Masayuki Shinmei

The role of interleukin-1 on proteoglycan metabolism of rabbit annulus fibrosus cells cultured in vitro

The effects of human recombinant interleukin-1 alpha (IL-1 alpha) on proteoglycan (PG) metabolism of rabbit intervertebral disc were investigated morphologically and biochemically using rabbit annulus fibrosus (AF) cells in culture. AF cells could maintain their differentiated phenotype well in our primary culturing condition. In this situation, IL-1 alpha stimulated the cells and induced marked increase of PG release. Dose dependency of IL-1 alpha on PG release was seen in the concentration range between 5-50 U/ml. Caseinolytic activity produced and secreted into the medium by AF cells was assayed and it was found that IL-1 alpha enhanced the enzyme activities in the medium. The effects of IL-1 alpha on PG and DNA synthesis were also studied. Slight depression was observed in PG synthesis but there was no effect on DNA synthesis. These data suggest that IL-1 alpha may play an important role in PG metabolism of intervertebral disc cells, especially in the catabolic pathway of PG.

Markers of cartilage matrix metabolism in human joint fluid and serum: the effect of exercise*

The concentrations of cartilage proteoglycan (aggrecan), stromelysin-1, tissue inhibitor of metalloproteinases-1 (TIMP-1) and procollagen II C-propeptide in knee joint fluid and the levels of aggrecan, hyaluronan and keratan sulfate in serum were measured before and after exercise in 33 healthy athletes. The samples before exercise were obtained after 24 h rest from running or soccer and the samples after exercise were obtained 30-60 min after the exercise. Nine athletes ran on a treadmill for 60 min, 16 ran on road for 80 min and 8 played one soccer game (90 min). A reference group of 28 patients with knee pain but not evidence of joint pathology or injury was used for comparison. In joint fluid no single marker from the degradative processes in cartilage matrix changed significantly with exercise but all showed a rising trend. All markers except stromelysin showed lower concentrations in athletes at rest compared to the reference group. In serum from runners before exercise the concentration of keratan sulfate was significantly higher than in both the soccer and reference groups and further increased after exercise. The increase in markers after exercise may reflect an effect of mechanical loading in combination with a possible high turnover rate of body cartilage matrix in these individuals.

Joint fluid carboxy-terminal type II procollagen peptide as a marker of cartilage collagen biosynthesis

Joint fluid levels of carboxy-terminal type II procollagen peptide (pCOL II-C) were measured in osteoarthritis, rheumatoid arthritis and traumatic arthritis by a newly developed one-step enzyme immunoassay (EIA). The detection limit of the new method was as low as 0.2 ng/ml. The levels of pCOL II-C were significantly (P < 0.001) higher in osteoarthritis and traumatic arthritis than in rheumatoid arthritis. In osteoarthritis, pCOL II-C levels were higher in moderately afflicted patients. Since type II collagen is a unique component of cartilage, pCOL II-C levels in joint fluids could reflect the synthetic activity of type II collagen of chondrocytes in the diseased joint and therefore could be utilized as a simple marker of type II collagen synthesis in articular cartilage in joint diseases.

Synovial fluid concentrations of the C-propeptide of type II collagen correlate with body mass index in primary knee osteoarthritis

OBJECTIVE To explore in a cross sectional study in patients with primary knee osteoarthritis (OA) the relations between body mass index (BMI), disease stage, and the concentrations of a putative joint fluid marker of type II collagen synthesis, procollagen II C-propeptide. PATIENTS AND METHODS The study included 142 patients with knee OA (median age 68, median BMI 24.1). OA was staged radiologically. The concentrations in synovial fluid of procollagen II C-propeptide were measured by a sandwich enzyme immunoassay. RESULTS Joint fluid concentrations of procollagen II C-propeptide were increased in knees with OA (median 3.7 ng/ml), compared with published reference values for knees in healthy adult volunteers (median 1.3 ng/ml). The concentrations of procollagen II C-propeptide were independently related to both OA stage and BMI (r s = 0.343, p < 0.0001 andr s = 0.253, p = 0.002, respectively). CONCLUSIONS Joint fluid concentrations of this putative marker of collagen II synthesis are high in early and mid-stage OA, but decrease in end stage disease. In addition and for the first time it was shown that the concentrations in synovial fluid of procollagen II C-propeptide increase with increasing BMI in primary knee OA. The increased joint fluid values of this marker in patients with primary knee OA and a high BMI, may reflect increased rates of collagen synthesis in their joint cartilage and could relate to the previously shown increased risk for disease progression in such patients.

The Mechanism of Cartilage Degradation in Osteoarthritic Joints

0 STEOARTHRITIS (OA) is primarily a disorder of cartilage, at least partially caused by neutral proteases. These enzymes are metalloproteinases and include collagenase, gelatinase, and stromelysin (matrix neutral metalloproteinase 3: MMP-3).‘,* Recent studies on cytokines in joint tissues suggest interleukin 1 (IL-l) and tumor necrosis factor alpha (TNFa) modulate synthesis of metalloproteinase by chondrocytes3’4 and synovial cells.5V6 Interleukin 1 and TNFa! may also be produced by synovial cells7 and chondrocytes.‘*’ Interleukin 6 (IL-6) “J has been detected in synovial fluid’2-‘4 and is also produced by both synovial cells’5U’7 and human articular chondrocytes.18-*’ This study is directed at correlating the distribution of MMP-3 and cytokines in cartilage, synovium, and synovial fluid of human OA.

