Cartilage Proteoglycan Synthesis Research Articles

AN N‐terminal peptide from link protein stimulates proteoglycan biosynthesis in human articular cartilage in vitro

Objective To determine the effects of a synthetic N-terminal peptide from link protein on the synthesis of proteoglycans by human articular cartilage. Methods Explants from adult knee cartilage were maintained for 4 days in serum-free Dulbecco's modified Eagle's medium. Peptides were added for the final 2 days of culture. Synthesis of proteoglycans and proteins was measured by the incorporation of 35S-sulfate and 3H-serine. The sizes, sulfation patterns, and serine: sulfate ratios of newly synthesized glycosaminoglycans were measured by gel chromatography, high performance liquid chromatography, and ion-exchange chromatography. Results The N-terminal peptide stimulated proteoglycan synthesis in cartilage from a wide age range of patients of both sexes. The newly synthesized glycosaminoglycans were identical in size and composition to those of control tissues, and their serine:sulfate ratios remained unchanged. Conclusion This N-terminal peptide, which can be liberated from proteoglycan aggregates by proteolysis, potently stimulated the synthesis of proteoglycans with normal glycosaminoglycan chains. The results suggest that the N-terminal peptide may have a regulatory role in maintaining the integrity of human cartilage matrix.

An N-terminal peptide from link protein stimulates proteoglycan biosynthesis in human articular cartilage in vitro.

To determine the effects of a synthetic N-terminal peptide from link protein on the synthesis of proteoglycans by human articular cartilage. Explants from adult knee cartilage were maintained for 4 days in serum-free Dulbecco's modified Eagle's medium. Peptides were added for the final 2 days of culture. Synthesis of proteoglycans and proteins was measured by the incorporation of 35S-sulfate and 3H-serine. The sizes, sulfation patterns, and serine: sulfate ratios of newly synthesized glycosaminoglycans were measured by gel chromatography, high performance liquid chromatography, and ion-exchange chromatography. The N-terminal peptide stimulated proteoglycan synthesis in cartilage from a wide age range of patients of both sexes. The newly synthesized glycosaminoglycans were identical in size and composition to those of control tissues, and their serine:sulfate ratios remained unchanged. This N-terminal peptide, which can be liberated from proteoglycan aggregates by proteolysis, potently stimulated the synthesis of proteoglycans with normal glycosaminoglycan chains. The results suggest that the N-terminal peptide may have a regulatory role in maintaining the integrity of human cartilage matrix.

Relations between functional, inflammatory, and degenerative parameters during adjuvant arthritis in rats

We assessed the time-course of adjuvant arthritis (AA) in Lewis rats, using biotelemetry to monitor the rat's spontaneous locomotor activity and body temperature, and studied the evolution of the arthritic index, circulating concentrations of inflammation-promoting cytokines, cartilage proteoglycan synthesis, and the effect of indomethacin as a cyclooxygenase inhibitor to evaluate prostaglandin (PG) contribution in AA. The injection of complete Freund's adjuvant on day 0 (D0) induced a marked, transient loss of locomotor activity (D1-D4; initial phase) and then a second phase of hypomobility peaking on D15 and thereafter irreversible (D16-D20; arthritic phase). Fever peaked first on D1 and again between D13 and D17. The primary hyperthermia was associated with increases in plasma interleukin-6 and tumor necrosis factor-alpha concentrations and seemed to be partly PG dependent. Proteoglycan synthesis inhibition in the patellar cartilage increased gradually, spreading from the injected paw to the contralateral paw. It was corrected on D20 by preventive and curative indomethacin treatments. Indomethacin also greatly relieved hypomobility during the systemic phase of AA (D10-D15). The combination of information about cartilage metabolism, body temperature, locomotor activity, and cytokine in this study permits analysis of analgesic, antipyretic, anti-inflammatory, and chondroprotective properties of drugs in the various phases of AA. Thus, using a new methodology, we have discriminated the different phases of the disease and confirmed the symptomatic and systemic inhibitory effect of indomethacin on fever, activity, and cartilage metabolism.

