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Hyaluronic acid (HA) exerts a critical role in the lubricating and buffering properties of synovial fluid in joints. The production of HA is regulated by growth factors, hormones, inflammatory cytokines and mechanical load. The reduction of HA contributes to the progression of osteoarthritis. Herein, we found that d-galactose (d-gal) induced the senescence of rabbit synovial membrane cells, accompanied by decreased HA production. The mRNA level of HA synthase 2 (HAS2) was downregulated by d-gal, as analysed by real-time polymerase chain reaction. Melatonin, an endocrine hormone, can regulate the homeostasis of bone and cartilage. We found that melatonin treatment attenuated d-gal-induced cell senescence and decreased the expression of p21, p16 and pp65 proteins. Melatonin could reverse HA production and maintain HAS2 expression. Furthermore, we revealed that Sirt1 signalling was required for melatonin effects. Sirt1 inhibitor could counteract melatonin-mediated HA production and HAS2 expression. Additionally, Sirt1 overexpression directly antagonized d-gal-induced cell aging and HA downregulation. Taken together, our results suggest that melatonin-Sirt1 signal has a protective effect on synovial membrane cells, enhancing HA synthesis and interrupting cell senescence., (© 2021 John Wiley & Sons Ltd.)

As an alternative to chondrocytes-based cartilage repair, stem cell-based therapies have been investigated. Specifically, human synovium-derived stem cells (hSSCs) are a promising cell source based on their highly capacities for chondrogenesis, but some methodological improvements are still required towards optimal cartilage regeneration. Recently, a small compound, TD-198946, was reported to promote chondrogenesis of several stem cells, but the effect on hSSCs is still unknown. This study aimed to examine the effects of TD-198946 on chondrocyte differentiation and cartilaginous tissue formation with hSSCs. A range of concentrations of TD-198946 were examined in chondrogenic cultures of hSSC-derived cell pellets. The effect of TD-198946 on glycosaminoglycan (GAG) production, chondrocyte marker expression, and cartilaginous tissue formation was assessed. At concentrations >1 nM, TD-198946 dose-dependently enhanced GAG production, particularly hyaluronan, whereas chondrocyte differentiation was not impacted. When combined with transforming growth factor β3 (TGFβ3), TD-198946 promoted chondrocyte differentiation and production of cartilaginous matrices at doses <1 nM as judged by SOX9, S100, and type 2 collagen upregulation. Conversely, doses >1 nM TD-198946 attenuated TGFβ3-associated chondrocyte differentiation, but aggrecan was efficiently produced at 1 to 10 nM TD-198946 as judged by safranin O staining. Thus, TD-198946 exhibited different dose ranges for either GAG synthesis or chondrocyte differentiation. Regarding use of TD-198946 for in vitro engineering of cartilage, cartilaginous particles rich in type 2 collagen and GAG were predominately created with TGFβ3 + 0.25 nM TD-198946. These studies have demonstrated that TD-198946 synergistically enhances chondrogenesis of hSSCs in a unique dose range, and such findings may provide a novel strategy for stem cell-based cartilage therapy., (© 2019 John Wiley & Sons, Ltd.)

Hyaluronan (HA) and superficial zone protein (SZP) distribute in joint structures and play a crucial role in joint lubrication. The aim of this study was to examine the effect of fluid flow on the synthesis of both HA and SZP in synovial membrane cells. Shear stress was applied by fluid flow to the rabbit synovial membrane cell line, HIG-82. The mRNA levels of HA synthase 2 (HAS2) , HA synthase 3 (HAS3), and SZP were examined by real-time PCR. The levels of HA and SZP protein were determined by sandwich ELISA and western blotting, respectively. The expression of SZP protein was increased by the application of low-magnitude shear stress, whereas high-magnitude shear stress decreased expression of SZP protein. Meanwhile, the level of HA protein in culture was decreased when high-magnitude shear stress was applied. The levels of both HAS2 and HAS3 mRNAs were down-regulated by high-magnitude shear stress, resulting in a significant decrease in HA concentration. In conclusion, it is shown that the application of shear stress to synovial membrane cells substantially affects the synthesis of both HA and SZP, which are inhibited if excessive stress is applied., (© 2013 Eur J Oral Sci.)

Objective: To clarify the roles of hyaluronan (HA) in joint inflammation and the process of joint destruction, using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, in a mouse model of collagen-induced arthritis (CIA) and in a monolayer culture of fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis., Methods: DAB/1J mice were immunized with type II collagen. The effects of 4-MU were evaluated by the physiologic arthritis score, paw swelling, the histologic arthritis score, and expression of matrix metalloproteinase 3 (MMP-3) and MMP-13 in chondrocytes and synovial tissue. In vitro, the effect of 4-MU on messenger RNA and protein expression of MMP-1 and MMP-3 was determined. The effects of 4-MU on HA deposition and on serum/medium concentrations of HA were analyzed using biotinylated HA binding protein staining and an HA binding assay, respectively., Results: Treatment with 4-MU in mice with CIA dramatically decreased the severity of arthritis (based on the arthritis score), paw thickness, and histopathologic changes. MMP-3 and MMP-13 expression in chondrocytes and synovial cells was significantly inhibited by 4-MU in vivo. Treatment with 4-MU also inhibited MMP-1 and MMP-3 expression in tumor necrosis factor α-stimulated FLS, in a dose-dependent manner. The 4-MU-induced decreases in the serum HA concentration in mice with CIA and in "medium" and "pericellular" HA concentrations in cultured FLS support the contention that the inhibitory mechanism of 4-MU is mediated by HA suppression., Conclusion: Reduced disease activity induced by 4-MU in mice with CIA revealed HA to be a crucial regulator in the course of arthritis. Therefore, 4-MU is a potential therapeutic agent in arthritis, and its inhibitory mechanism is possibly mediated by suppression of HA synthesis., (Copyright © 2013 by the American College of Rheumatology.)