Effects of Interleukin-1 and Anti-Inflammatory Drugs on the Degradation of Human Articular Cartilage

SummaryIt has been suggested that metalloproteases produced by chondrocytes play an important role in cartilage breakdown in joint diseases. The aim of this study was to investigate changes in enzyme activities in human rheumatoid and osteoarthritic articular cartilage.Cartilage fragments were incubated with various drugs for 48 hours. The concentrated culture media were used as enzyme solutions. Collagenase was assayed using FITC-collagen as the substrate. Proteoglycanase (PGase) was measured either by the release of 35Slabelled proteoglycans from cartilage into the medium, or by enzyme assay using proteoglycan monomer bound to fluorescein-conjugated hyaluronic acid as the substrate.Collagenase and proteoglycanase were found only in trace amounts in the concentrated media of healthy cartilage. Interleukin-1 (IL-1) enhanced the enzyme activities significantly. Marked increases of enzyme activities were observed in the concentrated media of rheumatoid (RA) and osteoarthritic (OA) cartilage. The sensitivity to interleukin-1 was also higher in OA and RA cartilage compared with healthy cartilage. Dexamethasone (106 mol/L) markedly depressed enzyme activity. Tiaprofenic acid (4 × 10−5 mol/L) also decreased enzyme activity, whereas indomethacin (4 × 10−6 mol/L) and naproxen (3 × 10−4 mol/L) had no effect.

Glycosaminoglycans in malignant diffuse mesothelioma.

Because of frequently encountered diagnostic difficulty due to a morphologic similarity between diffuse pleural mesothelioma and peripheral pulmonary adenocarcinoma, glycosaminoglycans (GAG) of human malignant diffuse mesothelioma were histochemically stained and chemically quantitated, and were compared with GAG of papillary adenocarcinoma of the lung. In all seven patients, the diagnosis of diffuse mesothelioma was confirmed morphologically by such findings as abundant bushy microvilli on cell surface and intermediate filaments in cytoplasm. The total GAG in mesothelioma obtained from fresh materials (5 cases) was significantly increased over that in pleural connective tissue (P < 0.01) and lung adenocarcinoma (P < 0.02). Two dimensional electrophoretic separation of GAG of mesothelioma and lung cancer showed hyaluronic acid, heparan sulfate, heparin, dermatan sulfate and chondroitin sulfate; among them, the two predominant fractions were hyaluronic acid and chondroitin sulfate. In the quantitative analysis, the hyaluronic acid content of mesothelioma averaged 57% of the total GAG, but that of lung adenocarcinoma, 38%. The results suggest that chemical analysis of GAG may be useful as supplementary diagnostic procedure to morphologic examination in the differentiation of diffuse mesothelioma from papillary adenocarcinoma of the lung.

Localization of Carboxy-terminal Type II Procollagen Peptide (pCOL-II-C) and Type II Collagen in the Repair Tissue of Full-thickness Articular Cartilage Defect

It is well established that a full-thickness articular cartilage defect is repaired with a fibrocartilaginous tissue of which cells are derived from undifferentiated mesenchymal stem cells in the bone marrow. To characterize the repair tissue immunohistochemically, full-thickness defects were created in rabbit knee joints, and the repair tissues immunostained at 3, 6, and 12 weeks after surgery. Well characterized polyclonal antibody against carboxyterminal type II procollagen peptide (pCOL-II-C) and monoclonal antibody against type II collagen were used to evaluate the repair tissue with regard to the metabolism of type II collagen. Immunohistochemistry revealed that pCOL-II-C was localized in or around most of the repair cells obtained at 3 and 6 weeks after surgery, while type II collagen distributed mainly in the pericellular matrix of metaplastic round-shaped repair cells. The results suggest that the repair cells taken at the early stage of the repair process of the defect could originally have more activity of type II collagen synthesis.

Localization of carboxy-terminal type II procollagen peptide (pCOL-II-C) in diseased cartilage

A well characterized rabbit polyclonal antibody against human carboxy-terminal type II procollagen peptide (pCOL-II-C) was used to study the immunolocalization of pCOL-II-C in articular cartilage obtained from patients with osteoarthritis (OA), rheumatoid arthritis (RA), and control non-diseased joints. In moderately degenerative OA cartilage, immunoreactive chondrocytes were observed in all layers, particularly along the margins of fibrillation and fissures, in chondrocyte clusters and in osteochondrophytes. The grade of immunostaining in OA correlated directly with Mankins histological-histochemical scores of 0–7, but there was an inverse correlation between grade of immunostaining and Mankins scores of 8–14. The grade of immunostaining was significantly higher in OA than in RA and normal control cartilage. Since type II collagen is a unique component of articular cartilage, localization of pCOL-II-C could reflect the increased synthesis of type II collagen by chondrocytes in diseased cartilage.