Inhibition Of Proteoglycan Synthesis Research Articles

Effects of murine recombinant interleukin 1 on synovial joints in mice: measurement of patellar cartilage metabolism and joint inflammation.

Murine recombinant interleukin 1 was injected intra-articularly into mice. It induced a clear effect on patellar cartilage within 24 hours. A low dose of interleukin 1 (1 ng) elicited a significant reduction in [35S]sulphate incorporation (50%) into proteoglycans and an accelerated breakdown (twofold) of 35S prelabelled proteoglycan. Proteoglycan breakdown returned to normal rates (approximately 10%/day) 48 hours after a single interleukin 1 injection. Recovery of proteoglycan synthesis was delayed by up to 72 hours, however, which implies that repair of the depleted cartilage matrix is retarded. Interleukin 1 induced only minor joint inflammation, too slight to be held responsible for the strong suppression of proteoglycan synthesis. Vehement joint inflammation was found after repeated interleukin 1 injections. The plasma extravasation and massive infiltration and exudation of leucocytes, predominantly polymorphonuclear leucocytes, were not a mere summation of single interleukin 1 effects, but point to interleukin 1 induced local hypersensitivity. The cartilage matrices of patella and femur were heavily depleted. Measurement of the extent of loss of 35S prelabelled proteoglycan and the prolonged inhibition of [35S]sulphate incorporation indicate that both inhibition of proteoglycan synthesis and enhanced loss of proteoglycan contributed substantially to this depletion.

Effects of β- d-xyloside on differentiation of the respiratory epithelium in the fetal mouse lung

Differentiation of respiratory endings in the fetal lung appears to be controlled by its surrounding mesodermal capsule. The capsule may exert its influence by controlling the composition of the epithelial basal lamina or of the extended extracellular matrix that is deposited during the period when alveolar sacs are formed. As a first step in testing this hypothesis, the effects of the drug, p-nitrophenyl-β- d-xylopyranoside (β-xyloside), an inhibitor of proteoglycan synthesis, and its inactive α anomer (α-xyloside) were examined. Lung primordia from mice at 16 days of gestation were tested for inhibition of morphological and functional differentiation as a result of drug treatment. Pseudoglandular lung epithelium did not form respiratory endings, contained fewer specialized cells, and accumulated little additional surfactant when treated with β-xyloside but developed normally when treated with α-xyloside or grown in control medium. The results are interpreted to suggest that deposition of an extracellular matrix rich in proteoglycan is required to support maturation of the respiratory epithelium.

CAMP-dependent protein kinase in chondrocyte cultures: Holoenzyme activation, phosphorylation of cellular proteins, effects of NSAIDs and possible role in proteoglycan synthesis

R ECENT EVIDENCE showed that chondrocytes treated in culture with nonsteroidal antiinflammatory drugs (NSAIDs) exhibited reduced eicosanoid production, which was coupled to reduced intracellular accumulation of cyclic adenosine monophosphate (CAMP).’ Typically, NSAIDs have variable effects on cartilage proteoglycan and collagen synthesis and catabolism.2*3 Some NSAIDs are known to stimulate proteoglycan synthesis. These include sulindac sulfide4 and benoxaprofen5 However, the majority, including fenoprofen4 ibuprofen4 indomethacin,2T6*7 phenylbutazone,8 salicylates,7~9~‘0 and tolmetin,” suppress proteoglycan synthesis. Piroxicam may enhance proteolycan synthesisI or be without effect 12,13 the same can be said of the effect of tiaprdfenic acid2 and in some cases, indomethatin and sulindac sulfoxide.4 Many of these results come from studies conducted on articular cartilage explant culturesI or on cultured chondrocytes’2q’3 where a concentration range of NSAIDs can be tested readily. A recent review has appeared on this subject.” Two types of CAMP-dependent protein kinase (cAMPPk), type 1 and type 2, resolved by differential elution from diethylaminoethylcellulose (DEAE-cellulose), have been shown to

Specific effects of ethanol on neurite-promoting proteoglycans of neuronal origin

