Extracellular matrix metabolism by chondrocytes. 5. The proteoglycans and glycosaminoglycans synthesized by chondrocytes in high density cultures.

Proteoglycans were extracted from the extracellular matrix of cultures of embryonic chick chondrocytes grown at high density and were purified by CsC1 density gradient centrifugation. The chemical, physical and hyaluronate binding properties of the proteoglycans were similar to those observed in proteoglycans from other hyaline cartilages. Proteoglycans in the media were also purified and on analysis showed three populations of proteoglycans to be present. One population had the physical characteristics of a typical proteoglycan subunit and bound hyaluronate, the other two populations were unable to complex with hyaluronate but one had the physical characteristics of the proteoglycan subunit and the other was of smaller molecular weight. The small molecular weight appears to be a product of the enzymatic degradation of the larger molecular weight species.

Extracellular matrix metabolism by chondrocytes. III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture.

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. Benzyl-beta-D-xyloside, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.

Inhibition of proteoglycan biosynthesis by hyaluronic acid in chondrocytes in cell culture.

The depression of proteoglycan synthesis in ten-day-old high density chondrocyte cultures was shown to be dependent on both the concentration and time of exposure of the cells to hyaluronic acid. Hyaluronic acid had no effect on the overall protein synthesis by the cultured cells. Using benzyl-beta-D-xyloside an exogenous acceptor, it was shown that glycosaminoglycan biosynthesis by the chondrocytes was not affected by hyaluronic acid. It was concluded that hyaluronic acid was effecting glycosaminoglycan chain initiation, hence proteoglycan biosynthesis, either by specifically depressing the synthesis of the core protein or by repressing the activity of the xylosyltransferase.

Effect of dibutyryl cyclic AMP on the sulphation of proteoglycans by chondrocytes.

The addition of a 1 mM N6, O2'-dibutyryl cyclic AMP to incubations of foetal calf articular cartilage for a 4 h period resulted in a 13 and a 15% decrease, respectively, in the rate of incorporation of [3H]glucosamine and [14C]glucose into glycosaminoglycans. Under the same conditions, a 41% increase in the rate of incorporation of [35S]sulphate into glycosaminoglycans was observed. Dibutyryl cyclic AMP had no effect on protein synthesis over a 4 h period or on the hydrodynamic size of the proteoglycan subunits or glycosaminoglycans synthesized by the cartilage. When the glycosaminoglycans were digested with chondroitin ABC lyase and the resulting disaccharides analysed on Dowex 1 (formate form), it was observed that dibutyryl cyclic AMP increased the degree of sulphation of the disaccharides. Furthermore, an over-sulphated disaccharide corresponding to chondroitin 4,6-disulphate was identified. It was concluded that dibutyryl cyclic AMP was stimulating the process of sulphation of glycosaminoglycans by cartilage.

Extracellular matrix metabolism by chondrocytes. 4. Role of glutamine in glycosaminoglycan synthesis in vitro by chondrocytes.

The rate of synthesis of glycosaminoglycans by cartilage was shown to be dependent on an exogenous source of L-glutamine. In the absence of L-glutamine the tissue and cellular levels of this amino acid were rapidly depleted. The levels of nucleotide sugars and their precursors were measured after separation on Dowex 1 (formate form) in cartilage incubated with and without L-glutamine. It was found that the levels of N-acetylhexoamine 6-phosphate and UDP-N-acetylhexosamine were decreased by 27 and 40% respectively. This demonstrates that L-glutamine is required as the amido group donor in the synthesis of glucosamine 6-phosphate and that the decrease in glycosaminoglycan synthesis is due to the limitation in synthesis of UDP-N-acetylhexoamine.

Extracellular matrix metabolism by chondrocytes. 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells.

The incorporation of [3H]glycine into acid-insoluble protein and of [3H]acetate into glycosaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gradients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of polyribosomes that was revered by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence of L-glutamine was also demonstrated for other avain connective tissues.

