O-Linked oligosaccharides of human articular cartilage proteoglycan.

O-linked oligosaccharides and keratan sulphate chains have been isolated from the proteoglycan subunits of human articular cartilage. The oligosaccharides possessed a size and chemical composition similar to the equivalent moieties present in the proteoglycan subunits of the Swarm rat chondrosarcoma. Furthermore, the size and chemical composition of the oligosaccharides showed little change with the age of the individual from whom the proteoglycan was obtained. In contrast, the keratan sulphate chains appeared to increase in chain length with increased age of the individual. The total number of keratan sulphate and oligosaccharide chains per core protein decreased with age, but it was not clear whether there was any change in the ratio of the two components with respect to one another.

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.

Characterization of lymphocyte subsets in spontaneous mouse mammary tumors and host lymphoid organs.

The subsets of small lymphocytes (surface Igm-positive or B, Thy-I-positive or T and "double-negative" or null) appearing within spontaneous mammary tumors of C3H retired breeder female mice and in various lymphoid organs of the host at different stages of tumor development were characterized directly using a radioautographic method. Tumor-bearing mice showed early transient splenomegaly and progressive atrophy of the thymus. The proportion of T small lymphocytes increased with tumor age within the tumor and in the spleen, blood and thymus; then, except in the spleen, these proportions declined again by 8 weeks of tumor growth. The incidence of B small lymphocytes showed no change in the tumor or thymus; there was an increase in the blood after 3 weeks, and a small decrease in the spleen after 7 weeks. At all the sites examined, the proportion of null small lymphocytes declined from an initial high level observed in the elderly tumor-free control mice, and then recovered to control levels after 7 weeks. The absolute numbers of T and null cells in the spleen changed in parallel to their proportions, while splenic B-cell numbers increased at 1-3 weeks. In the thymus the absolute numbers of all subsets decreased, with a late recovery of null cell numbers. Age-matched control mice showed higher proportions of null cells in the spleen, blood and thymus, and lower proportions of T cells in the blood and spleen, than did young normal mice. This spontaneous tumor appears to be characterized by an increase in T cells, rather than in null cells, as observed for rapidly growing transplanted tumors. The null cell rise in the present case takes place before the clinical appearance of tumors. In both cases, however, tumor-infiltrating B cells exhibited low levels of surface Ig, possibly related to a low level of maturation. The functional significance of these findings remains to be determined.

Comparison of proteoglycans extracted from high and low weight-bearing human articular cartilage, with particular reference to sialic acid content.

Proteoglycan subunits extracted in the presence of proteinase inhibitors from autologous knee and shoulder cartilage were examined in different donors of various ages, in order to determine the effect of weight bearing on the age-related changes in proteoglycan structure. At each age, high density proteoglycans from the two joints of the same individual were virtually identical with respect to chemical composition, hydrodynamic size, ability to aggregate with hyaluronic acid, and chondroitin sulfate chain length. The proteoglycan content of the cartilage decreased with age, as did the size of the proteoglycan subunits. In contrast, the proportions of keratan sulfate to chondroitin sulfate, protein to glycosaminoglycan, and chondroitin 6 sulfate to chondroitin 4-sulfate all increased with age. These changes occurred to the same extent in both knee and shoulder cartilage. There was no change in the ability of the proteoglycan to aggregate with hyaluronic acid. The sialic acid profile on Bio-Gel P-10 chromatography, following alkaline borohydride degradation, showed the presence of both keratan sulfate and oligosaccharide chains. The oligosaccharide content of the core protein of the proteoglycan appeared to decrease during the aging process, but it could not be ascertained whether this was related to the increased abundance of keratan sulfate.

The presence of a cartilage-like proteoglycan in the adult human meniscus.

Proteoglycans were extracted from the adult human meniscus under dissociative conditions and purified by CsCl-density-gradient centrifugation. The preparations of highest density contained proteoglycan that possessed the ability to interact with hyaluronic acid, was of large subunit size and was composed of chondroitin sulphate, keratan sulphate and sialic acid-containing oligosaccharides. This 'cartilage-like' proteoglycan also exhibited subunit and aggregate structures analogous to those of hyaline-cartilage proteoglycans when examined by electron microscopy. However, the composition of this proteoglycan was more comparable with proteoglycans from immature cartilage than from age-matched cartilage. The preparations from lower density, which were enriched in dermatan sulphate, contained smaller proteoglycan that was not able to interact with hyaluronic acid. This non-aggregating proteoglycan may be structurally distinct from the 'cartilage-like' proteoglycan, which does not contain dermatan sulphate.

