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.A receptor binding assay for IGF-1 was set up in normal articular chondrocytes to determine whether or not growth factors and cytokines found in inflammatory tissues could influence the response of these cells to IGF-1 through modulation of target receptors.The results of the binding assays show that IGF-1 receptors on rabbit articular chondrocytes are not down-regulated by inflammatory cytokines, and are only modulated by high concentrations of IGF-1, insulin and Nu-serum (of which IGF-1 and insulin are constituents). This suggests that if monolayer cultures of chondrocytes behave similarly to chondrocytes in cartilage, reduced responsiveness to IGF-1 is unlikely to be caused by a depletion of IGF-1 receptors.     

Enhancement of toluidine blue staining by transforming growth factor-beta, insulin-like growth factor and growth hormone in the temporomandibular joint of aged mice

Osteoarthritic lesions appear in the articular cartilage of the temporomandibular joint of mice aged 7 months and older. Reduced rate of proteoglycan (PG) synthesis leading to destruction of the articular cartilage was observed in this joint. The purpose of the present study was to test the ability of transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor-1 (IGF-1) and growth hormone (GH) to induce PG synthesis in joint cartilage of aged animals and to compare it with the effect of interleukin-1alpha (IL-1alpha). Mandibular condyle explants from 18-month-old mice were cultured up to 72 h in serum-free medium, supplemented with IL-1alpha (TGF-beta1 (0.1-5.0 ng/ml), TGF-beta1 (1.0 ng/ml) + IGF-1 (2 ng/ml) or GH (10 ng/ml). The incorporation of (35)S-SO(4) into sulfated PG was tested. Cartilage samples were processed for histomorphometry using sections stained with 0.1% toluidine blue (TB), pH 1.8. Results indicated that in cultures supplemented (48 h) with either TGF-beta, TGF-beta + IGF-1 or with GH, an increased height and area of TB-positive staining as well as increased incorporation of (35)S-SO(4) into sulfated PG were observed. In contrast, the cytokine IL-1alpha exerted an inhibitory effect on TB staining and on (35)S-SO(4) incorporation. The present study demonstrated that in vitro supplementation of IL-1alpha to mandibular condyle cartilage reduced the height and area of TB staining and incorporation of (35)S-SO(4), whereas TGF-beta1, TGF-beta1 + IGF-1 or GH increased the height and area of TB staining and increased incorporation of (35)S-SO(4). The two parameters used to identify increased PG synthesis were shown to reveal similar results and were useful for studying the dynamic events taking place in cartilage destruction and repair in osteoarthritis.     

Clinical diagnosis of potentially treatable early articular cartilage degeneration using optical coherence tomography

A series of bench to operating room studies was conducted to determine whether it is feasible to use optical coherence tomography (OCT) clinically to diagnose potentially reversible early cartilage degeneration. A human cadaver study was performed to confirm the reproducibility of OCT imaging and grading based on identification of changes to cartilage OCT form birefringence using a polarized OCT system approved for clinical use. Segregation of grossly normal appearing human articular cartilage into two groups based on the presence or absence of OCT form birefringence showed that cartilage without OCT form birefringence had reduced ability to increase proteoglycan synthetic activity in response to the anabolic growth factor IGF-1. The bench data further show that IGF-1 insensitivity in cartilage without OCT form birefringence was reversible. To show clinical feasibility, OCT was then used arthroscopically in 19 human subjects. Clinical results confirmed that differences to OCT form birefringence observed in ex vivo study were detectable during arthroscopic surgery. More prevalent loss of cartilage OCT form birefringence was observed in cartilage of human subjects in groups more likely to have cartilage degeneration. This series of integrated bench to bedside studies demonstrates translational feasibility to use OCT for clinical studies on whether human cartilage degeneration can be diagnosed early enough for intervention that may delay or prevent the onset of osteoarthritis.     