Hyaluronan (HA) fragments are able to induce inflammation by stimulating both CD44 and toll-like receptor 4 (TLR-4). CD44 and TLR-4 activation stimulates the liberation of NF-kB and pro-inflammatory cytokine responses. The aim of this study was to investigate the effects of hyaluronidase (HYAL) treatment, which depolymerises HA into small fragments, and of the addition of specific hyaluronan synthases-1, 2, and 3 small interference RNA (HASs siRNA), which silence HASs activity, on normal mouse synovial fibroblasts (NSF) and on rheumatoid arthritis synovial fibroblasts (RASF) obtained from mice subjected to collagen induced arthritis (CIA). The addition of HYAL to NSF and/or RASF significantly increased the TLR-4, CD44 and NF-kB activity, as well as the pro-inflammatory cytokines, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-33 (IL-33) in both groups, but to a greater extent in RASF. The addition to NSF and/or RASF of the HASs siRNA, which block HASs activity and therefore the availability of HA substrate for HYAL, was able to reduce HYAL effects in both NSF and RASF. Finally, the HA evaluation confirmed the increment of HA at low molecular weight after HYAL treatment., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Proline-hydroxyproline (Pro-Hyp) stimulated hyaluronic acid production in cultured synovium cells. It was detected in guinea pig blood after oral ingestion of collagen hydrolysates. Oral administration of collagen hydrolysates increased the amount of proteoglycans in the epiphyses. It also reduced the morphological changes associated with osteoarthritic cartilage destruction of the knee joint. The results suggest that collagen hydrolysates have therapeutic potential for treatment of osteoarthritis.

Synovial membrane consists of fibroblasts and macrophages forming the synovial lining supported by vascularized subsynovium. Each of these components may specifically react to a particular stimulus. Thus, reactions of isolated synovial cells may not correspond to that of intact tissue. We characterized the production of hyaluronan (HA) by rat synovial membrane exposed in vitro to pro- and anti-inflammatory cytokines and compared it with previous results obtained with isolated fibroblasts. Synovial membrane dissected from one knee joint served as a control to that from the opposite knee exposed to IL-1 beta, TGF-beta1, TNF-alpha, IFN-gamma or IL-4 for 24 h. The HA content was determined by ELISA, and hyaluronan synthase (HAS) mRNA by real-time PCR. The size distribution of the HA chain was evaluated by agarose gel electrophoresis. The HA content in the freshly dissected synovial membrane was approximately 1 microg and decreased to 0.1 microg after incubation, while in the medium it increased from 0 to 3 to 5 microg. All cytokines stimulated production of HA. The strongest effect was observed in the case of TNF-alpha. The level of HAS1 and HAS2 mRNA increased 2-fold during a 12-h incubation while that of HAS3 decreased. The distribution of the HA chain length did not differ in the medium from the control and stimulated membranes. Transfer of the synovial membrane from the HA-rich synovial fluid into the medium stimulated release of HA from the membrane and increased HAS expression and HA production. Thus, the synovial membrane acts as a sensor reacting to changes in HA concentration in its environment. Pro-inflammatory cytokines stimulate production of HA in intact synovial membranes similarly as in cultures of rheumatoid fibroblasts. In contrast, our results suggest that the response to anti-inflammatory cytokines (TGF-beta1 and IL-4) of the whole synovial membrane differs from that of isolated fibroblasts.

Objective: IA therapy with hyaluronan (HA) is reported to provide symptomatic relief and disease modification in OA. This study assessed the pathological changes in the synovium of an ovine model of OA and evaluated the effects of two HA preparations on this pathology., Methods: Eighteen sheep had bilateral lateral meniscectomy to induce OA. Four months post-surgery animals received IA saline or HA (Hyalgan) weekly for 5 weeks or three injections of an amide derivative of HA (HYADD 4-G) every 2 weeks (n = 6 per group). Six months after meniscectomy, sheep were killed, knee joint synovium processed, scored for pathological change and compared with synovium from non-operated animals. Sections of synovium from normal and treated joints were also immunostained for TNF-alpha, HSP-47, TGF-beta, CD44, connective tissue growth factor (CTGF) or iNOS. HA synthesis by synovial fibroblasts isolated from each OA joint was quantified., Results: Aggregate scores of pathological change were higher in OA joint synovia compared with controls, with individual measures of subintimal fibrosis and vascularity predominantly affected. Depth of intimal fibrosis was also significantly higher in meniscectomized joints. IA treatment with Hyalgan decreased aggregate score, vascularity and depth of fibrosis. HYADD 4-G treatment decreased vascularity, intimal hyperplasia and increased high-molecular weight HA synthesis by synovial fibroblasts. CD44, CTGF or iNOS expression was increased in the synovial lining of OA joints compared with normal, but there was no significant modulation of this increase by either HA preparation., Conclusion: Increased fibrosis and vascularity are hallmarks of pathological change in synovium in this meniscectomy model of OA. Both the IA HA and an amide derivative of HA reduced aspects of this pathology thus providing a potential mechanism for improving joint mobility and function in OA.