Quantitative analysis of proteoglycan synthesis and release by neurons indicated that of the total incorporation of 35SO 4 and [ 3H]glucosamine into proteoglycan, approximately 75% was chondroitin sulphate while approximately 25% was heparan sulphate. Using a biological assay it has been shown that heparan sulphate proteoglycans (HSPGs) in conditioned medium promote neurite outgrowth from sensory neurons when complexed to a laminin substrate. Culture media conditioned in the presence of ethanol did not enhance neurite formation over control levels. Co-incubation of neurons with β- d-xyloside, an inhibitor of proteoglycan synthesis, reduced neurite outgrowth after 20 h in culture and the combination of ethanol and β- d-xyloside produced no further inhibition than with either ethanol or β- d-xyloside used alone. If the laminin substrate was coated with medium conditioned by neurons, the direct inhibitory effects on process formation seen when ethanol was co-incubated with neurons were no longer observed. Ethanol inhibited the incorporation of 35SO 4 and [ 3H]glucosamine into HSPG by neurons while having little or no effect on incorporation into chondroitin sulphate. These results suggest that inhibition of neuronal synthesis of HSPGs by ethanol is responsible for the decrease in neurite promoting activity of medium conditioned in the presence of ethanol.

Etodolac: Efficacy in osteoarthritis and effects on chondrocyte function

Some nonsteroidal anti-inflammatory drugs (NSAIDs) used in the treatment of osteoarthritis (OA) may damage articular cartilage. This damage may be caused by suppression of proteoglycan synthesis or by altered collagen synthesis in the presence of prostaglandin E2 (PGE2) induced by interleukin-1 (IL-1). The clinical and biochemical effects of etodolac, a new NSAID, on the symptoms of OA and on cartilage metabolism are reviewed. Clinically, etodolac (200-600 mg/day) was more effective than placebo, and as effective as aspirin (3200-4800 mg/day), piroxicam (20 mg/day), naproxen (1000 mg/day), and diclofenac (150 mg/day) in relieving the symptoms of OA. In in vitro studies, proteoglycan synthesis was not affected by the presence of etodolac when human chondrocytes were grown in a three-dimensional culture. Etodolac preserved collagen phenotype in human chondrocytes cultured in a monolayer in the presence of IL-1. In contrast, the collagen phenotype of cells cultured in the presence of indomethacin and IL-1 changed. Under those conditions, less type II and type IX collagen were synthesized and more type I and type III collagen were synthesized. These findings suggest that etodolac, an effective agent in the treatment of OA, has the potential advantage of not damaging articular cartilage in vivo.

Passive loss of proteoglycan from articular cartilage explants

The addition of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 0.25 mM benzamidine hydrochloride, 6.25 mM EDTA, 12.5 mM 6-aminohexanoic acid and 2 mM iodoacetic acid) to explant cultures of adult bovine articular cartilage inhibits proteoglycan synthesis as well as the loss of the macromolecule from the tissue. Those proteoglycans lost to the medium of explant cultures treated with proteinase inhibitors were either aggregates or monomers with functional hyaluronic acid-binding regions, whereas proteoglycans lost from metabolically active tissue also included a population of monomers that were unable to aggregate with hyaluronate. Analysis of the core protein from proteoglycans lost into the medium of inhibitor-treated cultures showed the same size distribution as the core proteins of proteoglycans present in the extracellular matrix of metabolically active cultures. The core proteins of proteoglycans appearing in the medium of metabolically active cultures showed that proteolytic cleavage of these macromolecules occurred as a result of their loss from the tissue. Explant cultures of articular cartilage maintained in medium with proteinase inhibitors were used to investigate the passive loss of proteoglycan from the tissue. The rate of passive loss of proteoglycan from the tissue was dependent on surface area, but no difference in the proportion of proteoglycan aggregate to monomer appearing in the medium was observed. Furthermore, proteoglycans were lost at the same rate from the articular and cut surfaces of cartilage. Proteoglycan aggregates and monomer were lost from articular cartilage over a period of time, which indicates that proteoglycans are free to move through the extracellular matrix of cartilage. The movement of proteoglycans out of the tissue was shown to be temperature dependent, but was different from the change of the viscosity of water with temperature, which indicates that the loss of proteoglycan was not solely due to diffusion. The activation energy for the loss of proteoglycans from articular cartilage was found to be similar to the binding energies for electrostatic and hydrogen bonds.