转化生长因子-β及胰岛素样生长因子-Ⅰ修复关节软骨缺损的实验观察

目的观察转化生长因子-β（transforming growth factor-β,TGF-β）、胰岛素样生长因子-Ⅰ（insulin—like growth factor-Ⅰ,IGF-Ⅰ）对关节软骨缺损修复的作用。方法采用组织工程方法制备骨基质明胶（BMG）软骨细胞移植物。将40只4月龄的新西兰兔随机分为TGF-β组、IGF-Ⅰ组、TGF-β联合IGF-Ⅰ组、空白对照组（前三组为实验组）。各组制备关节软骨缺损模型,实验组兔膝关节腔注射对应等量人重组蛋白,对照组注射等量盐水。术后行组织学观察及免疫组化检测。结果TGF-β联合IGF-Ⅰ组软骨细胞生长较快,术后24周修复的软骨组织HE染色与正常关节软骨一致,软骨细胞呈柱状排列,免疫组化见Ⅱ型胶原染色较深;TGF-β组、IGF-Ⅰ组术后24周部分软骨细胞呈柱状排列,免疫组化见Ⅱ型胶原染色较浅;空白对照组未修复。结论联合应用TGF-β及IGF-Ⅰ可较好促进关节软骨缺损修复,其作用优于两者单独应用。     

The potential of IGF-1 and TGFbeta1 for promoting "adult" articular cartilage repair: an in vitro study

Research into articular cartilage repair, a tissue unable to spontaneously regenerate once injured, has focused on the generation of a biomechanically functional repair tissue with the characteristics of hyaline cartilage. This study was undertaken to provide insight into how to improve ex vivo chondrocyte amplification, without cellular dedifferentiation for cell-based methods of cartilage repair. We investigated the effects of insulin-like growth factor 1 (IGF-1) and transforming growth factor beta 1 (TGFbeta1) on cell proliferation and the de novo synthesis of sulfated glycosaminoglycans and collagen in chondrocytes isolated from skeletally mature bovine articular cartilage, whilst maintaining their chondrocytic phenotype. Here we demonstrate that mature differentiated chondrocytes respond to growth factor stimulation to promote de novo synthesis of matrix macromolecules. Additionally, chondrocytes stimulated with IGF-1 or TGFbeta1 induced receptor expression. We conclude that IGF-1 and TGFbeta1 in addition to autoregulatory effects have differential effects on each other when used in combination. This may be mediated by regulation of receptor expression or endogenous factors; these findings offer further options for improving strategies for repair of cartilage defects.     

Rapid degradation of articular cartilage proteoglycan by neutrophils: Comparison with macrophages and synovial fibroblasts

OBJECTIVE AND DESIGN: To determine and compare the proteoglycan degradative properties of neutrophils, macrophages and synoviocytes in cultures of articular cartilage. MATERIAL OF SUBJECTS: Bovine articular cartilage was aseptically isolated from metacarpopharyngeal joints.Neutrophils and macrophages were isolated from normal human blood and bovine synovial fibroblasts were isolated from explant cultures before being incubated with the cartilage. TREATMENT: Neutrophils, macrophages or synovial fibroblasts (1 x 10(6)-8 x 10(6)) were incubated with 35SO4 labelled cartilage for 2.5-72 h. METHODS: Cartilage degradation was measured as a loss of 35SO4 into the cartilage medium as a percentage of the total labelled proteoglycan in the cartilage slice. Statistical significances were determined using a 2-tailed unpaired Student's t-test. RESULTS: Neutrophils rapidly degraded articular cartilage. After 2.5 hours of culture, neutrophils degraded cartilage proteoglycan up to 28 times more than either macrophages or synovial fibroblasts. CONCLUSIONS: Neutrophils induce rapid damage to articular cartilage proteoglycan, whereas in comparison, macrophages and synovial fibroblasts degrade articular cartilage proteoglycans poorly. These findings indicate that at least under conditions where the influence of cellular-cellular interactions and soluble mediator action are excluded, adhesion of neutrophils to articular cartilage is sufficient to stimulate rapid and marked cartilage degradation compared to the other two cell types.     