Hyaluronan (HA) is central to joint function, contributing to synovial fluid retention, lubrication, matrix organisation and joint embryogenesis. HA synthesis by intimal synoviocytes is stimulated by stretch (SSHA), linking HA production to joint usage; but the signal transduction paths are unknown. Low passage rabbit synoviocytes (RS), cultured from micro dissected synovial intima, were subjected to 10min of 10% static stretch followed by 170-min relaxation, or to sustained stretch for 180min in a Flexcell 2000 apparatus. Medium HA content was analysed by a HA-binding assay. The roles of protein kinase C (PKC) isoforms, extracellular signal-regulated kinases (ERK1/2) and Ca(2+) signalling in SSHA were tested using kinase inhibitors, Ca(2+) chelators and Ca(2+) channel activators combined with Western blots for activated kinases. Stretch increased HA secretion by 57%, independently of stretch duration. PKCalpha translocated from cytosol to membrane and triggered the phosphorylation of ERK1/2. The PKC inhibitor bisindolylmaleimide (BIM) blocked both SSHA and ERK phosphorylation, as did Gö 6976, a specific inhibitor of Ca(2+)-dependent PKC. The Ca(2+) channel activator Bay K stimulated HA secretion and ERK phosphorylation. Extra- and intra-cellular Ca(2+) chelation by EGTA and BAPTA-AM (respectively) inhibited SSHA. SSHA was also blocked by the partially selective protein kinase A inhibitor, H-89. Connective tissue growth factor, CTGF, was not involved in SSHA. Thus, stimulation of synoviocyte HA secretion by static stretch is due at least in part the o activation of a Ca(2+) influx-dependent activation of the PKCalpha-MEK-ERK1/2 cascade. This is functionally important because it links joint lubrication to joint use.

The aim of the present study was to investigate the effects of (i) the pro-inflammatory cytokines IL (interleukin)-1beta, TNF-alpha (tumour necrosis factor-alpha), IFN-gamma (interferon-gamma) and anti-inflammatory cytokines IL-4 and IL-13, and (ii) NO (nitric oxide) donors on HA (hyaluronic acid) production by synovial cells from patients with rheumatoid arthritis. Synovial cells obtained from five patients with rheumatoid arthritis were incubated for 24 h without or with IL-1beta, TNF-alpha, IFN-gamma, or with this mixture for 24 h plus IL-4 or IL-13 for the last 6 h. The same cells were also incubated for 3-24 h without or with SNP (sodium nitroprusside) or SNAP (S-nitroso-N-acetyl-DL-penicillamine). HA secretion was determined by an immunoenzymic assay based on HA-specific binding by proteoglycan isolated from bovine cartilage. IL-1beta, TNF-alpha and IFN-gamma alone or in combination stimulated HA synthesis, whereas IL-4 and IL-13 dose-dependently inhibited HA production induced by Th1 cytokines. HA production was significantly increased by the presence of 1 mM SNP after 6 and 12 h (maximal effect). HA production was significantly increased by the presence of 0.01 and 0.1 mM SNAP after 12 h of incubation, and cells treated with 1 mM SNAP showed a maximal HA production after 24 h of incubation. In conclusion, the present study provides data concerning the regulatory role of pro- and anti-inflammatory cytokines and NO donors on HA metabolism in rheumatoid synovial cells and may help in understanding the pathophysiology of rheumatoid arthritis.

Many studies have highlighted the importance of movement-induced mechanical stimuli in the development of functional synovial joints. However, such phenomenological results have failed to provide a full explanation of the mechanism essential for the morphogenesis of fluid-filled joint cavities. We have previously demonstrated that the large glycosaminoglycan hyaluronan (HA), in association with its principal cell surface receptor CD44, plays a major role during the morphogenesis of chick joints. We have taken cells from the surface of recently cavitated joints and subjected them to a brief period of dynamic mechanical strain (3800 microE for 10 min) and measured changes in HA synthesis/release, CD44 expression and HA synthase gene expression. In addition, we subjected cells to matrix depletion prior to the application of mechanical strain in order to examine any potential modulatory function of the ECM during the cell response to strain. Removal of the cell-associated HA-containing matrix with hyaluronidase significantly increased the release of HA into tissue culture media over 24 h and is associated with increased CD44 expression, alterations in HA synthase gene expression and enhanced binding of HA to the cell surface. Such changes in HA release were shown to be blocked by addition of exogenous HA and synergistically enhanced by the application of dynamic mechanical strain. These results show that cell-matrix interactions modify the response of embryonic cells to mechanical strain and provide further insight into the mechano-dependent mechanism of joint cavity morphogenesis.

To gain further insight into the synthesis and structure of the synovial matrix of joints, we have established cell cultures from synovial specimens and elaborated their production of hyaluronan and proteoglycans. The cultures secreted mainly the small proteoglycan decorin, but also considerable amounts of the related biglycan and the large proteoglycan versican. Only minor amounts of heparan sulfate proteoglycans were found. All cultures also had a high production of hyaluronan, which highlights the important role for normal joint function of these cells. In joint diseases, a common feature is the presence of an extra chromosome 7 (trisomy 7) in the synovial cells. To study the possible consequences of trisomy 7 on the synovial cell function, we extended our study to cultures that had been sub-cloned to contain high amounts of trisomy 7-carrying cells. These cell cultures had approximately four times more versican than their disomic counterparts in the cell culture medium, indicating that versican may be a mediator in the processes of joint destructive disorders. To find an explanation for this increase in versican, we investigated the expression/secretion of PDGF-AA and IL-6, cytokines with their genes located to chromosome 7. Indeed, both these cytokines were increased in the cultures with high frequencies of trisomy 7. We then added the two cytokines to cell cultures of disomic synovial cells, but only cells treated with IL-6 displayed an increased amount of versican. Thus, we suggest that the increased amount of versican in cultures of trisomy 7-carrying cells relates to an autocrine loop involving an increased IL-6 production.