Metabolic effects of forskolin in chick chondrocytes

The effects of forskolin on parameters of energy metabolism and proteoglycan synthesis have been investigated in chick embryo sternal chondrocyte cultures. After 8 h exposure to 100 μM forskolin, ATP levels and oxygen consumption were unaltered. Protein synthesis was unaffected up to 50 μM forskolin and protein degradation was unaffected by forskolin up to 100 μM. In contrast, incorporation of the proteoglycan precursors, 35SO 4 and [ 3H]glucosamine, was more sensitive to forskolin. Inhibition was linear with dose between 10 and 100 μM, reaching 70% at 100 μM. Incorporation of 35SO 4 into glycosaminoglycan chains initiated on an artificial β-xyloside acceptor was inhibited in the same manner. cAMP accumulation was maximal at 10 μM forskolin, a concentration which did not alter proteoglycan synthesis. We conclude that a major, acute effect of forskolin in these short-term experiments is inhibition of proteoglycan synthesis in a cAMP-independent manner.

Mechanisms of human neutrophil‐mediated cartilage damage in vitro: The role of lysosomal enzymes, hydrogen peroxide and hypochlorous acid

Cartilage is a focal point of attack by cellular and molecular elements of the inflammatory response which occurs in arthritic diseases. Neutrophils damage articular cartilage by degrading matrix components and inhibiting their synthesis. The aim of this study was to elucidate mechanisms of this damage. Human neutrophils were isolated from blood by centrifuging through Ficoll-Hypaque and granule extract prepared from them. Articular cartilage from adult humans and cattle was maintained in organ culture. Cartilage degradation (release of 35S-labelled proteoglycan) or synthesis (incorporation of 35S into proteoglycan) was determined after various treatments. Human neutrophils and neutrophil granule extract degraded proteoglycan and inhibited proteoglycan synthesis. The specific leucocyte elastase inhibitor N-methoxysuccinyl-(ala)2-pro-val-chloromethylketone (MAAPVCMK) partially reversed these effects. H2O2, a product of the neutrophil respiratory burst, when added directly at 10(-6)mol/L, or generated by glucose oxidase (GO)/glucose inhibited proteoglycan synthesis but had no effect on degradation. Hypochlorous acid (OHCl), a product of the myeloperoxidase (MPO)/H2O2/Cl system at 50 mumol/L degraded proteoglycan and inhibited its synthesis. OHCl produced by granule extract (as a source of MPO) + GO-generated H2O2 + Cl- degraded proteoglycan. The results indicate that neutrophil-mediated proteoglycan degradation and inhibition of synthesis is largely attributable to elastase and secondarily to OHCl, whereas H2O2 impairs synthesis without affecting degradation of proteoglycan.

CHONDROCYTE NONRESPONSIVENESS TO INSULIN‐LIKE GROWTH FACTOR 1 IN EXPERIMENTAL ARTHRITIS

Inhibition of chondrocyte proteoglycan (PG) synthesis is one of the mechanisms leading to cartilage destruction in joint inflammation. Using murine cartilage from normal and arthritic knee joints, we examined this process. We found that for normal, anatomically intact murine articular cartilage, insulin‐like growth factor 1 (IGF‐1) is a potent anabolic factor. Recombinant IGF‐1 at physiologic concentrations in a completely synthetic medium sustained PG synthesis, at the in vivo rate, of patellar cartilage in organ culture. Using an experimental arthritis model, we found that cartilage from an arthritic joint could not be stimulated in vitro with IGF‐1. This nonresponsiveness was not caused by a generalized metabolic inhibition of the chondrocytes, because PG synthesis in arthritic cartilage could still be stimulated by forskolin, an activator of adenylate cyclase. Our data suggest that during joint inflammation, inhibition of chondrocyte PG synthesis is, at least partially, caused by a defect in the IGF‐1 responsiveness of the chondrocyte. We propose this finding as a possible pathogenetic mechanism for cartilage destruction in joint diseases.

Inhibition of proteoglycan synthesis alters extracellular matrix deposition, proliferation, and cytoskeletal organization of rat aortic smooth muscle cells in culture.