Restoration of the differentiated functions of serially passaged chondrocytes using staurosporine

Among the various directions explored in order to have a large number of differentiated articular chondrocytes easily available, the restoration of the differentiated properties after cell multiplication in monolayer has been proposed. It has been clearly shown that the synthesis of cartilage proteoglycans and type II collagen synthesis is coincident with the presence of a faint microfibrillar architecture but is absent in chondrocytes showing well-defined actin cables. Staurosporin, mainly described as a protein kinase C inhibitor, has also been shown to rapidly induce the disruption of the actin microfilaments. The purpose of this paper was to investigate whether properties of differentiated chondrocytes were reinitiated upon staurosporin treatment of serially passaged chondrocytes. Results showed, after staurosporine treatment of cells at Passage two for 5 d, complete suppression of type I and type III collagen synthesis and induction of type II collagen synthesis and of Alcian blue stainable matrix. Additionally, we showed that staurosporin restored metabolic responses that chondrocytes in primary culture exhibit upon interleukin-1 beta treatment (decrease of Alcian blue- positive cells, induction of expression of the 92 kDa gelatinase, nitric oxide production). We conclude that staurosporin is a potent redifferentiating agent of articular chondrocytes that have been subcultured up to Passage two for multiplication. Taking into account that the cellularity of cartilage is very low, staurosporine-treated chondrocytes could be useful as an alternative cellular model to evaluate pharmacotoxicological effects of drugs.

An N-terminal peptide from link protein stimulates synthesis of cartilage proteoglycans.

An N-terminal peptide from link protein stimulates synthesis of cartilage proteoglycans.

Bone morphogenetic protein 2 stimulates articular cartilage proteoglycan synthesis in vivo but does not counteract interleukin-1alpha effects on proteoglycan synthesis and content.

To study the effect of bone morphogenetic protein 2 (BMP-2) on articular cartilage proteoglycan (PG) synthesis in vivo and to investigate whether BMP-2 is able to counteract the effects of interleukin-1 (IL-1) on articular cartilage PG synthesis and content. BMP-2 alone or in combination with IL-1alpha was injected into murine knee joints. PG synthesis was measured by 35S-sulfate incorporation using an ex vivo method or autoradiography. Cartilage PG content was analyzed by measuring Safranin O staining intensity on histologic sections. BMP-2 appeared to be a potent stimulator of articular cartilage PG synthesis in vivo. However, BMP-2 was not able to counteract the deleterious effects of IL-1alpha on articular cartilage PG synthesis and content. In addition, intraarticular injections of BMP-2 induced chondrophytes. Although BMP-2 is a very potent stimulator of cartilage PG synthesis in vivo, the therapeutic applications of BMP-2 are limited due to the inability of BMP-2 to counteract the effects of IL-1 and the induction of chondrophytes.

Prostaglandin E2 stimulates insulin-like growth factor binding protein-4 expression and synthesis in cultured human articular chondrocytes: Possible mediation by Ca++ -calmodulin regulated processes

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism whose action is mediated by high affinity cell surface receptors and bioactivity and bioavailability regulated, in part, by IGF-1 binding proteins (IGFBPs). Prostaglandin E2 (PGE2) stimulates collagen and proteoglycan synthesis in cartilage via an autocrine feedback loop involving IGF-1. We determined whether the eicosanoid could regulate IGFBP-4, a major form expressed by chondrocytes and, as such, act as a modifier of IGF-1 action at another level. Using human articular chondrocytes in high-density primary culture, Western and Western ligand blotting to measure secreted IGFBP-4 protein, and Northern analysis to monitor IGFBP-4 mRNA levels, we demonstrated that PGE2 provoked a 2.7 +/- 0.3- and 3.8 +/- 0.5- (n = 3) fold increase in IGFBP-4 mRNA and protein, respectively. This effect was reversed by the Ca(++) channel blocker, verapamil, and the Ca(++)/calmodulin inhibitor, W-7. The Ca(++)ionophore, ionomycin, mimicked the effects of PGE2. The phorbol ester, PMA, which activated phospholipid-dependent protein kinase C (PKC) in chondrocytes, had no effect on IGFBP-4 production. Cyclic AMP mimetics and PKA activators, IBMX, and Sp-cAMP, inhibited the expression of the binding protein as did the PGE2 secretagogue, interleukin-1 beta (IL-beta). The inhibitory effect of the latter cytokine was mediated by a erbstatin/genistein (tyrosine) sensitive kinase. Dexamethasone, an inhibitor of cyclooxygenase (COX-2) expression and PGE2 synthesis, down-regulated control, constitutive levels of IGFBP-4 mRNA and protein, eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE2-receptor signalling pathways. The results suggest that extracellular signals control IGFBP-4 production by a number of different transducing networks with changes in Ca(++) and calmodulin activity exerting a strong positive influence, possibly maintaining the constitutivity of IGFBP-4 synthesis under basal conditions. PGE2 activation of the IGF-1/IGFBP axis may play a pivotal role in the metabolism of cartilage and possibly connective tissues in general. Eicosanoid biosynthesis may be a rate-limiting step in cartilage repair processes.