We examined influences of increased soluble Fas (sFas) and hyaluronan in uremia on apoptosis and peripheral blood mononuclear cell (MNC) adhesiveness. Synovocytes, human umbilical cord endothelial cells (HUVEC), human coronary artery smooth muscle cells (CASMC), and MNC were prepared in this study. In cultures of synovocytes, HUVEC, and CASMC, sFas or high molecular hyaluronan was added to media at medium change. After 1 day, Fas-positive cells were calculated by fluorescence-activated cell sorting. Uremic level of sFas enhanced Fas-positive cells in all cell lines (P < 0.01) not in CASMC. On the contrary, hyaluronan inhibited Fas expression in all cell lines (P < 0.05). In culture with uremic serum, Fas were induced in all cell lines. At this time, the hyaluronan levels of the supernatant were measured and hyaluronan production was estimated. In contrast to the results using sFas supplement, hyaluronan production was increased in culture with sFas and uremic sera. MNC adhesiveness was increased in synovocytes and HUVEC lines by adding hyaluronan or sFas. Higher adherent cell numbers were recognized when both sFas and hyaluronan were added to the media. A most remarkable increase in cell numbers was observed in uremic MNC suspension as compared with that of MNC from healthy subjects. In conclusion, these results indicate that increased sFas in uremia stimulates apoptosis and hyaluronan production. Both sFas and hyaluronan are responsible for accelerated MNC adhesiveness in uremia.

To elucidate the mechanism of accumulation and fragmentation of hyaluronic acid (HA) under inflammatory conditions, we investigated the effect of proinflammatory cytokines on hyaluronic acid synthetase (HAS) mRNA expression using cultured rabbit synovial membrane cells. HASs mRNA levels were determined by real-time PCR. HAS2 mRNA expression was maximally enhanced 3.3- and 2.8-fold after 3-hour stimulation with IL-1beta (1 ng/ml) and after 1-hour stimulation with TNF-alpha (10 ng/ml). HAS3 mRNA expression was increased by a maximum of 4.3 times after 3-hour stimulation with IL-1beta (10 ng/ml), whereas 1-hour stimulation with TNF-alpha (10 ng/ml) and IFN-gamma (10 ng/ml) induced around a 2.5-fold increase in HAS3 mRNA. Although IFN-gamma (1-100 ng/ml) alone showed little effect on HAS2 mRNA expression, the effect was synergized by combined with both IL-1beta and TNF-alpha, substantially increasing HAS2 mRNA expression. These results suggest that proinflammatory cytokines regulate the HAS expression, and consequently may contribute to the accumulation and fragmentation of HA.

High-molecular-weight hyaluronic acid (HA) produced by the synovium may function physiologically to aid preservation of cartilage structure and prevent arthritic pain; both the size and concentration of HA in synovial fluid are diminished in osteoarthritis (OA). Glucosamine therapy for OA can be expected to increase synovial HA production by providing rate-limiting substrate. In addition, certain sulfated glycosaminoglycans and polysaccharides - including chondroitin sulfate (CS), dermatan sulfate, and pentosan polysulfate - stimulate synovial HA production, apparently owing to a hormone-like effect triggered by the binding of these polymers to membrane proteins of synovial cells. Surprisingly, a significant proportion of orally administered CS is absorbed as intact polymers - apparently by pinocytosis. These considerations may rationalize clinical studies concluding that oral CS provides slow-onset but durable pain relief and functional improvement in OA. The possibility that oral glucosamine and CS may interact in a complementary or synergistic fashion to improve synovial fluid HA content in OA should be assessed in clinical studies, and the potential of adjunctive CS administration to improve the clinical response achievable with optimal intakes of glucosamine should likewise be evaluated. In light of the fact that the synovium virtually functions as a 'placenta' for cartilage, focusing on synovium as the target for therapeutic intervention in OA may be a rational strategy., (Copyright 2000 Harcourt Publishers Ltd.)

Objectives: To investigate the regulatory roles of interleukin 1 beta (IL1 beta), tumour necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma) or transforming growth factor beta 1 (TGF beta 1) on hyaluronan (HA) synthesis by human fibroblastic synovial lining cells., Methods: Concentrations of HA in culture supernatants of fibroblastic synovial lining cell line (RAMAK-1 cell line) with or without stimulation by IL1 beta, TNF alpha, IFN gamma or TGF beta 1 were measured by sandwich binding protein assay. Levels of HA synthase mRNA of the cells with or without stimulation were detected by reverse transcribed polymerase chain reaction. Molecular weights of HA in the culture supernatants of the cells with or without stimulation were measured using high performance gel permeation liquid chromatography., Results: HA synthesis by the cells was not significantly augmented by TNF alpha or by IFN gamma. It was significantly stimulated by IL1 beta but inhibited by TGF beta 1. Molecular weights of HA in the culture supernatants of the cells were unchanged by stimulation with TNF alpha. They were remarkably increased by stimulation with IL1 beta and IFN gamma, but reduced with TGF beta 1., Conclusion: IL 1 beta is an up regulator of HA synthesis, while TGF beta 1 is a down regulator. HA production in the synovial lining cells of inflamed joints (for example, rheumatoid arthritis) might be regulated by the balance of these cytokines.