Arterial proteoglycans have been implicated in several important physiological processes ranging from lipid metabolism to regulation of smooth muscle cell growth. Vascular smooth muscle (VSM) cells are the major producers of proteoglycans in the medial layer of blood vessels. To study functional consequences of alterations in VSM proteoglycan metabolism we used 4-methylumbelliferyl-beta-D-xyloside to inhibit proteoglycan synthesis in primary and early passage cultures of rat aortic smooth muscle cells. Biochemical analysis of cultures labeled with 35SO4 showed the drug inhibited synthesis of different classes of proteoglycans by 50 to 62%. Inhibition of proteoglycan synthesis resulted in reduced accumulation of extracellular matrix, as shown by immunofluorescent staining with antibodies to chondroitin sulfate, fibronectin, thrombospondin, and laminin. There was also an inhibition of postconfluent (multilayered) growth of the smooth muscle cells, and a change in the morphology of the cells, with no apparent effect on subconfluent growth. In addition, in drug-treated cells there was a reduction in the number of cytoskeletal filaments that contained alpha-actin, the actin subtype synthesized by differentiated VSM cells. This occurred even though the total content of alpha-actin in the cells was not reduced. The effects of the inhibitor on growth and morphology could be reversed by switching the cultures to normal medium and could be prevented by growing the cells on preformed VSM extracellular matrix. These observations suggest the vascular extracellular matrix may play a role in regulating the growth and differentiation of smooth muscle cells.

Effect of interleukin-1-beta and tumor necrosis factor-alpha on cartilage proteoglycan metabolism in vitro.

The activities of recombinant interleukin-1-beta (IL-1) and recombinant tumor necrosis factor-alpha (TNF) on cartilage proteoglycan metabolism were compared in an organ culture system. IL-1, 1 to 100 ng/ml, and TNF, 10 to 1,000 ng/ml, increased proteoglycan degradation. The concentration-response curves were parallel. The timecourse for degradation was similar for the two cytokines during a 6 day incubation. Both cytokines inhibited the synthesis of new proteoglycan as measured by 35S incorporation. The inhibition curves were parallel and concentration-related between 1 and 10 ng/ml for IL-1 and between 10 and 100 ng/ml for TNF. Maximal inhibition was 60% in the presence of IL-1 (10 ng/ml) or TNF (100 ng/ml), and plateaued at higher concentrations. IL-1 was ten fold more potent than TNF in stimulating proteoglycan breakdown and inhibiting proteoglycan synthesis. Degradation in response to TNF, but not to IL-1, could be blocked by a polyclonal antibody to TNF. A polyclonal antibody to IL-1 could block proteoglycan breakdown in response to both cytokines suggesting that TNF may be mediating proteoglycan degradation by inducing the production of interleukin-1.

Independent effects of interleukin-1 on proteoglycan breakdown, proteoglycan synthesis, and prostaglandin E2 release from cartilage in organ culture.

Exposure of bovine nasal cartilage in culture to interleukin-1 (IL-1) leads to a time- and concentration-dependent stimulation of proteoglycan breakdown and prostaglandin E2 (PGE2) release, and to inhibition of proteoglycan synthesis. The threshold levels of IL-1 required for initiating these effects were different, and IL-1 was 10 times more potent in inhibiting synthesis than in stimulating breakdown of proteoglycan. Kinetic studies indicated that the effects on proteoglycan metabolism occurred earlier (16-24 hours) than those for PGE2 release (48 hours). Selective effects were observed with inhibitors. Nonsteroidal antiinflammatory drugs blocked PGE2 production in response to IL-1, but had no effect on proteoglycan metabolism, and the antiarthritic drugs that blocked IL-1-stimulated breakdown augmented the inhibition of proteoglycan synthesis. We suggest that the effects of IL-1 on proteoglycan breakdown, proteoglycan synthesis, and PGE2 release are mediated by independent post-receptor mechanisms.