Elevated levels of insulin-like growth factor (IGF) binding protein-3 in rheumatoid arthritis synovial fluid inhibit stimulation by IGF-I of articular chondrocyte proteoglycan synthesis

The objective of this study was to quantify insulin-like growth factor (IGF) binding proteins (IGFBPs) in the synovial fluid (SF) and plasma of patients with rheumatic diseases and to study the role of these proteins in the regulation of cartilage proteoglycan (PG) synthesis. Immunological determination of IGFBP-2, IGFBP-3, IGF-I, IGF-II, interleukin-1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF alpha) was undertaken in the SF and plasma of 115 patients with rheumatoid arthritis (RA; n = 53), osteoarthritis (OA; n = 44) and other rheumatic disorders. We also determined the effects of SF on bovine cartilage PG synthesis in culture. IGFBP-2 and IGFBP-3 were elevated in the plasma (by 38% and 28%, respectively) and SF (by 56% and 59%, respectively) of patients with RA compared to age- and sex-matched OA controls (determined by RIA and confirmed by Western ligand blot). IGF-I and IGF-II did not differ significantly between the two groups. OA SF, and, to a lesser extent, RA SF stimulated cartilage PG synthesis in culture, and more than 60% of this activity was neutralised by a specific monoclonal anti-IGF-I antibody. Human IGFBP-3 dose-dependently inhibited the stimulation of cartilage PG synthesis effected by SF or human IGF-I. In RA patients, the SF concentration of IGFBP-3 was positively correlated with SF levels of IL-1 beta and TNF alpha, with the serum levels of C-reactive protein and with the erythrocyte sedimentation rate. We concluded that IGF-I is, under the conditions studied, the most important anabolic factor in human SF with respect to articular cartilage PG synthesis. The bioactivity of IGF-I in joints is modulated by IGFBP-3, which is elevated in RA SF compared to OA SF. Elevated IGFBP-3 in RA SF may reduce the availability of IGF-I to articular chondrocytes, thus interfering with cartilage PG synthesis in RA.

Glucosamine may retard atherogenesis by promoting endothelial production of heparan sulfate proteoglycans

Heparan sulfate proteoglycans produced by vascular endothelium may function physiologically to restrain the migration, multiplication, and phenotypic transition of vascular smooth-muscle cells, and to maintain an anticoagulant luminal surface by bonding and activating antithrombin III. Thus, ample production of heparan sulfate proteoglycans may act to prevent atherosclerosis and its thrombotic complications. The ability of exogenous heparin to stimulate synthesis of heparan sulfate proteoglycans by vascular endothelium may be largely responsible for the positive outcomes of most controlled evaluations of low-dose heparin as a long-term therapy for coronary disease. Glucosamine, a biosynthetic precursor of mucopolysaccharides, can substantially enhance mucopolysaccharide production when added to cultured fibroblasts or chondrocytes; the clinical utility of oral glucosamine in osteoarthritis may reflect increased synthesis of cartilage proteoglycans. It is reasonable to speculate that exogenous glucosamine will likewise enhance heparan sulfate proteoglycans production by vascular endothelial cells, and, when administered orally in regimens comparable to those effective in osteoarthritis, will thereby act to retard atherogenesis.