Objective: To evaluate the influence of exogenous hyaluronan (HA) on in vitro synthesis of HA and collagenase by equine synoviocytes from normal and inflamed joints., Animals: 9 adult horses., Procedure: Synoviocytes for culture were taken from the middle carpal joint of 3 horses with normal joints (control) and 6 horses with osteochondral fractures (principal). Synoviocytes were propagated in monolayer cultures and were incubated with 3 commercial HA products at concentrations of 0, 200, 400, and 1,500 micrograms/ml. Newly synthesized HA was radiolabeled with [3H]glucosamine and quantified by cetylpyridinium chloride precipitation and liquid scintillation counting. The hydrodynamic size of radioactive HA was determined by high-performance liquid chromatography, and collagenase activity was evaluated by use of a quantitative radioactive collagen film assay., Results: Exogenous HA influenced neither the rate of synthesis nor the hydrodynamic size of the newly produced HA by control or principal cell cultures. Culture supernatants from abnormal synovium, exposed to 400 and 1,500 micrograms of exogenous HA/ml, contained significantly more collagenase activity than did those exposed to lower concentrations., Conclusion: Although HA is thought to have beneficial effects in equine arthropathies, the principal mechanisms of action of HA do not appear to be stimulation of synthesis of HA of augmented molecular weight or marked inhibition of collagenase synthesis.

Anecdotal reports of rapid symptomatic response to high-dose glucosamine in osteoarthritis are not credibly explained by the traditional view that glucosamine promotes synthesis of cartilage proteoglycans. An alternative or additional possibility is that glucosamine stimulates synovial production of hyaluronic acid (HA), which is primarily responsible for the lubricating and shock-absorbing properties of synovial fluid. Many clinical and veterinary studies have shown that intra-articular injections of high-molecular-weight HA produce rapid pain relief and improved mobility in osteoarthritis. HA has anti-inflammatory and analgesic properties, and promotes anabolic behavior in chondrocytes. The concentration and molecular weight of synovial fluid HA are decreased in osteoarthritis; by reversing this abnormality, high-dose glucosamine may provide rapid symptomatic benefit, and in the longer term aid the repair of damaged cartilage.

Objectives: To isolate and characterize human synovial endothelial cells and to determine the effects of cytokines and fibroblast growth factor on human synovial endothelial (HSE) cell hyaluronic acid production., Methods: Endothelial cells were isolated from primary cultures of human synovial cells by fluorescent activated cell sorting based on the incorporation of a fluorescent derivative of acetylated low-density lipoprotein (DiI-Ac-LDL). Identity of endothelial cells was confirmed by positive immunostaining for von Willebrand factor (vWf), cytokeratins, endoglin, and reactivity with the lectin ulex europeans agglutinin (UEA). Hyaluronic acid production was measured by a radioligand-binding assay., Results: HSE cells were isolated and maintained in long-term culture. The identity of the cultured cells as endothelial was based on uniform uptake of a (DiI-Ac-LDL), immunoreactivity for vWf, and endoglin and the binding of the lectin UEA. In addition, small blood vessels in the synovium were stained selectively with anticytokeratin antibodies K462 (cytokeratin 19 specific) and K8.13 (reactive for cytokines 1, 5, 6, 7, 8, 10, 11, and 18). Isolated HSE cells also demonstrated immunoreactivity with these cytokeratin antibodies. The cytokeratins identified by the monoclonal antibody clone K8.13 demonstrated a diffuse, fibrillar staining pattern. The cytokeratin distribution revealed with monoclonal antibody K4.62 (cytokeratin 19) was also fibrillar; however, the majority of cells also demonstrated numerous punctuate cytoplasmic vesicular structures. Treatment of HSE cells with interleukin-1 alpha (IL-1 alpha) or acidic fibroblasts growth factor (aFGF), but not tumor necrosis factor (TNF alpha), dramatically reduced the vesicular structures staining with the K4.62 antibody. HSE cells produced hyaluronic acid (HA) at a constitutive rate of 200-800 ng/10(5) cells/24 h, which could be upregulated when the cells were incubated with either IL-1 alpha or aFGF. HA production was not significantly increased when HSE cells were incubated with TNF alpha, IL-4 or interferon-gamma., Conclusions: Synovial microvascular endothelial cells produce and secrete HA and endothelial HA secretion is upregulated by IL-1 and aFGF. IL-1 and aFGF also reduce the number of vesicular-like structures immunoreactive with a monoclonal antibody to cytokeratin 19. These studies suggest that cytokine stimulation of local endothelial secretion and/or accumulation of HA may influence leukocyte adhesion to the synovial endothelium.

1. When intra-articular pressure is raised to pathological values (> 9 cmH2O) by saline, the hydraulic conductance of the synovial lining increases manyfold. The increase at 25 cmH2O is only partially accounted for by stretching of the tissue and has been ascribed to washout and/or dilution of interstitial matrix biopolymers. This suggestion was tested in this study by sampling synovium from control joints (rabbit knees) and from joints perfused with saline to 25 cmH2O, and analysing them quantitatively for collagen, chondroitin sulphate, heparan sulphate and hyaluronan. 2. Pressure and trans-synovial flow measurements showed that in samples taken at 25 cmH2O the conductance of the synovial lining had increased by a factor of 5.23 +/- 1.5 (mean +/- S.E.M.) over the conductance at low pressures (just above atmospheric pressure). 3. The tissue concentrations of collagen and the sulphated glycosaminoglycans (GAGs) were reduced by similar amounts after perfusion to 25 cmH2O, namely to 62.8-70.4% of control. The hyaluronan concentration by contrast was not significantly reduced (106% of control). 4. The reduction in collagen concentration (fixed material) indicated increased interstitial hydration. The closely similar reduction in sulphated GAGs indicated that dilution rather than washout of these components was occurring. The hyaluronan results could be explained by synthesis in vivo at a rate of > or = 91 micrograms h-1 (ml synovium)-1 (possibly a non-basal rate under the conditions of the experiment, i.e. raised pressure and a stretched hydrated membrane). 5. Because interstitial hydraulic drag is related to biopolymer concentration by a power function, the overall matrix dilution observed here was more than sufficient to explain the rise in synovial lining hydraulic conductance at 25 cmH2O when taken in conjunction with stretching of the synovial lining (increased area, reduced thickness).

