Effects of human recombinant tumor necrosis factor-alpha and interleukin 1 on the synthesis of glycosaminoglycan and DNA in cultured rat costal chondrocytes

We examined effects of human rTNF alpha on the synthesis of glycosaminoglycan and DNA in cultured rat costal chondrocytes. The effects of human recombinant IL-1 alpha and IL-1 beta were also given attention. rTNF alpha, as well as rIL-1 alpha and rIL-1 beta, decreased the incorporation of [35S]sulfate into glycosaminoglycan to about 10% of the levels in the control. The half-maximal doses of rTNF alpha, rIL-1 alpha or rIL-1 beta required for the suppression of glycosaminoglycan synthesis (by rTNF alpha, rIL-1 alpha, and rIL-1 beta) were 2 ng/ml, 30 ng/ml, or 5 ng/ml, respectively. rTNF alpha stimulated incorporation of [3H]thymidine in the chondrocytes in a dose- and time-dependent manner. DNA synthesis was increased to about threefold over the control cultures in the presence of 1 microgram/ml rTNF alpha for 72 hr. The stimulatory effect of rTNF alpha on DNA synthesis was observed in both subconfluent and confluent cultures, whereas rIL-1 alpha and rIL-1 beta had no stimulatory activity on DNA synthesis. The addition of rTNF alpha to the cultures of chondrocytes stimulated DNA synthesis, even in medium containing no fetal calf serum. The fetal calf serum acted synergistically with rTNF alpha in increasing DNA synthesis. We propose that both TNF and IL-1 may be involved in inflammatory diseases of cartilage, and that TNF alpha, but not IL-1, may have some physiologic growth factor function for chondrocytes.     

Modulation of human chondrocyte metabolism by recombinant human interferon gamma: in-vitro effects on basal and IL-1-stimulated proteinase production, cartilage degradation and DNA synthesis

Human articular chondrocytes in monolayer culture and fragments of human articular cartilage were treated with recombinant human interferon gamma (IFN-gamma) both alone and in combination with interleukin 1 (IL-1). IFN-gamma alone inhibits metalloproteinase production, as measured in the caseinase assay, and decreases glycosaminoglycan release from cartilage fragments in culture. The synthesis of DNA, as measured by [3H]thymidine incorporation, is stimulated by IFN-gamma. Similar effects are seen in the presence of IL-1. Thus, IFN-gamma opposes the stimulatory effect of IL-1 on caseinase production and decreases IL-1-stimulated cartilage degradation, as measured by glycosaminoglycan release. In contrast, IFN-gamma has no effect on IL-1-stimulated prostaglandin production, and acts synergistically with IL-1 to cause a large stimulation of DNA synthesis. These results show that IFN-gamma has a number of effects on articular chondrocytes in-vitro and suggest a possible role for IFN-gamma in limiting cartilage degradation in inflammatory joint conditions.     

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.     

Stimulation of hyaluronan synthesis by interleukin-1beta involves activation of protein kinase C betaII in fibroblasts from patients with Graves' ophthalmopathy

Hyaluronan accumulation in the retroorbital connective tissue is one of the pathological features of Graves' ophthalmopathy. Interleukin-1beta (IL-1beta) is known to stimulate hyaluronan synthesis in orbital fibroblasts. In the present study, the intracellular signal transduction pathways involved in this stimulatory effect were investigated in cultured human retroorbital fibroblasts from patients with Graves' ophthalmopathy. IL-1beta-induced hyaluronan synthesis was significantly inhibited by pretreatment of the cells with two protein kinase C (PKC) inhibitors, chlerythrine chloride and H-7. In addition, treatment with phorbol 12-myristate 13-acetate (PMA), a direct PKC activator, also resulted in increased hyaluronan production. IL-1beta- or PMA-stimulated hyaluronan synthesis was blocked by the protein synthesis inhibitor, cycloheximide. Moreover, the intracellular Ca(2+) concentration of the orbital fibroblasts was also involved in the IL-1beta induced transduction pathway, the effect being completely inhibited by BAPTA, an internal calcium chelator. In addition, A23187, a calcium ionophore, increased hyaluronan synthesis in unstimulated cells. These results suggest that the Ca(2+)-dependent PKC signal transduction pathway plays an important role in the IL-1beta-induced hyaluronan synthesis. Moreover, IL-1beta treatment resulted in increased PKC activity and the rapid translocation of PKC betaII from the cytoplasm to the plasma membrane. These results indicate that cytosolic Ca(2+) and PKC betaII are involved in IL-1beta-induced hyaluronan synthesis in cultured orbital fibroblasts from patients with Graves' ophthalmopathy.     