Induction of articular cartilage degradation by recombinant interleukin 1 alpha and 1 beta

The purpose of this study was to compare the effects of human recombinant interleukin 1, alpha and beta, on articular cartilage. The effects of rIL-1 alpha and rIL-1 beta on proteoglycan degradation and synthesis following treatment of bovine articular cartilage in serum-free organ culture were quantified. Purified human IL-1 and both rIL-1 alpha and rIL-1 beta induced a two-fold or greater increase in glycosaminoglycan (GAG) release from cultured articular cartilage. Levels or rIL-1 alpha as low as 15 pM induced increased proteoglycan degradation whereas identical levels of rIL-1 beta did not. Killing of the cartilage cells abolished induced GAG release by all forms of IL-1. Analysis of proteoglycan size following IL-1 treatment showed limited degradation of material released into the culture medium or remaining within cartilage. Both forms of recombinant IL-1 inhibited GAG synthesis when continually present in the culture medium. Actinomycin D and cycloheximide inhibited IL-1 dependent cartilage destruction whereas indomethacin did not.     

Mutant WISP3 triggers the phenotype shift of articular chondrocytes by promoting sensitivity to IGF-1 hypothesis of spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA)

This article introduces the hypothesis that mutant WISP3 (Wnt1 inducible secreted protein-3) triggers the phenotype shift of the chondrocytes, especially in the articular chondrocytes, by promoting sensitivity to IGF-1 (insulin-like growth factor 1), and results in chondrocytes apoptosis in SEDT-PA. SEDT-PA is also referred to as progressive pseudorheumatoid dysplasia (PPD), arthropathy progressive pseudorheumatoid of childhood (APPRC). Evidence for the hypothesis is based on the following indications: (1) SEDT-PA is caused by mutations of the WISP3 gene. WISP3 encodes a domain that bears homology to the amino-terminal domain of the insulin-like growth factor binding proteins (IGFBPs). (2) IGF-1 enhances chondrocyte hypertrophy by insulin-like actions. WISP3 can up-regulate the expression of type II collagen. When chondrocytes become hypertrophic, they reduce the expression of types II and IX collagen. (3) The chondrocytes in the normal articular cartilage maintain a stable phenotype. These cells exhibit no mitotic activity, low matrix synthesis and low degradation. But articular chondrocytes could react to certain stimuli such as IGF-1. (4) The loss of WISP3 expression alters the phenotype of the breast epithelium and promotes motility and invasion. The WISP3-deficient cells are extremely sensitive to the growth stimulatory effects of IGF-1. (5) The action of IGF-I is inhibited by IGFBPs, both in articular chondrocytes and in the normal breast epithelium. In conclusion, the mutant WISP3 lose is the function of inhibiting IGF-1 and disturbs the maintenance of a stable phenotype in articular chondrocytes. So, the articular chondrocytes undergo hypertrophic and terminal differentiation apoptosis. The precise mechanism of WISP3 function during postnatal cartilage growth and homeostasis is not clear yet. This hypothesis provides a new clue on the present mechanism study on SEDT-PA. If verified, this new concept may lead to a novel pathogenesis of SEDT-PA.     

Anatomical localization of cartilage degradation markers in a surgically induced rat osteoarthritis model

Osteoarthritis (OA) is a degenerative disease characterized by an irreversible loss of articular cartilage. Although surgically induced animal OA models are commonly used in drug efficacy assessment, degradation of type II collagen, an important component of articular cartilage is not routinely evaluated. Here, the medial meniscectomy surgical model (MMT) in Lewis rats was evaluated for proteoglycan loss with toluidine blue staining and collagen degradation with immunohistochemical staining for a collagen cleavage C-neoepitope, using a novel anti-type II collagen neoepitope antigen (TIINE) antibody. Femorotibial joints were collected for histology at 0 (no surgery), 3, 7, 14, 21, 28, 35, and 42 days postsurgery. Following MMT surgery, the medial tibial articular cartilage had proteoglycan matrix loss by day 3 that reached subchondral bone by days 28-42. Femoral cartilage damage occurred by day 14. TIINE staining was present at basal levels in growth plates and articular cartilage of all joints while all MMT-treated animals had increased intensity and area of staining in erosions that colocalized with proteoglycan loss. The MMT model produces a progressive pattern of cartilage damage resembling human OA lesions, making it useful, when evaluated with cartilage biomarkers, for assessing changes in cartilage degradation.     