Objective: To investigate the role of cytokines and growth factors in the regulation of hyaluronan synthesis in human synovial lining cells., Methods: Synovial lining cells were obtained from human knee joints, isolated by the explant method, and characterized by immunocytochemistry using monoclonal antibodies against monocyte/macrophage markers as well as antibodies against hyaluronan synthase. After stimulation by cytokines and growth factors, hyaluronan was measured by radiometric assay. The molecular weight distribution of the hyaluronan synthesized was determined by high-performance gel-permeation liquid chromatography. To test the effect of oxygen-derived free radicals, the concentration and molecular weight distribution of hyaluronan were determined in the presence and absence of catalase and superoxide dismutase., Results: Hyaluronan synthesis was stimulated in synovial lining cells by transforming growth factor beta 1 (TGF beta 1), interleukin-1 beta (IL-1 beta), and to a lesser extent by tumor necrosis factor alpha (TNF alpha). Analysis of the molecular weight distribution of hyaluronan after stimulation of synovial lining cells with TGF beta 1, IL-1 beta, and TNF alpha indicated that hyaluronan is synthesized in a high molecular weight form and might be degraded in the course of inflammatory processes by oxygen-derived free radicals., Conclusion: Our findings suggest that TGF beta 1 is a major stimulator of hyaluronan synthesis in human synovial lining cells and might be involved in the pathogenic mechanisms of joint swelling in inflammatory and degenerative joint diseases.

Although synovial lining cells (SLC) have been implicated in the production of hyaluronan (HA), which is found at particularly high concentrations in synovial fluid, the degree to which individual cells within the synovium are adapted to this particular function remains to be elucidated. Uridine diphosphoglucose dehydrogenase (UDPGD) activity is the irreversible, rate-limiting step in the production of UDP-glucuronate, an essential monosaccharide in the synthesis of HA. We have assessed the UDPGD activity, microdensitometrically, in individual lining cells of normal and rheumatoid (RA) synovium, using a modified quantitative cytochemical method. In normal synovium, high activity was confined to the cells of the lining with negligible activity in the deeper subintima. The mean UDPGD activity/cell in lining cells of rheumatoid synovium was significantly lower than the activity in normal SLC. In some samples of RA and normal synovium, a bimodal distribution of cells was evident in the lining on the basis of UDPGD activity, a zone of cells in the basal layers with high UDPGD activity and a separate population of cells in more superficial layers with relatively low UDPGD activity. The results suggest that a particular population of cells is present, consistently in normal and more variably in RA synovial lining, which have high UDPGD activity/cell and may be involved in the production of HA. Furthermore, in RA synovium both the UDPGD activity/cell and the relative proportion of these cells within the lining appear to be decreased.

The effect of methotrexate (MTX) on proliferation and on interleukin 1 stimulated secretory activities of human synovial fibroblasts in culture was investigated. MTX caused a dose dependent inhibition of growth over the concentration range 0.07-2.2 microM with a half-maximal effect at 0.37 microM. INhibition was competitively relieved by coaddition of leucovorin. Cell growth was fully restored after MTX pretreatment of 24 h but not after 48 h, even on subsequent leucovorin addition. Cell viability was unaffected by MTX treatment. MTX had no effect on interleukin 1 stimulated production of prostaglandin E, hyaluronic acid and collagenase. Our results raise the possibility that one of the mechanisms contributing to the therapeutic effects of MTX in patients with rheumatoid arthritis may involve modulation of synovial fibroblast growth.

Human and bovine bone matrices were extracted with salt solutions of different composition and the extracts tested for stimulation of incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the hyaluronic acid and chondroitin sulfate of the cell pellet, the cell surface and the medium fractions of human synovial cells in culture. Stimulatory activity was extracted with a solution of 0.3 M EDTA in 2.5 M NaCl from bovine but not human bone. Subsequent extraction of the residues with 4 M guanidinium hydrochloride yielded activity from both matrices. A major stimulation of incorporation of radioactivity was observed in the cell surface fractions. Human synovial cells constitute a more sensitive assay system for the stimulatory activity than rabbit synovial cells.

Individual canine synovial villi were used to establish short-term synovial organ cultures. These villi incorporated 3H-glucosamine into highly-polymerized 3H-hyaluronic acid (3H-HA), which was the only 3H-glycosaminoglycan identified in the culture medium. Some 3H-HA, and larger amounts of other 3H-glycosaminoglycans, were recovered from cultured tissues. Culture medium 3H-HA content was proportional to the surface area of cultured villi. Organ cultures of nonvillous synovium were compared with villi; nonvillous cultures synthesized less 3H-HA per mm2 of their synovial intimal surface than villi. These cultures complement cell culture techniques for in vitro studies of synovial lining cell function.

1 Cultured human synovial fibroblasts were 'activated' by leucocytes or poly (I) . poly (C) to overproduce hyaluronic acid. 2 Diflunisal in concentrations of 30, 15 and 10 microgram/ml completely abrogated the overproduction of hyaluronic acid induced by leucocytes. 3 Diflunisal (10 microgram/ml), indomethacin (2 microgram/ml) and aspirin (200 microgram/ml) partially inhibited the overproduction of hyaluronic acid induced by poly (I) . poly (C), although none of them decreased it to control non 'activated' levels. 4 Diflunisal 25 microgram/ml completely abrogated hyaluronic acid overproduction induced by poly (I) . poly (C). 5 This model could be an in vitro indicator for in vivo pharmacological levels of anti-inflammatory drugs.