Molecular size distribution of proteoglycan subunits and glycosaminoglycans synthesized by chondrocytes under conditions of reduced proteoglycan synthesis.

The rate of proteoglycan synthesis by chondrocytes in vitro was depressed by either omitting L-glutamine from the incubation medium or by addition of proteoglycan subunit to the medium. The molecular size distribution on Sepharose 2B of the proteoglycan subunits synthesized by the chondrocytes under these conditions of reduced proteoglycan synthesis was found to be the same as those synthesized by the control cells. Likewise, the molecular size distribution on Sepharose 6B CL of the glycosaminoglycan chains synthesized by the depressed cells was found to be similar to that observed in untreated chondrocytes. This work demonstrates that, under conditions of reduced proteoglycan synthesis, fewer proteoglycan subunits are synthesized by chondrocytes and that the molecular size distribution of these macromolecules is similar to those synthesized by untreated cells.

Degradation of proteoglycan in articular cartilage.

Adult rabbit articular cartilage was labelled in vivo over 48 h with [35S]sulphate and was then incubated in organ culture at pH 7.2. Approx. 65% of the tissue content of [35S]proteoglycan was released into the culture medium during the first 48 h of incubation. The average molecular size of the released proteoglycans, as assessed by fractionation on Sepharose 2B/CL and 4B/Cl, was only slightly smaller than that of the proteoglycans extracted from non-cultured cartilage with 4 M guanidine HCl. The percentage of released proteoglycans and extracted proteoglycans which formed aggregates with hyaluronic acid was approx. 25% and 75%, respectively. The results indicate that proteoglycan degradation in adult articular cartilage is initiated by a limited proteolysis of subunit core protein, with the production of non-aggregating species which diffuse readily from the tissue.

Extracellular matrix metabolism by chondrocytes. VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes.

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[3H]proline into hydroxyproline and [3H]acetate into glycosaminoglycans, was shown to be depressed by 58% and 39%, respectively, by the addition of exogenous proteoglycan at a concentration of 10 mg/ml growth media. The incorporation of L-[3H]proline into acid-insoluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity on the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-translational modification of collagen and proteoglycan.

Characterization of the collagen synthesized by cultured cartilage cells.

Cartilage cells from embryonic chick cartilage were grown in primary cultures. The cell layer was sequentially extracted with neutral saline, mercaptoethylamine and pepsin which revealed that these cells produced salt-soluble and salt-insoluble collagen. The alpha1- to alpha2-chain ratio was determined for the collagen extracted from the cultured cells and was found to be 13 to 1. Further analysis of the molecule was carried out by CNBr cleavage of the salt-extracted collagen and separation of resulting peptides by ion-exchange chromatography. It was shown that the cultured cartilage cells synthesize collagen of the type (alpha1[II])3.

Inhibition of proteoglycan synthesis by hydrogen peroxide in cultured bovine articular cartilage.

Oxygen-derived reactive species, generated enzymatically by the action of xanthine oxidase upon hypoxanthine, significantly inhibit proteoglycan synthesis by cultured bovine articular cartilage (Bates, E.J., Lowther, D.A. and Handley, C.J. (1984) Ann. Rheum. Dis. 43, 462-469). Here we extend these investigations and show, through the use of catalase and the specific iron chelator diethylenetriaminepentaacetic acid, that the active species involved is H2O2 and not the hydroxyl radical. Incubations of cartilage with H2O2 at concentrations of 1 X 10(-4) M and above are also inhibitory to proteoglycan synthesis. Subsequent recovery of the tissue is dependent upon the initial dose of xanthine oxidase or H2O2. Xanthine oxidase at 84 mU per incubation results in a prolonged inhibition of proteoglycan synthesis which is still apparent after 14 days in culture. Lower concentrations of xanthine oxidase (21-66 mU) are inhibitory to proteoglycan synthesis, but the tissue is able to synthesise proteoglycans at near normal rates after 3 days in culture. The inhibition of proteoglycan synthesis by 1 X 10(-4) M H2O2 is completely reversed after 5 days in culture, whereas 1 X 10(-3) M H2O2 results in a more prolonged inhibition. The synthesis of the proteoglycan core protein is inhibited, but the ability of the newly formed proteoglycans to aggregate with hyaluronic acid is unimpaired.