结缔组织生长因子与关节软骨的修复

背景：结缔组织生长因子有刺激间充质细胞向软骨细胞分化的潜能,能促进软骨细胞的增殖和分化,可促进关节软骨细胞Ⅱ型胶原及蛋白多糖的表达,与其他生长因子一起在关节软骨修复的过程中发挥着重要作用。目的：重点就结缔组织生长因子的结构,在关节软骨修复中的功能,与其他物质的相互作用方面作一综述。方法：以“connective tissue growth factor,connective tissue growth factor and articular cartilage,articular cartilage damage,articular cartilage repairment”为英文检索词,以“关节软骨损伤”为中文检索词,检索PubMed数据库、中国知网-cnki数据库1980年1月至2014年7月有关关节软骨损伤修复的文献,排除与软骨损伤的修复重建相关性不强、以及内容重复、陈旧的文献。共保留32篇文献进行综述。结果与结论：结缔组织生长因子有刺激间充质细胞向软骨细胞分化的潜能,能促进软骨细胞的增殖、分化和成熟,可维持胞外基质合成以及平衡,可促进关节软骨细胞Ⅱ型胶原及蛋白多糖的表达,与其他生长因子一起在关节软骨修复的过程中发挥着重要作用。结缔组织生长因子是软骨细胞生长、增殖、分化的关键生长因子之一,贯穿软骨修复整个过程。研究发现,骨性关节炎患者的关节软骨细胞对成纤维细胞生长因子1和结缔组织生长因子表达呈现明显相关性增加。结缔组织生长因子通过对关节软骨组织的信号通路及与其他组织内的细胞因子彼此作用对关节软骨细胞及基质发挥作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Effect of construct properties on encapsulated chondrocyte expression of insulin-like growth factor-1

Hydrogels are a promising type of biomaterial for articular cartilage constructs since they have been shown to enable encapsulated chondrocytes to express their predominant phenotypic marker, type II collagen. Endogenously expressed signaling molecules, such as insulin-like growth factor-1 (IGF-1), are also known to facilitate the retention of this chondrocytic phenotype. Recent investigations have attempted to enhance the ability of encapsulated chondrocytes to regenerate cartilage through delivery of exogenous signaling molecules. However, we hypothesize that by altering construct properties, such as cell density and polymer concentration, we can augment the expression of endogenous IGF-1 in chondrocytes. To this end, bovine articular chondrocytes were encapsulated within alginate hydrogels at two different cell densities (25,000 and 100,000 cells/bead) and various alginate concentrations (0.8%, 1.2%, and 2.0% w/v). These parameters were chosen to simultaneously investigate cell-to-cell distance on paracrine signaling and water content on IGF-1 diffusion by chondrocytes. At 1, 4, and 8d, chondrocytes were analyzed for protein and mRNA expression of IGF-1 as well as type II collagen. Results suggest that cell density and alginate concentration at high cell density can significantly affect the endogenous IGF-1 expression by chondrocytes. Therefore, these results indicate that construct properties can impact chondrocyte gene expression and should be considered in order to create a proper engineered articular cartilage construct.     

Modulation of intracellular reactive oxygen species level in chondrocytes by IGF-1, FGF, and TGF-beta1