Relaxin as an Aetiological Factor in Diabetic Embryopathy

In diabetic pregnancy, the fetus is at risk for congenital malformation, stillbirth, and perinatal death. Although the incidence of stillbirth and perinatal death has decreased as control of diabetes in pregnancy has improved, the incidence of malformation has not decreased and may even have increased. The present authors review some of the experimental work that has been undertaken to determine the cause of this impasse. At or near term, the mammalian placenta is impermeable to insulin, which cannot, therefore, be the cause of congenital malformations. The authors favor an alternative explanation, based on the hypothesis that an “insulin-like” substance is present in the fetoplacental unit at an appropriate stage in embryogenesis. By “like” substance, they mean, not an analogue, but an insulin homologue, specifically designated relaxin. Relaxin is related to insulin through a common ancestral polypeptide resembling proinsulin. Its usual source is the corpus luteum, but it is found also in the decidua and the term placenta. Among other functions, relaxin enhances secretion of collagenase and plasminogen. This property may lead to collagen degradation in fetal membranes and to eventual rupture at parturition. Relaxin influences the distensibility of the nonpregnant uterus in animals without ovaries maintained on estrogen, but has little effect alone. For maximum effect and greatest increase in the collagen framework, relaxin must be combined with estrogen and progesterone in accurate dosage. In pregnant women, one stimulus to relaxin production is hCG. The serum profiles of relaxin and hCG are similar. Moreover, in nonpregnant women, injection of hCG during the late follicular phase causes relaxin secretion. In women having cesarean sections, a linear relation was found between progesterone and relaxin concentrations in the serum of blood from the ovarian vein on the side of the corpus luteum. The authors studied the known functions of relaxin in relation to the development of the embryonic palate. During fetal development, the palatal shelves undergo reorientation from their initial vertical position lateral to the tongue to a horizontal alignment cephalad to it and fuse to form the secondary palate. Swelling of the shelves is generally attributed to production of sulphated proteoglycans in the matrix, and shelf remoulding and direction of movement are physically controlled by concomitantly developed collagen, arranged in a specific way. Cleft palate may be produced experimentally by drugs that inhibit proteoglycan synthesis (such as cortisone, chlorcyclizine, and diazonorleucine) and by those that cause abnormalities of fetal collagen. This effect is not limited to the palate but involves the whole embryo. When acting with specific amounts of estrogen and progesterone, relaxin has effects on collagen that resemble those of substances causing cleft palate in laboratory animals. If, in normal circumstances, a high relaxin secretion is avoided during the stage of organogenesis, disharmony among the three hormones could give rise to numerous malformations other than cleft palate. Among diabetic women studied before the 10th week of pregnancy, investigators found that those in whom diabetes was not well controlled had estradiol, prolactin, and hCG levels below the range associated with normal pregnancy, and that these rose to normal after careful control of the diabetes with insulin. It is possible, therefore, that administered insulin, while correcting maternal plasma glucose, may, by raising circulating hCG and estrogen concentrations (and thus increasing relaxin levels), trigger a teratogenic response in certain predisposed embryos.

Ultrastructural changes accompany inhibition of proteoglycan synthesis in chondrocytes by cyclofenil diphenol

Cyclofenil diphenol, a weak non-steroidal oestrogen, profoundly inhibits [35S]proteoglycan synthesis in cultures of Swarm chondrosarcoma chondrocytes under conditions in which protein synthesis is only marginally reduced. In the present experiments it was shown that after a 40-min treatment with Cyclofenil diphenol (90 micrograms ml-1) most of the normally abundant Golgi stacks in these cells disappeared and after 60 min they were absent. After 2-3 h treatment the cisternae of the endoplasmic reticulum (ER) were grossly distended and transformed into large ribosome-studded vesicles containing flocculent and filamentous material. These changes were dependent on the concentration of Cyclofenil and were fully reversible within 21 h of withdrawing the drug. The ultrastructural changes differed in some aspects if protein synthesis was blocked with cycloheximide for 15 min or 180 min before and during treatment with Cyclofenil. The Golgi disappeared but the ER cisternae, though distended, formed a continuous network and swollen ribosome-studded vesicles did not develop. However, non-membrane-bounded structures containing lipid droplets and material of low electron density developed in the cytoplasm under these conditions. The ultrastructural changes induced by Cyclofenil differ from those induced by monensin and diethylcarbamazine, suggesting that the drug acts at a different point in the secretory pathway for macromolecules.