Recombinant human osteogenic protein 1 is a potent stimulator of the synthesis of cartilage proteoglycans and collagens by human articular chondrocytes.

To study the effects of recombinant human osteogenic protein-1 (rHuOP-1; bone morphogenetic protein-7) on proteoglycan and collagen synthesis by human articular chondrocytes. Articular chondrocytes from fetal, adolescent, and adult human donors were cultured in alginate beads for 4 days in a mixture of Ham's F-12, Dulbecco's modified Eagle's medium, 10% fetal bovine serum (FBS), then for an additional 3-10 days in the presence and absence of rHuOP-1, with and without FBS. Chondrocyte synthetic activity was measured as the amount of incorporation of 35S-sulfate into proteoglycans and 3H-proline into hydroxyproline. Sieve chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed to identify specific proteoglycans and collagens. Recombinant human OP-1 markedly stimulated the synthesis of proteoglycans (mostly aggrecan) and collagens (predominantly type II) by all chondrocyte preparations. This did not require the presence of FBS and was associated with continued expression of the chondrocyte phenotype. Recombinant human OP-1 is a more potent stimulator of the synthesis of cartilage-specific molecules by human articular chondrocytes than are other factors tested for comparison, including TGF beta 1 and activin A.

Prevention and reversal of cartilage degradation in rheumatoid arthritis by interleukin‐10 and interleukin‐4

Inflammation-induced articular cartilage degradation is a major problem in rheumatoid arthritis (RA). Type 1 T cell activity (characterized by interferon-gamma/interleukin-2 [IL-2] production), and consequently, the production of the proinflammatory cytokines IL-1 and tumor necrosis factor alpha (TNF alpha), have been reported to play a major role in cartilage damage. IL-10 and IL-4, both produced by type 2 T cells, are cytokines with the capacity to down-regulate proinflammatory responses. The present study was undertaken to investigate the way in which these cytokines affect activated mononuclear cells (MNC) of RA patients in relation to human articular cartilage degradation in vitro. MNC from synovial fluid and peripheral blood of RA patients were stimulated with bacterial antigen and treated with IL-10 and/or IL-4. Bacterial antigen is known to activate type 1 T cells and to induce proinflammatory IL-1/TNF alpha-dependent cartilage damage. Cytokine production and effects of conditioned media, as well as effects of IL-10 and IL-4 on proteoglycan (PG) turnover (as a measure for cartilage damage), were determined. IL-10 and IL-4 inhibited proinflammatory cytokine production of stimulated RA MNC and completely reversed inhibition of cartilage PG synthesis induced by these stimulated RA MNC. IL-10 was more potent than IL-4 in this respect, and the combination of IL-10 and IL-4 had an additive effect. In addition, IL-10 directly stimulated cartilage PG synthesis. IL-10 reverses the cartilage degradation induced by antigen-stimulated MNC, and IL-4 has an additive effect on this process. Furthermore, IL-10 has a direct stimulatory effect on PG synthesis, and IL-4, as a growth factor for type 2 T cells, can reduce the ratio of type 1 to type 2 T cell activity. These results provide evidence in favor of the use of a combination of the two cytokines in the treatment of RA.

Monoclonal antibodies that detect biochemical markers of arthritis in humans.

To evaluate the potential of using monoclonal antibodies (MAb) 3-B-3(-) and 7-D-4 to detect biochemical markers of altered cartilage metabolism in human arthritides. Fifty-five samples of normal articular cartilage (subjects' age range 18 weeks of gestation to 83 years of age) and 89 samples of arthritic cartilage (patients' age range 20-81 years) were collected, and their proteoglycans were extracted and analyzed for the presence of the epitopes recognized by MAb 3-B-3 and 7-D-4. Native 3-B-3(-) mimotope was expressed at a high incidence in proteoglycans extracted from the cartilage of patients with most of the arthritic diseases examined (osteoarthritis, juvenile rheumatoid arthritis, rheumatoid arthritis, avascular necrosis, and degenerative meniscal tears). Its expression in normal cartilage specimens was very low or absent, occurring mainly in the young, skeletally immature individuals. In contrast, expression of the 7-D-4 epitope was more variable in patients with different arthritides and was also frequently found in normal cartilage specimens. Immunohistochemical analyses with both 3-B-3(-) and 7-D-4 showed strong focal positive staining in superficial areas, where cartilage degeneration, remodeling, and repair were greatest. The biochemical markers recognized by MAb 3-B-3(-) and 7-D-4 are indicative of altered proteoglycan synthesis and metabolism in human articular cartilage. The data suggest that in human cartilage, the 3-B-3(-) epitope might be a better marker of biochemical changes than the 7-D-4 epitope.