Lectin-binding patterns of small lymphocytes in bone marrow, thymus and spleen: demonstration of lymphocyte subsets by quantitative radioautography.

Cells from mouse bone marrow, thymus and spleen were exposed to 125I-labeled concanavalin A (Con A). Lens culinaris lectin (LCL), soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), phytohemagglutinin-P (PHA-P), peanut agglutinin (PNA), or wheat germ agglutinin (WGA) in a range of concentrations and examined radioautographically. Small lymphocytes in the three organs differed in the minimal concentration of each lectin which gave detectable surface labeling, while at optimal lectin concentrations, their labeling intensity profiles differed markedly. Inhibition by sugars demonstrated the labeling specificity. Major populations of bone marrow small lymphocytes bound WGA strongly, while Con A, SBA, HPA, PHA-P and LCL were bound only weakly, and PNA binding was lacking. Most thymus cells bound Con A, SBA, HPA, PHA-P and PNA strongly, WGA and LCL weakly. Subsets of bone marrow and thymus small lymphocytes differed from the major populations in their lectin-binding intensities. Spleen small lymphocytes were heterogeneous in the binding of each lectin. However, a major population bound LCL exceptionally strongly, while few cells bound PNA. Using a panel of lectins under standardized conditions, these studies show distinctive lectin-binding patterns for small lymphocytes in the bone marrow, thymus and spleen, respectively. Major and minor cell populations are distinguishable in each organ, providing an approach to discriminating lymphocyte lineages, subtypes and differentiation stages.

Proteoglycans from normal and degenerate cartilage of the adult human tibial plateau.

Proteoglycans were extracted from normal and degenerate cartilage of the human tibial plateau. Both areas possessed proteoglycans of similar chemical composition, though the degenerate cartilage contained a greater proportion of molecules of lower buoyant density and enriched in keratan sulfate. There was no evidence for the changes in glycosaminoglycan synthesis that have been described for clinically osteoarthritic cartilage, or for changes in the ability to aggregate with hyaluronic acid.

Enzymic iodination. A probe for accessible surface proteins of normal and neoplastic lymphocytes.

1. Radioactive iodide was covalently bound to living cells from normal mouse spleen and a variety of lymphoid tumours by a system consisting of lactoperoxidase, hydrogen peroxide and iodide. 2. About 3x10(5)-6x10(5) molecules of [(125)I]iodide/cell could be incorporated without affecting cell viability. 3. Electron-micrographic radioautography showed that the radioactive label was associated with the outer surfaces of the cells. 4. Radioiodinated proteins were solubilized in 9m-urea-0.2m-mercaptoethanol and analysed by gel-filtration and disc electrophoresis. 5. Comparison of distinct tumour lines by disc electrophoresis showed qualitative and quantitative differences in protein distribution patterns.

Mononuclear cell factors stimulate the concomitant secretion of distinct latent proteoglycan, gelatin and collagen degrading enzymes from human skin fibroblasts and synovial cells.

Human skin fibroblasts or synovial cells exposed to conditioned medium from human peripheral blood mononuclear cells release distinct latent enzymes capable of degrading collagen, proteoglycan (PG) and gelatin at neutral pH. The PG-degrading activity also degrades casein. These enzymes require calcium, are activated by 4-aminophenylmercuric acetate and can be inhibited by ethylenediamine tetraacetic acid, but not by phenylmethylsulfonyl fluoride, iodoacetamide or pepstatin. Simultaneous secretion of these proteinases after exposure to conditioned medium from peripheral blood mononuclear cells may be an important mechanism by which connective tissue extracellular matrix is destroyed during chronic inflammation in diseases such as rheumatoid arthritis.

Pregnancy-related changes in the connective tissue of the ovine cervix.