β-Xyloside effects on basal lamina structure and anionic site distribution in the embryonic mouse submandibular salivary gland

β- d-Xyloside is a proteoglycan biosynthesis inhibitor. Previous studies on embryonic salivary glands have demonstrated that 0.5 mM β-xyloside (1) inhibits proteoglycan synthesis by 50%; (2) severely depresses sulphated glycosaminoglycan deposition at the basal epithelial surface, and (3) dramatically inhibits epithelial branching morphogenesis. Electron microscopy revealed a conventional three-layered basal lamina that is altered in the presence of β-xyloside by a 35% reduction in the number of tannic acid-resolved particles in the lamina densa. Basal lamina anionic sites, resolved with ruthenium red (RR) and polyethyleneimine (PEI) cationic probes, were also reduced in the presence of β-xyloside. PEI particles were reduced by 28%, and RR particles by 24%, per two-dimensional unit of basal lamina. These β-xyloside effects on anionic sites are consistent with an hypothesis that sulphated glycosaminoglycans account for 50% of the basal lamina anionic sites and a predicted 25% decrease in anionic sites in the presence of β-xyloside.

Effects of Interleukin-1 and Lipopolysaccharides on Protein and Carbohydrate Metabolism in Bovine Articular Cartilage Organ Cultures

The long term (18 day) metabolic response of bovine articular cartilage to treatment with either E. Coli lipopolysaccharide (LPS) or interleukin 1 was studied. For LPS treatment, incorporation of [35S]sulfate into the large proteoglycan population was inhibited 80% while that into the small interstitial proteoglycans was only inhibited 40%. Incorporation of [3H]serine into the large proteoglycan population was inhibited approximately 72% while incorporation into other protein was inhibited only 16%. Furthermore, the rate of catabolism of [3H]serine labeled proteoglycans was increased 2-fold by LPS treatment while the rate of 3H-labeled general protein catabolism was not affected. Incorporation of [3H]glucosamine into hyaluronate was increased; however a correction for changes in the specific activity of the intracellular [3H]glucosamine precursor pool in LPS-treated cultures indicated that the net amount of hyaluronate synthesized was not altered by LPS treatment. The 3H/35S ratios in isolated chondroitin sulfate disaccharides labeled with [35S]sulfate and [3H]glucosamine precursors were significantly changed during long term LPS treatment, suggesting that general carbohydrate pathways are altered. The 3H/35S changes were larger in the disaccharides isolated from the small proteoglycans indicating that different precursor pools, probably in different cell populations, preferentially synthesize this proteoglycan population. Interleukin-1 affected the same chondrocytic pathways as LPS as shown by a) the extent of inhibition of proteoglycan synthesis, b) the selective inhibition of synthesis of the large proteoglycan species, c) acceleration of proteoglycan catabolism, d) net depletion of proteoglycans from the tissue, e) increases in guanidine HCl extractable [3H]hyaluronate, f) increases in levels of prostaglandin E2 synthesis, g) changes in 3H/35S ratios in glycosaminoglycan chains and, h) minimal effects on general protein synthesis.

Extracellular matrix synthesis is required for the movement of sclerotome and neural crest cells on collagen

Abstract During early embryogenesis cells of several different populations disperse by active cell movement from one location to another. Preexisting extracellular materials are major determinants of these dispersal patterns, but the cells are also able to modify their substrata by synthesizing and secreting extracellular matrix molecules as they move. In order to determine the contribution made by these deposited materials, several tissues from the early chick embryo have been cultured in the presence of inhibitors of extracellular matrix synthesis and secretion. The tissues examined were sclerotome cells from differentiated somites and neural crest cells. For comparison, undifferentiated somites were also cultured. The movement of these cells was compared in type I collagen gel culture and in conventional culture on artificial substrata. Inhibitors of collagen synthesis were used ( cis-hydroxy proline and l-azetidine-2-carboxylic acid) in addition to a proteoglycan inhibitor (p-nitrophenyl-xylo-pyranoside) and a secretion inhibitor (monensin). Results indicate that sclerotome cells require collagen synthesis for movement in a collagen matrix. Reversal of the effects of collagen inhibitors, by proline and type II collagen, suggest that sclerotome cells normally condition the type I matrix in order to move in it. Inhibition of proteoglycan synthesis produced the greatest effect on the movement of neural crest cells regardless of the substratum, confirming an important role for these molecules in the crest migratory routes. The attachment of all cells to collagen was highly sensitive to the presence of monensin, which is known to reduce the deposition of glycosaminoglycans and fibronectin. These results suggest that conditioning of the extracellular matrix by newly synthesized material is required for cell attachment and movement during early development.