Increased proteoglycan synthesis in cartilage in experimental canine osteoarthritis does not reflect a permanent change in chondrocyte phenotype.

To determine whether chondrocytes in early experimental osteoarthritic (OA) cartilage continue to show increased synthesis and turnover of proteoglycans (PGs) during explant culture. A comparison was also made between the responsiveness of experimental OA and control cartilage to interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) after 1 day and 3 days in culture. OA was induced in mature animals by sectioning of the anterior cruciate ligament followed by 3 months of normal exercise. PG synthesis in the articular cartilage was determined by measuring 35S-sulfate incorporation during explant culture over 1-3 days. Inhibition of PG synthesis was also determined with various concentrations of IL-1 beta and TNF alpha after 1 and 3 days in culture. PGs extracted from the articular cartilage over 1-3 days in culture were examined by agarose-polyacrylamide gel electrophoresis. Up to 24 hours after excision from the joint, PG synthesis was higher in experimental OA cartilage than in control cartilage. It was also less sensitive to inhibition by TNF alpha. These differences were no longer detected after 48-72 hours in culture. There were no changes in the relative proportions of aggrecan and decorin/biglycan extracted from and synthesized by control and experimental OA cartilage over the 3 days in culture. Previous results indicated that PG synthesis and turnover in articular cartilage was increased for many months after induction of experimental OA. Our present results show that the enhanced rate of PG synthesis and turnover were evident in freshly explanted tissue, but the differences were lost over 3 days in culture. A decreased responsiveness to TNF alpha was also lost. The hypermetabolic activity of experimental OA chondrocytes was thus reversible and not a permanent change in chondrocyte phenotype.

Insulin-like growth factor 1 and parathyroid hormone effects on the growth of fetal rat metatarsal bones cultured in serum-free medium.

Insulin-like growth factor 1 (IGF-1) has roles in bone growth, and parathyroid hormone (PTH) is suspected of having effects on bone, perhaps mediated by IGF-1. The purpose of this study was to determine the individual and combined effect of PTH and IGF-1 on fetal long bone metabolism. Three medial metatarsal bones were dissected from Sprague-Dawley rat fetuses harvested at the 19th day of gestation, then grown in serum-free MEM. IGF-1 (group II) or PTH (group PP) were added at the dose of 100 ng/ml for 8 days. In a third group (PI), bones were preincubated for 4 days with PTH followed by a 4-day incubation with IGF-1. Both hormones stimulated endochondral (longitudinal) growth, the highest effect was observed with IGF-1 (II: 3.11 +/- 0.06 vs. 2.16 +/- 0.08 mm in controls). The length elicited by the PI treatment ranged between those measured with IGF-1 (II) and PTH (PP) given alone (PI: 2.80 +/- 0.04 mm; PP: 2.57 +/- 0.06 mm). In addition, both hormones enhanced periosteal growth (endomembranous ossification), as measured by the width of bones (II: 0.39 +/- 0.02 mm; PP: 0.34 +/- 0.02 mm; PI: 0.38 +/- 0.02 vs. 0.29 +/- 0.01 mm in controls). On the other hand, IGF-1 but not PTH caused a significant increase in 35S incorporation (as an indicator of sulfated proteoglycan synthesis in cartilage (percent of incorporating activity; II: 0.18 +/- 0.04%; PI: 0.09 +/- 0.01 vs. 0.03 +/- 0.01% in controls). Nevertheless, both IGF-1 and PTH enhanced osteoblastic activity as shown by increased alkaline phosphatase activity in treated bones (II: 1.18 +/- 0.00 mumol/bone; PP: 0.50 +/- 0.00 vs. 0.28 +/- 0.00 mumol/bone). In conclusion, IGF-1 had the greatest effects on growth in bone length (endochondral osteogenesis) and bone width (intramembranous osteogenesis) and appeared to stimulate both chondrogenesis and osteogenesis. It also increased growth but appeared to have greater effects on osteogenesis than on chondrogenesis. Pretreatment with IGF-1 followed by PTH produced effects that were intermediate between the groups treated with IGF-1 and PTH alone.