During the course of gestation, changes in the metabolism and chemical composition of the cervical connective tissue occur. The concentrations of collagen, proteoglycan and hyaluronate in the cervix decrease with time of pregnancy, while there is a small but significant increase in tissue hydration. The collagen network loses its dense, ordered appearance and assumes a loose, frayed, disoriented structure. A small molecular weight dermatan sulphate proteoglycan with Kav = 0.48 on Sepharose CL-4B has been isolated from pregnant and nonpregnant ovine cervix. Proteoglycans isolated from dilated cervix yield a second peak at the excluded volume of a Sepharose CL-4B column which may represent a new proteoglycan species, or represent a proteoglycan monomer with larger glycosaminoglycan constituents. The rate of synthesis of proteoglycan is enhanced during pregnancy, and doubles in the time between 140 days and term (dilated). The increased rate of proteoglycan synthesis in the presence of a diminishing hexuronate concentration in the pregnant cervix is indicative of enhanced proteoglycan turnover. This may provide some insight into the mechanism whereby a new matrix with altered mechanical properties is produced, since a rapid turnover of matrix components enables a remodeling of the connective tissue framework. This reorganization of the matrix is discussed with respect to parturition.

Antigen-induced arthritis. Decreased proteoglycan content and inhibition of proteoglycan synthesis in articular cartilage.

An arthritis was induced in rabbits immunized with human serum albumin by injection of antigen into the hind knee joint. Histological changes in the synovial membrane and an increase in polymorphonuclear granulocytes in the synovial fluid indicated an inflammatory response similar to that described with fibrin as antigen (1). The arthritis was accompanied by no significant change in the collagen content, but a marked decrease in the proteoglycan content of the cartilage was noted. Cartilage from inflamed joints generally exhibited a decreased ability to synthesize proteoglycan in vitro.

Changes in the host natural killer cell population in mice during tumor development. 1. Kinetics and in vivo significance.

Our earlier studies revealed an increase in the level of null (surface IgM-negative, Thy 1-negative) lymphocytes in mice shortly after tumor transplantation and before the clinical appearance of spontaneous mammary tumors. The present study examined the splenic natural killer (NK) cell activity as well as the incidence of NK lineage cells in these hosts, since NK cells are considered to be a subset of null lymphocytes. Splenic NK activity against YAC-1 lymphoma targets was measured with a 4-hr 51Cr-release assay in CBA mice transplanted ip with 10(6) Ehrlich ascites tumor (EAT) cells, in elderly C3H mice prior to and during the growth of spontaneous mammary tumors (SMT) and in young C3H mice transplanted sc with 5 X 10(6) SMT cells or 10(6) cells from two syngeneic mammary tumor lines (T-58 and MT-2) of recent origin. In EAT-transplanted mice total NK activity in the spleen increased rapidly to a peak (11-fold) at 3 days, coincident with the null cell rise, but then declined to subnormal levels by Day 7 when the null cell level was still high. A similar pattern of activity was exhibited by intratumor lymphocytes isolated from the EAT. In SMT-transplanted mice splenic NK activity showed a small rise at Day 3, followed by a drop to below normal at Day 7, subnormal levels lasting for the tumor life span. Similar results were noted in T-58- or MT-2-transplanted mice. Null lymphocytes recovered during the peak NK activity from the spleen of 3-day EAT-bearing mice, when mixed with 10(6) EAT cells at 25:1 E:T ratio and adoptively transferred into fresh mice in a Winn type assay either ip or sc, completely prevented tumor development indicating a high enrichment of NK cells functionally effective in vivo. Elderly clinically tumor-free C3H mice showed measurable NK activity, which dropped after the appearance of spontaneous mammary tumors to very low levels, the magnitude of decline rising with increasing tumor age (1-11 weeks) or size. The incidence of NK lineage cells was measured from the tumor target (YAC-1 lymphoma)-binding ability of the splenic null cells, identified with a radioautographic technique. Null target-binding cells (TBC) were NK-1+ and included both active as well as inactive NK lineage cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Carrageenin-induced arthritis. IV. Rate changes in cartilage matrix proteoglycan synthesis.

A localized inflammatory response was initiated by both single and repeated injections of carrageenin into femorotibial joints. Histologic changes were observed 24 hours after a single intraarticular injection, and an inhibition in the in vitro rate of proteoglycan synthesis was detected 72 hours after the injection. This inhibition was relieved in vitro by the addition of beta-D-xyloside, an exogenous initiator of glycosaminoglycan biosynthesis. Following repeated carrageenin injections, most cells appeared to be dead on histologic examination and no in vitro proteoglycan synthesis could be detected; nor could any stimulation be achieved by adding exyloside.