The oxidant hypochlorite (OCl-), a product of the myeloperoxidase system, degrades articular cartilage proteoglycan aggregate.

The myeloperoxidase-derived oxidant, hypochlorite (OCl-) was shown to be able to degrade proteoglycan aggregate prepared from bovine articular cartilage. Exposure of proteoglycan aggregate to OCl- concentrations less than 10(-4) M resulted in a decrease in the size of the constituent proteoglycan monomers, which were unable to reaggregate with hyaluronate due to the loss of the hyaluronic acid binding region as indicated by immunoblotting using the monoclonal 1-C-6 antibody. Analysis of the [35S]-labeled core proteins by SDS/polyacrylamide electrophoresis and fluorography indicated a decrease in the size of the core protein. These data suggest that concentrations of OCl- below 10(-3) M results in the cleavage of the proteoglycan core protein in or near the hyaluronic acid binding region. The physiological consequences of these data are discussed. Exposure to higher concentrations (greater than 10(-3)) of OCl- caused more extensive degradation of the core protein; however, there was no evidence to suggest that OCl- cleaves glycosaminoglycan (GAG) chains.

Carrageenin-induced arthritis: V. A morphologic study of the development of inflammation in acute arthritis.

Following a single injection of the polysaccharide carrageenin into the rabbit knee joint, a rapid inflammatory process occurs in the joint space and synovial membrane, followed by changes in the articular cartilage. Initially there is an influx of cells, mainly PMNs, into the synovial fluid, accompanied by proliferation of the synovial lining cells and infiltration of the synovial membrane. The numbers of synovial fluid cells decline gradually after 24 hr. The reaction in the synovial membrane is greatest at day 7, and inflammation is still evident at day 21. Initially, the infiltrate consists mainly of PMNs, but by day 7 it is predominantly mononuclear, with small clusters of lymphocytes. The articular cartilage shows loss of metachromasia with toluidine blue at 3-14 days after injection, but stains normally after day 21. Electron microscopy shows damage to the chondrocytes at day 1 and 7, with complete destruction of cells in the surface layer. At day 7 cells in the deeper layers have lost the apparatus required for proteoglycan synthesis, but at day 21 the cells appear virtually normal. There was no evidence for a direct inhibitory effect of carrageenin on proteoglycan biosynthesis. Most labeled carrageenin was rapidly cleared from the joint space, but about 10% was retained in the synovial membrane and 0.6% in articular cartilage at 48 hr after injection. Since the increase and decline in PMN numbers respectively precede the cartilage damage and recovery, it is suggested that there may be a correlation between the clinical activity of arthritis and the number of PMNs in the synovial fluid.

Manganese levels and the morphology of the epiphyseal plate in broilers with slipped tendons.

Manganese deficiency in chickens results in perosis and a higher incidence of slipped tendon. Perosis is associated with a disorganization of the epiphyseal growth plates and changes in the chemical composition of the cartilage matrix. Male broilers with slipped tendons were selected from a commercial broiler farm over a 9 week growing period. Their growth rates, epiphyseal cartilage histology and tissue manganese concentrations were examined and compared with (a) normal broilers from the same farm, and (b) similar broilers raised on control and manganese deficient diets. Field broilers with slipped tendons showed no evidence of abnormality in the histological appearance of the proximal tibial growth plate at any of the ages examined. The manganese content of liver and epiphyseal cartilage from field broilers showing slipped tendon was comparable with that present in tissue from normal broilers wither from the field or raised on a chemically defined diey supplemented with managese. The slightly retared growth rate seen in the field broilers was attributed to feeding problems associated with the lameness condition. These results provide conclusive evidence that slipped tendon in field broilers is not dur to a manganese deficiency in the tissues, nor does it appear to be associated with abnormal proliferation of the chondrocytes in the epiphyseal plate.