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. $\text{Benzyl}\text{-β-}\text{D}\text{-}\text{xyloside}$, 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.     

Nitric oxide mediates interleukin-1 induced inhibition of glycosaminoglycan synthesis in rat articular cartilage

Interleek-1beta (IL-1) is a key mediator of cartilage matrix degradation in osteoarthritis and rheumatoid arthritis. It was found that the IL-1-induced suppression of glycosaminoglycan (GAG) synthesis in rat articular cartilage occurred simultaneously with the accumulation of nitrite (a metabolite of nitric oxide (NO) in aqueous milieu) in the culture medium. NO-synthase inhibitors, L-NMMA and L-NIO, inhibited both these IL-1 effects. Dexamethasone suppressed GAG synthesis additively to IL-1, but did not alter nitrite accumulation. Three NO-donors (GEA 3175, SNAP and SIN-1) also had an inhibitory effect on cartilage GAG synthesis. Therefore, it is concluded that IL-1 induced suppression of GAG synthesis in rat articular cartilage is mediated by the production of NO.     

Effect of a interleukin-1 like factor (mononuclear cell factor) on proteoglycan synthesis in cultured human articular chondrocytes

A partially purified monocyte factor, with Interleukin-1 properties (MCF/IL-1), enhances the proteoglycan synthesis of human neonatal articular chondrocytes in culture, and changes the repartition of these macromolecules between medium and cell layer. The size of the proteoglycan monomers, the length of the glycosaminoglycan chains and the respective levels of chondroitin-6-sulfate, chondroitin-4-sulfate and non sulfated chondroitin remain unchanged under MCF/IL-1 exposures. The addition of indomethacin reduces the stimulation effect by 60-70% only, suggesting that the MCF/IL-1 action is partially dependent on prostaglandins but seems also related to mechanisms distinct from the cyclooxygenase pathway.     

Effects of glutathione depletion on the synthesis of proteoglycan and collagen in cultured chondrocytes

We studied the effect of the depletion of glutathione on the synthesis of proteoglycan and collagen in cultured chick chondrocytes. When the cultured chondrocytes were incubated with 1 mM buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase, the intracellular glutathione level markedly dropped within 12 h with no loss of cell viability. Incorporation of 35SO2-4 into proteoglycan was lowered in the presence of BSO. When the 35S-labeled proteoglycans were separated into two fractions by glycerol density gradient centrifugation, the inhibitory effect of BSO on the synthesis of proteoglycan was greater in the fast-sedimenting proteoglycan fraction, which consisted mainly of cartilage specific large proteoglycan (PG-H), than in the slowly sedimenting proteoglycan fraction. The inhibition by BSO of the synthesis of core protein-free glycosaminoglycan chains primed by p-nitrophenyl-beta-D-xyloside was smaller than the inhibition of the synthesis of proteoglycan. Analysis of glycosaminoglycans labeled with [3H]glucosamine indicated that the treatment of chondrocytes with BSO resulted in a small increase in the proportion of synthesis of hyaluronic acid to the synthesis of total glycosaminoglycan. The incorporation of [3H]proline into collagen was also inhibited by BSO. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the 3H-labeled collagen showed that, in the presence of BSO, processing of Type II collagen appeared to slow down and the proportion of Type X collagen synthesis was reduced.     

Effects of adeno-associated virus (AAV) of transforming growth factors β1 and β3 (TGFβ1,3) on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type Ⅱ were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type Ⅱ, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.     

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.     

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-β-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.     