The pronounced synovial hyperplasia often found in the joints of patients with rheumatoid arthritis could be explained partially by the action of monocyte-macrophage polypeptides (monokines). This report demonstrates that two cytokines which may be derived from monocyte-macrophage populations, namely platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), stimulate the DNA synthesis and proliferation of human synovial fibroblast-like cells cultured in low (i.e., 1%) fetal bovine serum. Epidermal growth factor, insulin-like growth factor-I, insulin-like growth factor-II (multiplication stimulating activity) and substance P were inactive. Unlike IL-1, PDGF and FGF do not also stimulate PGE2, plasminogen activator, and hyaluronic acid levels. Thus PDGF and FGF, arising from stimulated monocyte-macrophages, may play a role in the stimulation of mesenchymal cell proliferation that often accompanies chronic inflammatory arthritic disease. The synovial cells respond to a variety of cytokines in different ways suggesting multiple-signaling pathways.

Recombinant human cytokines were compared for their effects on glycosaminoglycan (GAG) synthesis in human synovial fibroblast cultures and human articular cartilage explant cultures. In fibroblast cultures, recombinant human interleukin-1 alpha (rHuIL-1 alpha), rHuIL-1 beta, and recombinant human tumor necrosis factor alpha (rHuTNF alpha) stimulated hyaluronic acid (HA) production and, to a lesser extent, sulfated GAG production, while recombinant human gamma-interferon did not have a significant effect. Half-maximal stimulation of HA by rHuIL-1 beta was 0.14 pM, while stimulation for rHuIL-1 alpha and rHuTNF alpha was 1.6 pM and 32 pM, respectively. Indomethacin (10 micrograms/ml) had no influence on HA stimulation by cytokines, while hydrocortisone (2-10 micrograms/ml) caused a significant reduction. In articular cartilage cultures, the cytokines inhibited production of sulfated GAGs. The activity of rHuIL-1 beta was greater than that of rHuIL-1 alpha (half-maximal inhibition at 0.71 pM and 4.7 pM, respectively) and both were considerably more active than rHuTNF alpha; gamma-interferon again had no significant effect. Neither indomethacin nor hydrocortisone influenced cytokine-induced inhibition by either rHuIL-1 preparation. These studies indicate that cytokines released during an inflammatory process may affect GAG synthesis in human joint tissues and may have opposite effects on GAG synthesis in different types of connective tissues.

The effect of various sulfated glycosaminoglycans on glycoconjugates syntheses in synovial membranes of rabbit knee joints in culture was investigated by two different approaches. In the first approach, synovial membranes isolated from rabbit knee joints were cultured in the presence of sulfated glycosaminoglycans and [14C]glucosamine. In the second approach, solutions of sulfated glycosaminoglycans were injected into rabbit knee joints and synovial membranes isolated from the joints were cultured in the presence of [14C]glucosamine. The major part of [14C]glucosamine-labeled glycoconjugates associated with the synovial membranes and secreted into culture medium was hyaluronic acid. Of the natural glycosaminoglycans tested, dermatan sulfate gave the maximum stimulation of hyaluronic acid synthesis followed by chondroitin 4- and 6-sulfate. Heparin, heparan sulfate, keratan sulfate, keratan polysulfate, and hyaluronic acid had no significant effect. Of the chemically polysulfated glycosaminoglycans, GAGPS (a persulfated derivative of chondroitin sulfate) gave high stimulation but N-acetylchitosan 3,6-disulfate had no effect. The effect of sulfated glycosaminoglycans on hyaluronic acid synthesis was the same in both experimental approaches. The increase in the amount of secreted hyaluronic acid in culture medium paralleled that in synovial membranes. The results indicate that the galactosamine-containing sulfated glycosaminoglycans have a specific stimulatory effect on hyaluronic acid synthesis. A high degree of sulfation of the molecules appeared to potentiate the stimulatory effect.

Some in-vitro effects of the arthritogenic polysaccharide carrageenin were studied on cells from human synovium. Synovial cells were isolated from intact human knee joints, and cell lines were developed by passaging with trypsin. Carrageenin was ingested by the cells but did not significantly affect cell growth, numbers of lysosomes, intracellular lysosomal enzyme activity (N-acetyl-beta-D-glucosaminidase), or release of lysosomal enzyme from cells. Carrageenin produced a reduction in net hyaluronic acid synthesis. It also induced a striking morphological change in a high proportion of synovial cells, characterised by increased spreading over the culture surface and apparent condensation of the cytoplasm into a pattern of ridges. Nonrheumatoid and rheumatoid synovial cells behaved similarly to one another.

The effects of peripheral blood monocyte and rheumatoid synovial fluid macrophage conditioned media were studied on hyaluronic acid (HA) metabolism of normal synovial fibroblasts. Both media stimulated HA synthesis about two-fold compared to controls (1% fetal calf serum). The activated mononuclear phagocyte conditioned media did not contain HA-degrading activity in these experiments. The effects of various concentrations of interleukin-1 (IL-1) on HA synthesis and proliferation of synovial fibroblasts were studied. Even at very low concentrations (0.1 IU IL-1/ml) HA synthesis was stimulated. With increasing concentrations HA synthesis did not increase but proliferation was stimulated. Stimulated fibroblasts synthesized mainly high molecular weight HA. Thus with IL-1-activation, normal synovial fibroblasts could not produce increased amounts of abnormal HA with decreased molecular weight.