Concentration and size distribution of IGF-I in human normal and osteoarthritic synovial fluid and cartilage

The role of free insulin-like growth factor (IGF-I) in stimulating proteoglycan (PG) synthesis in cartilage has been well documented (e.g., McQuillan et al., 1986). Equally well documented is the enhancing effect of serum addition to the incubation medium. However, it is by no means clear what, if any, is the connection between the latter two: thus in scrum IGF-I is not present in free form but exists in the form of complexes, mainly as a “large complex” of MWt around 140 kD and to a lesser extent as a “small complex” of MWt around 50 kD. Molecules of this size are largely excluded from the matrix of normal human articular cartilage. Moreover, it should be remembered that cartilage in vivo is not in contact with blood, but with synovial fluid (SF). Information about IGF-I in SF is scarce: there is only one set of data available (Schalkwijk et al. 1989) and these only for total IGF-I content.

The endocrinology of growth in carp and related species

The focus of this review is on various aspects of the endocrine control of growth by the brain neuroendocrine-growth hormone (GH)-insulin-like growth factor (IGF) axis in carp and other cyprinids, with reference to other species, including mammals, if there are particular gaps in knowledge. The neuroendocrine regulation of GH secretion in goldfish and other carp is multifactorial, with a balance of stimulatory and inhibitory inputs to somatotrophs. Somatostatin is the primary inhibitor of basal and stimulated growth hormone (GH) secretion. GH secretion is stimulated by GH-releasing factor, gonadotropin-releasing hormone, dopamine, neuropeptide Y, thyrotropin-releasing hormone and cholecystokinin. Sex steroids, in particular estradiol, influence the responsiveness of the somatotrophs to neuroendocrine factors; the responsiveness to gonadotropin-releasing hormone, neuropeptide Y, and thyrotropin-releasing hormone is increased by estradiol, whereas the responsiveness to dopamine and cholecystokinin is greatest in sexually regressed goldfish. Growth hormone, a 188-amino-acid peptide with 5 cysteine residues, stimulates growth through direct actions on some tissues, as well as by stimulation of production of insulin-like growth factor (IGF) production. The liver has a high number of GH receptors and is a primary target organ; GH binding sites have also been demonstrated in gill, intestine, kidney and gonads. GH increases the efficiency of food conversion; GH stimulates intestinal amino acid transport and intestinal mass, which may be one mechanism for the effects on food conversion. A number of factors influence GH receptor number, including GH itself, nutritional status and other hormones. IGFs have been characterized in several salmonid species using molecular biology techniques. Production of IGFs is under stimulation of GH, insulin and other hormones, and is also influenced by nutritional status and metabolic factors. The liver contains the highest concentrations of IGF, although IGFs have been found in a number of other tissues. IGFs appear to travel in the blood bound to specific binding proteins. IGF receptors have been demonstrated in only one teleost species to date. IGF-I stimulates cartilage proteoglycan synthesis; GH is dependent on IGF-I for this action. Growth rates of cultured fish may be stimulated by neuroendocrine factors added to food, administration of GH or recombinant GH preparations, or by producing GH transgenic lines of fish. Techniques for enhancing growth rates of cultured fish are in an experimental stage.

Insulin-like growth factor I accelerates recovery of articular cartilage proteoglycan synthesis in culture after inhibition by interleukin 1.