The application of the research work of James Clerk Maxwell in electromagnetics to industrial frequency problems.

Faraday's work inspired the development of electrical motors and generators. Until Maxwell pointed out the significance of Ampere's Law, there was no rigorous design method for magnetic devices. His interpretation strongly influenced the creation, by others, of the 'magnetic circuit' approach, which became the seminal design technique. This, utilizing the concept of reluctance, led to the design method for magnetic machines that is still widely in use today. The direct solution of the Maxwell equations (less the displacement current term) had to await the development of modern continuum methods to yield the field everywhere in, and around, the devices of interest, and this then permitted the application of the Maxwell stress tensor. This final refinement yielded forces and torques, and this resulted in the accurate prediction of electrical machine performance.

Receptor-mediated binding of leukocyte elastase by chondrocytes.

Studies on the effect of leukocyte elastase on the metabolism of chondrocytes in culture have demonstrated that these cells possess a specific cell surface receptor for leukocyte-derived elastase. Purified elastase from rabbit and human leukocytes is capable of modulating the metabolism of the cell by causing a marked decrease in both proteoglycan and protein biosynthesis. Addition of 125I-labeled elastase to chondrocytes maintained in suspension culture has shown that binding occurs, and that it is saturable and is inhibited by the addition of unlabeled enzyme. We ascertained that the active site of the enzyme was necessary for binding to the chondrocyte, since phenylmethylsulfonyl fluoride-inactivated leukocyte elastase failed to bind. Pancreatic elastase had only a slight affinity for the receptor, whereas trypsin and bovine serum albumin failed to bind to any significant extent. Autoradiographic studies and the use of inhibitors of endocytosis, such as dansyl cadaverine, confirmed that endocytosis of elastase was the secondary event after cell binding.

Fluorimetric determination of DNA in papain digests of cartilage, using ethidium bromide.

A fluorimetric procedure for the determination of the DNA content of cartilage is described. The tissue is initially solubilised by digestion with papain, and ethidium bromide is used for the subsequent quantitation of DNA. The basis of the procedure is the enhancement of fluorescence which occurs when ethidium bromide complexes with native nucleic acids, fluorescence due to DNA being distinguished from that due to RNA through the use of ribonuclease. The method provides reproducible results, allowing determination of DNA in papain digests containing greater than 1.25 microgram DNA/ml, and is a rapid alternative to more laborious colorimetric or fluorimetric methods, which require the separation of DNA from other tissue components. The procedure is highly specific for DNA and is useful in metabolic studies in which various parameters of chondrocyte activity are being studied.

The effect of chondroitin sulphate-protein on the formation of collagen fibrils in vitro.

1. It was found that the precipitation of collagen fibrils at 37 degrees from mixtures of chondroitin sulphate-protein and tropocollagen at physiological ionic strength and pH takes place in two distinct phases. The first occurs immediately on mixing either at 4 degrees or at 37 degrees , and the second occurs only at 37 degrees and after a lag phase whose magnitude depends on the proportions of components. 2. When the second stage of precipitation was inhibited by mixing the reactants at 4 degrees , the initial precipitate was found to contain ;native-type' collagen fibrils and chondroitin sulphate-protein. 3. On the basis of kinetic experiments it was concluded that aggregates of chondroitin sulphate-protein and tropocollagen form instantaneously and that these act as sites for the second stage of precipitation of fibrils. 4. The gels that result after continued incubation at 37 degrees are fibrous in appearance if formed in the presence of the initial precipitate of chondroitin sulphate-protein and tropocollagen. 5. On the basis of these experiments in vitro the authors propose a sequence of events for collagen fibrogenesis in vivo.