Interleukin-1 beta is a potent growth inhibitor of adult rat hepatocytes in primary culture

Interleukin-1 beta (IL-1 beta) strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin and epidermal growth factor (EGF). Its effect was dose-dependent and was maximal at 2 ng/ml. IL-1 beta had no cytotoxic effect but changed the cells from a flat to a spindle shape as shown by phase-contrast microscopy. The inhibition of DNA synthesis by IL-1 beta was closely correlated with a decrease in the labeling index. This inhibitory effect was observed only when IL-1 beta was added for 10 h to cultured hepatocytes in the G1 phase within 12 h after addition of insulin and EGF: it was not observed in the S phase, which starts about 24 h after addition of the mitogens. Exposure of the hepatocytes to IL-1 beta for two 1-h periods, one at an early stage (0-6 h) and one at a late stage (6-12 h) of the G1 phase, resulted in the same marked inhibition of DNA synthesis as exposure to IL-1 beta for 10 h in the G1 phase. This requirement of IL-1 beta at two stages in the G1 phase for inhibition of DNA synthesis of hepatocytes is different from that with transforming growth factor-beta, which is required for only 1 h in the early G1 phase for a similar inhibition. These findings suggest that IL-1 beta acts at two distinct stages in the G1 phase and that its cooperative actions are necessary to inhibit growth of adult rat hepatocytes in primary culture. Other cytokines, such as IL-6/B-cell stimulating factor-2, were less potent, but caused significant inhibition of DNA synthesis of adult rat hepatocytes at 2 ng/ml, whereas IL-2 and tumor necrosis factor did not affect hepatocyte growth. From these results it is suggested that Kupffer cells in liver lobules and macrophages in the blood may play important roles, mainly via IL-1, in repair of liver damage and regeneration.     

Human recombinant interleukin-1 receptor antagonist inhibits lymphocyte blastogenesis induced by concanavalin A. Restorative effect of hrIL-1.

Interleukin-1 (IL-1), mainly produced by monocyte-macrophages, is a polypeptide cytokine with pleiotropic biological effects. IL-1 plays an important role in mediating immune response and inflammation. Recently a natural inhibitor to IL-1 has been discovered, interleukin-1 receptor antagonist (IL-1ra), produced by human monocytes cultured on adherent IgG which binds to the IL-1 receptors. In our study we found that the pretreatment of cells with serial dilutions of IL-1ra (250 ng/ml-2.5 pg/ml) inhibits, in a dose-dependent manner, lymphocyte DNA synthesis stimulated with Con A (10 micrograms/ml). IL-1ra did not have any effect on resting peripheral blood mononuclear cells (PBMC). Time course experiments show that IL-1ra at 250 ng/ml has its maximum inhibitory effect on lymphocyte blastogenesis when cells are pretreated 2 h before Con A. No effect was found when hrIL-1ra was added after Con A. Moreover, hrIL-1ra also inhibits the enhancing effects of exogenous hrIL-1 (400, 200, 100 and 50 ng/ml) on lymphocytes stimulated with Con A; while when hrIL-1ra was used on cells treated with only Con A, the inhibition was more pronounced. When PBMC were removed from monocytes, by adherence, the Con A-treated lymphocytes were not influenced by 2 h pretreatment of hrIL-1ra; while a strong inhibition was found when exogenous hrIL-1 was added at different concentrations. In addition, hrIL-1ra also inhibits the enhancing effect of hrIL-2 on lymphocyte DNA synthesis. In another set of experiments PBMC were pretreated with hrIL-1ra (250 ng/ml) for 2 h and then added LPs (10 ng/ml) and IL-1 alpha generation was determined using ELISA. In these experiments IL-1ra completely abolished the generation of IL-1 alpha. These data suggest that hrIL-1ra exhibits a dose-response inhibition of lymphocyte blastogenesis induced by Con A, probably through the down-regulation of IL-1 synthesis necessary as an early signal for T-cell activation and IL-2 production.     

Anti-inflammatory effects of hepatocyte growth factor: induction of interleukin-1 receptor antagonist

Hepatocyte growth factor (HGF) prevents liver failure in various animal models including endotoxin-induced acute liver failure. We were interested to find out whether human HGF exerts anti-inflammatory effects by modulation of cytokine synthesis. Therefore, human HepG2 cells were cultured with increasing concentrations of HGF. HGF dose-dependently upregulated the production of interleukin-1 receptor antagonist (IL-1Ra). Incubation of HepG2 cells with interleukin-1beta (IL-1beta) caused an increase in IL-1Ra levels, while interleukin-6 (IL-6) had no effect on IL-1Ra synthesis. Co-stimulation of HepG2 cells with HGF + IL-1beta resulted in a synergistic effect on IL-1Ra mRNA and protein expression. Stimulation of freshly isolated mouse hepatocytes from male C57 BL/6 mice with HGF increased IL-1Ra mRNA and protein synthesis dose-dependently. A co-stimulation with HGF and IL-1beta had a synergistic effect on IL-1Ra mRNA expression but only a partially additive effect on IL-1Ra protein synthesis. HGF-induced IL-1Ra production was significantly decreased by the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Accordingly, HGF stimulation specifically increased MAPK-dependent signalling pathway (p42/44). In contrast, in preactivated PBMC mRNA expression and protein synthesis of IL-1Ra, interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) were unaffected after stimulation with HGF. In conclusion, our data suggest that HGF exerts anti-inflammatory effects by modulating the signal transduction cascade leading to increased expression of IL-1Ra, which might explain the protective and regenerative properties of this cytokine in animal models of liver failure.     

Effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix formation by bovine corneal endothelial cell cultures

The effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix (ECM) formation by cultured bovine corneal endothelial (BCE) cells was investigated. BCE cells actively proliferating on plastic dishes produced in the absence of xyloside an ECM containing various proteoglycans. Heparan sulfate was the main 35S-labeled glycosaminoglycan component (83%). Dermatan sulfate (14%) and chondroitin sulfate (3%) were also present. Exposure of actively proliferating BCE cells to xyloside totally inhibited synthesis of proteoglycans containing dermatan sulfate or chondroitin sulfate and caused an 86% inhibition of heparan sulfate proteoglycan synthesis. The heparan sulfate proteoglycans that were extracted from the ECM produced by BCE cells exposed to xyloside had a smaller size and a reduced charge density compared to their counterparts extracted from the ECM of cultures not exposed to xyloside. In contrast to the inhibitory effect of the xyloside on proteoglycan synthesis, exposure of actively proliferating BCE cells to xyloside stimulated synthesis of free chondroitin sulfate and heparan sulfate chains. All of the xyloside-initiated glycosaminoglycan chains were secreted into the culture medium. The proteoglycan-depleted matrices produced by BCE cells exposed to xyloside were used to study the effect of these matrices on proteoglycan synthesis by BCE cells. BCE cells growing on proteoglycan-depleted ECM showed a considerable increase in the rate of proteoglycan synthesis compared to BCE cells growing on normal ECM. Moreover, the pattern of glycosaminoglycan synthesis by BCE cells growing on proteoglycan-depleted ECM was changed to one which resembled that of BCE cells actively proliferating on plastic dishes. It is postulated that BCE cells are able to recognize when an ECM is depleted of proteoglycan and to respond to it by increasing their rate of proteoglycan synthesis and incorporation into the ECM.     

Cytoskeleton and differentiation: effects of cytochalasin B and colchicine on expression of the differentiated phenotype of rabbit costal chondrocytes in culture

Cytochalasin B changed the shape of cultured rabbit costal chondrocytes from polygonal to nearly spherical and stimulated glycosaminoglycan synthesis, which is a differentiated phenotype of chondrocytes, whereas colchicine changed them from polygonal to flattened and inhibited glycosaminoglycan synthesis. These morphological changes occurred parallel with the changes in glycosaminoglycan synthesis. Induction of ornithine decarboxylase by parathyroid hormone, which is a good marker of differentiated chondrocytes, was markedly potentiated in the spherical cells which had been pretreated with cytochalasin B, whereas pretreatment with colchicine inhibited the induction of the enzyme. Both cytochalasin B and colchicine inhibited DNA synthesis. The inhibitions were observed after the appearance of changes in the morphology of the cells and glycosaminoglycan synthesis. These findings suggest that intactness of microtubules and disruption of microfilaments are involved in regulating the expression of the differentiated phenotype of chondrocytes in culture.     

Effects of somatomedin and other factors on glycosaminoglycan synthesis in cultured rat chondrocytes

We have investigated the effects of hormones and serum on glycosaminoglycan (GAG) synthesis, using cultured rat chondrocytes isolated from growing cartilage. Somatomedin A stimulated GAG synthesis at a physiological concentration, however in the case of insulin the dose required to stimulate GAG synthesis was 500 times as great as the physiological concentration. Parathyroid hormone also increased GAG synthesis. In contrast, hydrocortisone inhibited GAG synthesis at a pharmacological dosage. None of the following had any effect on GAG synthesis: epidermal growth factor, fibroblast growth factor, triiodothyronine, growth hormone, sex steroid or vitamin D3. Human serum up to a concentration of 1% stimulated GAG synthesis. Serum from patients with acromegaly stimulated GAG synthesis more than that from those with hypopituitarism.     

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.