Interleukin 1 (IL-1) is a cytokine which induces cartilage proteoglycan (PG) depletion by inhibiting PG synthesis and increasing PG breakdown. Insulin-like growth factor I (IGF-I), in contrast, is known to promote matrix formation. We examined the effects of both mediators in a bovine tissue culture model. IL-1 dose-dependently inhibited PG formation of articular cartilage [half-maximal effect (EC50) at 4 ng/ml], while PG synthesis was increased by IGF-I (EC50 = 15 ng/ml). After inhibition of PG formation with IL-1 for 2 days and subsequent removal of free IL-1, addition of IGF-I dose-dependently accelerated restoration of the original rate of synthesis with a half-maximal effect at 20 ng/ml and a maximal effect at 50 ng/ml. The IGF-I concentration required to elicit a half-maximal effect on cartilage PG synthesis remained constant in the absence or presence of IL-1. We therefore conclude that inhibition of cartilage PG synthesis by IL-1 is not effected by damage to the IGF receptor. Synovial fluid (SF) of 40 patients with rheumatoid arthritis (RA) was found to contain 64 +/- 6 ng IGF-I/ml (mean +/- SEM). The reported effects of IGF-I in vitro therefore occurred at concentrations comparable to those present in joints in vivo. IL-1 beta was detectable (> 0.5 pg/ml) in 38 of 40 RA-SF samples (mean 28 +/- 6 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo protection against interleukin-1-induced articular cartilage damage by transforming growth factor-beta 1: age-related differences.

Transforming growth factor-beta (TGF-beta) has been shown to antagonise interleukin-1 (IL-1) effects in different systems. Investigations were carried out to study whether TGF-beta 1 modulates IL-1 induced inflammation and IL-1 effects on articular cartilage in the murine knee joint. IL-1, TGF-beta 1 or both factors together were injected into the knee joint. Inflammation was studied in whole knee histological sections. Patellar cartilage proteoglycan synthesis was measured using 35S-sulphate incorporation while patellar cartilage glycosaminoglycan content was determined with automated image analysis on joint sections. Co-injection of TGF-beta 1 and IL-1 resulted in synergistic attraction of inflammatory cells. In contrast, TGF-beta 1 counteracted IL-1 induced suppression of articular cartilage proteoglycan synthesis. Proteoglycan depletion was similar shortly after the last injection of IL-1 or IL-1/TGF-beta 1, but accelerated recovery was found with the combination at later days. This protective effect of TGF-beta 1 could not be demonstrated in older mice. TGF-beta 1 aggravates IL-1 induced knee joint inflammation, but counteracts the deleterious effects of IL-1 on articular cartilage proteoglycan synthesis and content. The data indicate that TGF-beta 1 could play an important part in articular cartilage restoration after IL-1 induced proteoglycan depletion.

Antisense oligonucleotides, a novel tool for the control of cytokine effects on human cartilage. Focus on interleukins 1 and 6 and proteoglycan synthesis.

To prevent the negative effects of interleukin-1 (IL-1) and IL-1-induced IL-6 on cartilage proteoglycan (PG) synthesis, we used an antisense oligonucleotide (ASO) specific for IL-6 messenger RNA (mRNA) to inhibit IL-6 production. Explants of human articular cartilage were cultured in the presence or absence of IL-6-ASO, IL-1, and exogenous IL-6. As metabolic parameters, cartilage production of IL-6 was determined in the B9 bioassay and PG as incorporation of 35SO4. The IL-6-ASO prevented IL-1-induced production of IL-6 in the cartilage explants, as well as IL-1-induced inhibition of PG synthesis. This inhibition was restored, despite the presence of IL-6-ASO, when exogenous IL-6 was added. A control ASO (not specific for IL-6 mRNA) was not effective. The IL-6-ASO used can penetrate the extracellular matrix of articular cartilage, enter the chondrocytes, and inhibit the IL-1-induced production of IL-6. Furthermore, IL-6-ASO can prevent the IL-1-induced inhibition of cartilage PG synthesis. The effect of exogenous IL-6 shows that IL-1 requires IL-6 for inhibition of PG synthesis.

Regulation of IGF-1 receptors on rabbit articular chondrocytes by inflammatory mediators

Insulin-like growth factor 1(IGF-1) is a known stimulator of proteoglycan synthesis in articular cartilage. However, arthritic cartilage shows a reduced responsiveness to IGF-1, resulting in depletion of proteoglycan moieties.