We have previously reported that naturally occurring sulfated glycosaminoglycans having a chondroitin-type structure and glycosaminoglycan polysulfate (GAGPS, a persulfated derivative of chondroitin sulfate) caused a specific stimulation of hyaluronic acid synthesis in rabbit knee synovial membranes [H. Nishikawa et al. (1985) Arch. Biochem. Biophys. 240, 146-153]. In the present study, the interaction of [3H]GAGPS and the surface of the rabbit knee synovial membranes and the relationship between this interaction and the stimulatory effect of GAGPS on the hyaluronic acid synthesis were examined in order to define the stimulatory mechanism of hyaluronic acid synthesis by GAGPS. A part of the [3H]GAGPS taken up by the synovial membranes was released from the membranes by trypsin treatment. The interaction of [3H]GAGPS and the surface of the isolated synovial membranes was diminished by pretreatment of the membranes with proteases or chelating reagents. Pretreatment of synovial membranes with trypsin or ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid had little effect on the basal hyaluronic acid synthesis but caused the loss of GAGPS-induced stimulation of hyaluronic acid synthesis accompanied by significant decrease (20% P less than 0.05-P less than 0.01) in the interaction between GAGPS and the surface of the synovial membranes. Dermatan sulfate having a chondroitin-type structure also stimulated hyaluronic acid synthesis but this effect was not additive to the stimulation of hyaluronic acid synthesis by GAGPS. Heparin had no effect on either the basal hyaluronic acid synthesis or the GAGPS-induced stimulation of hyaluronic acid synthesis. These results indicate that binding of GAGPS to certain distinct protein components on the surface of synovial membranes is involved in the stimulatory mechanism of hyaluronic acid synthesis by GAGPS, and that the binding may be mediated by Ca2+ ion. The binding was also found to be specific for sulfated glycosaminoglycans having a chondroitin-type structure.

The quantitative radiochemical methodology described in this report allows a major increase in information generation, increased experimental flexibility, improved statistical control, and increased diversity of information per culture. Other advantages relate to economies of technical time, supplies, cells, and test materials per individual culture. Microcultures of human synovial cells incorporate [14C]glucosamine into hyaluronic acid that accumulated primarily in the media and to a lesser extent in the cell mass. CTAP-I (from lymphoid cells), CTAP-III (from human platelets), PGE2, dibutyryl cAMP, and poly(I) . poly(C) markedly stimulated hyaluronate synthesis, whereas cortisol, cycloheximide, and tunicamycin inhibited stimulated synthesis. Time studies with cycloheximide indicated that translation, essential for the activation of synovial cells, was completed by 17 h postexposure to CTAP-I. Tunicamycin also seemed to inhibit CTAP-I induced activation primarily by interfering with translation; however, tunicamycin also caused modest post-translation inhibition of hyaluronate synthesis in activated adult human synovial cells.

Myristyl-gamma-picolinium chloride (MGP) is a novel constituent of methylprednisolone acetate suspensions used for intra-articular corticosteroid therapy. Hyaluronic acid synthesis in organ cultures of normal canine synovial villi incubated with MGP was reduced in proportion to MGP concentration. Similar dose-dependent inhibition of sodium sulfate S 35-labeled glycosaminoglycan synthesis was observed in organ cultures of canine articular cartilage treated with MGP. In comparison, the intact cartilage of cultured femoral condyles appeared less sensitive to MGP inhibition of glycosaminoglycan synthesis. Cartilage and synovium were examined 4 hours after an intra-articular injection of either MGP or its vehicle into canine stifles. No differences in Na2(35)SO4-glycosaminoglycan synthesis were observed between cartilage organ cultures prepared from the MGP-treated and the contralateral vehicle-treated joints, but in four of the five dogs studied less hyaluronic acid was synthesized (P less than 0.05) by synovial cultures from joints exposed to MGP than by cultures from the contralateral joints. Synovium from the MGP-treated joints remained capable of phagocytosis. These observations indicate that at clinically relevant concentrations MGP can inhibit synovial hyaluronic acid synthesis, but has little metabolic effect on normal articular cartilage.

In studies designed to establish the safety and efficacy of laser treatment of arthritis, the in vitro effect of Nd:YAG laser radiation on normal, osteoarthritic and rheumatoid synovial tissue and synovial fibroblast metabolism has been addressed. Defined energy levels of normal pulsed radiation (TEMoo mode) consistently up-regulated synovial tissue explant culture hyaluronic acid and protein synthesis. In contrast, high intensity Q-switched delivery down-regulated the response. The application of normal pulsed radiation had a variable effect on synovial fibroblast synthesis but the response of any given cell isolate was consistent. DNA synthesis and cell viability were not affected.

The studies reported here were undertaken to determine if Erysipelothrix crude extract (CE) could elicit consistent, detectable alterations in synovial cells in vitro. Synovial cells of rabbits were cultured in vitro and data about normal synovial cells in vitro were presented. Such cells were consistently and characteristically affected by exposure to small amounts of CE; an effect that was neutralized in the presence of specific and anti-CE anti-serum. With an increase in the amount of CE added to the cells in culture, a decreasing number of cells was formed but protein content remained relatively constant.

Organ cultures and primary cell cultures were established from synovial tissue collected from patients with rheumatoid arthritis. Hyaluronic acid measured by the incorporation of [3H]glucosamine into the polysaccharide was found to be synthesised in the cultures immediately after transfer from in-vivo to in-vitro conditions. This was in contrast to the primary cultures established from cells isolated from normal joints. The latter cells did not synthesise any detectable hyaluronate. 90-100% of the cells in primary culture were found to be esterase positive, indicating their macrophage nature. The molecular weight of the hyaluronate produced by the pathological cells was low (approximately 50 000) compared with the molecular weight of hyaluronate found in joint fluid from normal or rheumatoid joints. Cell lines of fibroblasts established from rheumatoid joints and studied after four or seven passages also produced hyaluronate of low molecular weight. It is known that similar cell lines from normal joints produce a high molecular weight polymer.