Effects of murine recombinant interleukin 1 on intact homologous articular cartilage: a quantitative and autoradiographic study.

Murine recombinant interleukin 1 (IL1) was tested for its ability to affect intact murine articular cartilage. IL1 caused enhanced proteoglycan degradation and severe inhibition of chondrocyte synthetic function at a concentration of 3 U/ml (100 pg/ml). Inhibition of proteoglycan synthesis appeared to be delayed in onset but occurred consistently after 24 hours. Pulse chase experiments made it clear that proteoglycan degradation and inhibition of proteoglycan synthesis are two distinct actions of IL1. No indications were obtained for selective degradation of either newly synthesised or processed proteoglycan. Moreover, chondrocyte synthetic activity appeared to be inhibited uniformly throughout the cartilage matrix, i.e., no evidence was found for selective suppression of cells in certain regions. IL1 uptake measurement in the cartilage, using [125I]IL1, yielded a partition coefficient far below 1, and autoradiography demonstrated a faint but even distribution within the cartilage matrix. The coordinated induction of enhanced breakdown of proteoglycan and inhibition of proteoglycan synthesis, with such low concentrations of IL1 reaching the chondrocytes, underlines the impressive destructive potential of IL1.

Effects of nonsteroidal anti-inflammatory drugs on chondrocyte metabolism in vitro and in vivo

Salicylates and several other nonsteroidal anti-inflammatory drugs (NSAIDs) that are commonly employed in the treatment of osteoarthritis effectively decrease joint pain and increase mobility. Results from in vitro studies indicate that, in addition, some of these compounds affect proteoglycan metabolism of articular cartilage. Data from in vivo studies suggest that salicylate administration may accelerate articular cartilage damage in several animal models of osteoarthritis. At in vitro concentrations comparable to those that are achieved in the synovial fluid of patients treated with the drug, several NSAIDs suppress proteoglycan synthesis by the chondrocyte. Salicylate has been shown to inhibit the enzymes involved in the early stages of chondroitin sulfate biosynthesis. These NSAIDrelated effects on chondrocyte metabolism appear unrelated to inhibition of prostaglandin synthetase, and are much more profound in osteoarthritic cartilage than in normal cartilage, due to enhanced uptake of NSAIDs by the osteoarthritic cartilage. Depletion of matrix proteoglycans appears to be a major factor in the increased vulnerability of chondrocytes in degenerating cartilage to effects of NSAIDs. Some NSAIDs may be bound to matrix components. If similar changes occur in the cartilage of patients with arthritis treated with NSAIDs, despite the symptomatic improvement that these drugs produce, cartilage degeneration could be accelerated.

Xyloside inhibits synthesis of the class II-associated chondroitin sulfate proteoglycan and antigen presentation events.

An alternative form of the human invariant chain exists as a chondroitin sulfate proteoglycan (CSPG) with invariant chain as the core protein. The selective inhibitor of proteoglycan synthesis, p-nitrophenyl beta-D-xyloside was used to study the role of this CSPG in class II biology. At xyloside concentrations of 2.5 and 5.0 mM, CSPG synthesis was completely inhibited with marginal inhibition of protein synthesis. The inhibitory effect on CSPG synthesis was completely reversible. The number of class II molecules on the cell surface was not affected by xyloside, but biosynthesis and appearance of newly synthesized class II molecules at the cell surface were both decreased by xyloside. Recognition of influenza virus-infected cells by class II-restricted, virus-specific cytotoxic T lymphocytes was not diminished by the presence of xyloside in the effector phase of the cytotoxicity assay. However, sensitization of target cells was markedly inhibited when target cells were exposed to virus in the presence of xyloside. These results are consistent with the hypothesis that the CSPG form of invariant chain has a role in antigen processing.