Growth factors are important in the development, maintenance and repair of cartilage. The principal aim of this study was to test the capacity of three growth factors with established roles in cartilage, namely insulin-like growth factor (IGF)-1, fibroblast growth factor (FGF) and transforming growth factor (TGF)-beta 1, to alter intracellular reactive oxygen species (ROS) levels. Explants of articular cartilage from young, mature, and aged rats were pretreated with IGF-1, FGF, or TGF-beta 1 and intracellular ROS levels were quantified using the free radical sensing probe dihydrorhodamine 123 (DHR 123), confocal microscopy, and densitometric image analysis. Viability of chondrocytes following ROS stress and growth factor treatment was assessed using the live/dead cytotoxicity assay, and the activities of the antioxidant enzymes--catalase (CAT), total superoxide dismutase (SOD), and glutathione peroxidase (GPX)--were measured spectrophotometrically by decay of the substrate from the reaction mixture. The effect of IGF-1 on ROS levels in cultured human chondrocytes also was examined. In rat cartilage, FGF did not significantly affect ROS levels or antioxidant enzyme activity in any age group. TGF-beta1 significantly increased cellular ROS levels in mature and old cartilage whereas in marked contrast, IGF-1 significantly and age-dependently reduced ROS levels. IGF-1 also had a potent antioxidant effect on cultured human chondrocytes. Pretreatment of rat cartilage with IGF-1 significantly enhanced the activity of GPX, without altering the activity of SOD or CAT, and protected chondrocytes against ROS-induced cell death. TGF-beta 1 had no significant effect on the activity of the antioxidant enzymes. Despite promoting ROS production, TGF-beta 1 was not cytotoxic. We concluded that TGF-beta 1 exhibits an acute pro-oxidant effect in cartilage that is not cytotoxic, suggesting a role in physiological cell signalling. In marked contrast, IGF-1 is a potent antioxidant in mature and aged rat and human chondrocytes, protecting cells against ROS-induced cell death probably through the enhancement of the activity of the antioxidant enzyme GPX.     

The role of insulin-like growth factor-I in hyaluronan mediated repair of cultured cartilage explants

Objective and Design:To test whether hyaluronan (HA) restores proteoglycan (PG) in fibronectin fragment (Fn-f) damaged cartilage through activities of insulin-like growth factor 1 (IGF-1). Material:Bovine cartilage explants were studied. Methods:Cartilage PG content was measured by a dye binding assay. PG synthesis was measured by incorporation of radiolabeled sulfate. Release of IGF-1 from explants was measured by ELISA. Results:Higher mass HA forms were more reparative than smaller and enhanced PG synthesis in explants, but not in monolayer cultures. Higher mass HA enhanced PG synthesis and retention of IGF-1 within the explants while IGF-1 binding proteins decreased activities of HA. Conclusions:The reparative effects of HA appear to involve enhanced PG synthesis through IGF-1 and requires cartilage matrix.Received 28 July 2003; returned for revision 3 November 2003; accepted by W. B. van den Berg 29 March 2004     

Changes in collagen fibril network organization and proteoglycan distribution in equine articular cartilage during maturation and growth

The aim of this study was to record growth‐related changes in collagen network organization and proteoglycan distribution in intermittently peak‐loaded and continuously lower‐level‐loaded articular cartilage. Cartilage from the proximal phalangeal bone of the equine metacarpophalangeal joint at birth, at 5, 11 and 18 months, and at 6–10 years of age was collected from two sites. Site 1, at the joint margin, is unloaded at slow gaits but is subjected to high‐intensity loading during athletic activity; site 2 is a continuously but less intensively loaded site in the centre of the joint. The degree of collagen parallelism was determined with quantitative polarized light microscopy and the parallelism index for collagen fibrils was computed from the cartilage surface to the osteochondral junction. Concurrent changes in the proteoglycan distribution were quantified with digital densitometry. We found that the parallelism index increased significantly with age (up to 90%). At birth, site 2 exhibited a more organized collagen network than site 1. In adult horses this situation was reversed. The superficial and intermediate zones exhibited the greatest reorganization of collagen. Site 1 had a higher proteoglycan content than site 2 at birth but here too the situation was reversed in adult horses. We conclude that large changes in joint loading during growth and maturation in the period from birth to adulthood profoundly affect the architecture of the collagen network in equine cartilage. In addition, the distribution and content of proteoglycans are modified significantly by altered joint use. Intermittent peak‐loading with shear seems to induce higher collagen parallelism and a lower proteoglycan content in cartilage than more constant weight‐bearing. Therefore, we hypothesize that the formation of mature articular cartilage with a highly parallel collagen network and relatively low proteoglycan content in the peak‐loaded area of a joint is needed to withstand intermittent stress and shear, whereas a constantly weight‐bearing joint area benefits from lower collagen parallelism and a higher proteoglycan content.     

