which also contains a portion of the interglobular domain to which KS is bound. These hybridoma responses to G1 are enhanced by partial removal of KS by the endoglycosidase keratanase or by cyanogen bromide cleavage of core protein. KS removal results in increased cellular uptake by antigen-present cells in vitro. After removal of KS by keratanase, G1 alone can induce a severe erosive polyarthritis and spondylitis in BALB/c mice identifying it as an arthritogenic domain of aggrecan. The presence of KS prevents induction of arthritis presumably as a result of an impaired immune response as observed in vitro. These observations not only identify the arthritogenic properties of G1 but they also point to the importance of glycosylation and proteolysis in determining the arthritogenicity of aggrecan and fragments thereof.

Persistent gene lymphadenopathy has been well described in patients with seropositivity to the human immunodeficiency virus (HIV). Moreover, isolated enlargement of the parotid gland and parotid lymphadenopathy have been noted much more frequently over the past few years. Histologically, these lesions demonstrate follicular hyperplasia, cystic dilatation of the ducts fined by pseudo-stratified squamous epithelium, and lymphocytic infiltrates. They are generally considered to be benign lymphoepithelial lesions of the parotid or hyperplastic periparotid lymph nodes. The relationship of this entity to the AIDS-related complex (ARC) and the subsequent development of AIDS is not clear.Over the past 7 years, we have seen 50 patients with parotid enlargement in whom the diagnosis of benign lymphoepithelial lesion was made. Fine-needle aspiration was performed in 32 patients. Although not conclusively diagnostic, needle aspirates ruled out primary salivary glandular pathology. Most patients gave a history of intravenous drug abuse. HIV tests have been performed on a routine basis only in the last 2 years, and these were positive in the majority of the patients.Thirty-five patients underwent surgical excision. In the initial 20 patients, we routinely performed parotid exploration, identification of the facial nerve, and superficial parotidectomy. In the last 15 patients, we changed our surgical approach to parotid exploration and excision of the mass in the tail of the parotid. The exposure of the posterior belly of the digastric muscle, with identification and removal of the deep jugular node, has become routine. In each case, we found an enlarged lymph node in the deep jugular region, which was not clinically palpable preoperatively. The rate of surgical complications was minimal, and, after resection of the mass, patients improved symptomatically. If the patient shows obvious signs of AIDS, a nonsurgical approach with repeated aspirations should be considered, and treatment with zidovudine offered.

Hyperimmunization with chondroitin sulfate-depleted fetal human cartilage proteoglycan (HFPG) leads to the development of peripheral arthritis and spondylitis in BALB/c mice. Chondroitin-sulfate-depleted adult human cartilage proteoglycan (HAPG) is much less effective at inducing arthritis. These observations suggest age differences in the presence of arthritogenic proteoglycan (PG) epitopes. Earlier studies from this laboratory have indicated an important role for PG-reactive T cells in the pathogenesis of this arthritis model. To investigate further the cellular immunity to PG in mice, two T cell lines, JY.A and JY.D, and two T cell hybridomas, TH5 and TH14, were isolated from mice with PG-induced arthritis and characterized. Two patterns of reactivity to PG emerged from the analysis of these T cells. One pattern, as demonstrated by the T cell line JY.D and the two T cell hybridomas, TH5 and TH14, was characterized by reactivity to HFPG, HAPG, chondroitin sulfate-depleted bovine cartilage PG, the G1 domain (hyaluronate binding region) of bovine cartilage PG and bovine link protein. The epitope(s) recognized by these T cells appear to be part of the homologous regions shared between the G1 domain and the link protein. The second pattern of reactivity, as demonstrated by the T cell line JY.A, was characterized by reactivity to HFPG but not to HAPG or the other PG Ag or bovine link protein. All the T cell lines and hybridomas had a CD4+, CD8- phenotype, possibly belonged to the TH1 subset (IL-2+, IL-4-), and were MHC class II restricted. These studies indicate that HFPG has T cell epitopes in common with HAPG (such as in the G1 domain) and different than those in HAPG. The significance of this data in terms of PG structure, changes with age, and induction of arthritis remains to be established.

Peripheral arthritis is produced in BALB/c mice after hyperimmunization with the cartilage proteoglycan aggrecan (PG). Adoptive transfer studies have suggested the roles of T cells including CD8+ T cells in the disease process. To evaluate the roles of CD4+ and CD8+ T cell subsets in vivo in the induction of this disease by immunization, PG-immunized mice were treated with isotype-controlled rat IgG2b monoclonal anti-CD4 or anti-CD8 antibodies, or were left untreated. CD4+ T cell depletion resulted in total inhibition of the disease with markedly decreased anti-PG antibody responses. CD8+ T cell depletion, however, significantly enhanced the severity of the disease without affecting peak anti-PG antibodies, as compared to the control mice. These results demonstrate a crucial role for CD4+ T cells in the pathogenesis of this disease. However, CD8+ T cells do not seem to be required for the induction of arthritis by immunization but instead may play an immunoregulatory role.

The introduction of a new microanalytical approach has made it possible to investigate the molecular structure and content of proteoglycans within the extracellular matrix of the physis when the volumes of the matrix and the cells change before mineralization of the extracellular matrix. By using extracts of serial frozen sections with a combination of gel chromatography and chemical, radioimmunoassay, and morphometric analyses, we demonstrated that, in the bovine fetal physis, the content of large aggregating proteoglycans reaches a maximum in the extracellular matrix of the hypertrophic zone, in the region where calcification begins, at the time when calcification starts. Morphometric analyses revealed that, at this time, the volume of matrix is reduced to a minimum. There was no evidence that the remaining proteoglycans were reduced in size, nor was aggregation changed at the time when calcification was initiated. Thus, contrary to earlier reports, there is no indication that these molecules need to be degraded and removed for calcification to begin, although a progressive loss of proteoglycans clearly occurs as the volume of the matrix is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

The value of needle aspiration biopsy in the evaluation and management of salivary gland pathology is controversial. The major reasons for this controversy are the difficulty in cytologic evaluation and the fact that the extent of surgery can be easily defined based on clinical judgement. However, a preoperative diagnosis is helpful in discussions with patients regarding the extent and type of surgery. Apart from the fact that needle biopsy can distinguish benign from malignant conditions, it is also very useful in distinguishing between salivary and other nonsalivary pathology. Over the past 7 1/2 years, we have performed 160 needle aspirations of parotid, submandibular, and submucosal lesions. Adequate specimens for cytologic evaluation were obtained in 155 patients (97%). A total of 84 parotid lesions, 70 submandibular lumps, and 6 submucosal abnormalities were detected. A cytologic diagnosis of benign pathology was made in 120 patients. Twelve patients had lymphoma and the diagnosis was suspected based on needle aspiration. There were 10 patients with tuberculosis and 30 patients with hyperplastic lymph nodes or benign lymphoepithelial disease of the parotid. There were three false-positive and two false-negative reports. No complications such as hematoma, nerve injury, or infection developed. The major difficulty was in distinguishing between malignancy and obstructive sialadenitis in the submandibular region. Needle aspiration was helpful in evaluating lesions in the tail of the parotid and submandibular area. The cytologic distinction between salivary and nonsalivary pathology was useful in planning the appropriate surgery and the extent of surgical resection. From a clinical standpoint, the distinction between benign and malignant salivary and nonsalivary pathology was very helpful. Preoperative diagnosis of Warthin's tumor, lymphoma, or benign lymphoepithelial disease was essential to the correct management of these patients.

A mouse monoclonal antibody (AN9P1) to keratan sulphate is described. In a competitive-inhibition solution-phase radioimmunoassay employing 125I-labelled intact proteoglycan, it reacts preferentially with keratan sulphate bound to the core protein of adult human articular-cartilage proteoglycan and to a much lesser degree with keratan sulphate purified from this proteoglycan. Proteolytic cleavage of the proteoglycan by pepsin and trypsin has little effect on antibody binding, but treatment with papain decreases binding considerably and more than does treatment with keratanase. An even greater decrease in binding is observed after treatment with alkaline borohydride. A comparison of binding of antibody AN9P1 with that of another previously described monoclonal antibody, 1/20/5-D-4, to keratan sulphate [Caterson, Christner & Baker (1983) J. Biol. Chem. 258, 8848-8854] revealed similar binding characteristics, both showing much diminished binding after papain digestion of proteoglycan and even less with purified skeletal keratan sulphate. Removal of the Fc piece of antibody AN9P1 had no significant effect on the differential binding of divalent F(ab')2 fragment to proteoglycan, to papain-digested proteoglycan and to keratan sulphate, although there was a small decrease in binding to papain-digested proteoglycan. Conversion of the antibody into univalent Fab fragment with removal of the Fc piece resulted in diminished binding to proteoglycan, compared with that observed with IgG, and in enhanced binding to free keratan sulphate and to papain-digested proteoglycan. These results suggest that close proximity of keratan sulphate chains on the core protein of proteoglycans favours preferential reactivity of bivalent antibody with these species through cross-bridging of chains by antibody. Conversely, much decreased binding to keratan sulphate on proteoglycan core-protein fragments and to free keratan sulphate results from a lack of close proximity of keratan sulphate. By using univalent Fab fragment in these assays these differences in binding are minimized by preventing cross-bridging and thereby enhancing detection of smaller fragments without sacrificing too much sensitivity of detection of larger proteoglycan species. The persistent preferential binding of Fab fragment to proteoglycan is probably in part the result of the increased epitope density in the intact molecule compared with keratan sulphate in a more disperse form.

The large aggregating cartilage proteoglycans can be detected immunologically using antibodies directed against the hyaluronic acid-binding region, core protein, keratan sulfate, chondroitin sulfate, and hyaluronic acid. With radioimmunoassays we have detected proteoglycans released from adult human articular cartilage in vitro and in vivo. In vitro cleavage appears to occur mainly adjacent to the hyaluronic acid binding region with the release of the rest of the molecule. Two distinct populations have been observed, the larger of which is chondroitin sulfate-rich and the smaller keratan sulfate-rich. Although they are a little smaller and probably aggregate less with hyaluronic acid, these populations correspond in size and composition to two similar populations detected in situ in healthy adult human articular cartilage. In vivo studies of synovial fluid have revealed that the proteoglycan fragments are smaller and the most commonly detected fragment is the hyaluronic acid binding region. This suggests that in vivo further degradation occurs, probably due to degradation mediated by the inflamed synovium and polymorphonuclear leucocytes present in the synovial fluid of inflamed joints.