Structure and regulation of articular cartilage proteoglycan expression]

Beyond aggrecan, the major proteoglycan present in articular cartilage that confers resistance to compressive load and viscoelasticity to the tissue, other proteoglycan families have been described in cartilage. Among them, decorin, biglycan and fibromodulin which belong to the small leucine-rich proteoglycans family bind to matrix components, specially to collagen fibrils and thus regulate fibrillogenesis in cartilage and matrix integrity. These small proteoglycans can also interact with TGF-beta and modulate its bioavailability and stability. The third family is composed by cell surface proteoglycans as syndecans, glypican-1 and betaglycan. These molecules interact with various components of cell environment (growth factors, proteases, matrix components, etc.) and mediate numerous cell functions. Some modifications of one of these proteoglycan expression occur during degenerative pathologies and may lead to alteration of the functional properties of the tissue as well as variations in growth factor bioavailability. These factors are involved in the attempt of cartilage repair initiated by chondrocytes in the early stages of osteoarthritis.     

Influence of cartilage proteoglycans on type II collagen fibrillogenesis

The effects of various proteoglycan samples, isolated from human articular cartilage of different ages, on the rate of the lateral growth phase of the fibril formation of collagen type II were studied by turbidimetry. In general, proteoglycan aggregates accelerate fibrillogenesis, whereas non-aggregating proteoglycans retard this process. The only exception were non-aggregating proteoglycans from very young cartilage, which stimulated the fibril formation strongly. The extent of stimulation by proteoglycans from hip and knee cartilage were compared. The effects of non-aggregating proteoglycans dominate those of aggregated proteoglycans. Chondroitinase ABC digestion of proteoglycan samples did not change the effects on the fibrillogenesis of collagen type II, when these samples were isolated from 18 years-old knee cartilage. The collagen fibril formation was less stimulated in the presence of ABC-ase digested proteoglycan samples from 0-3 month-old knee cartilage, suggesting a primary role for keratan sulphate and a possible influence of chondroitin sulphate when keratan sulphate is not present. Only proteoglycans from very old cartilage were able to reduce the amount of collagen fibrils formed in vitro. Proteoglycans could not be detected bound to the fibril pellet despite the fact that part of the pellet was not dissolvable in acetic acid. It is concluded that proteoglycans may play a regulatory role in collagen type II fibril formation in articular cartilage.     

Influence of antigen-induced arthritis on connective tissue cell metabolism

Loss of hyaline articular cartilage during chronic joint inflammation may be due to enzymatic breakdown of cartilage proteoglycans and inhibition of proteoglycan biosynthesis. In vivo study in the mouse on the influence of antigen-induced arthritis on articular cartilage chondrocyte function revealed that proteoglycan synthesis was severely inhibited during active joint inflammation. In addition, autoradiographs showed that inhibition of chondrocyte synthetic function and chondrocyte death at later stages of the arthritis were most pronounced in the central part of patellar hyaline articular cartilage without pannus tissue being present nearby.     

关节软骨损伤后体外培养软骨细胞的功能变化

背景：关节软骨损伤可以影响软骨细胞功能,诱发创伤性骨关节炎。 目的：观察关节软骨损伤后体外培养的软骨细胞功能的变化。 方法：通过酶消化法分离培养高能量、低能量撞击后和正常兔膝关节透明软骨细胞,观察创伤能量对软骨细胞生存能力的影响;检测软骨细胞合成蛋白多糖和Ⅱ型胶原能力,检测细胞中白细胞介素1β和核转录因子κB mRNA表达水平,检测细胞合成白细胞介素1β和基质金属蛋白酶1的表达。 结果与结论：高能量和低能量关节软骨损伤后,软骨细胞的存活率下降,原代细胞的贴壁细胞数量减少,贴壁时间延长,生长曲线下移,细胞甲苯胺蓝染色异染反应减弱,Ⅱ型胶原免疫组化染色强度减弱,软骨细胞中白细胞介素1β和核转录因子κB mRNA表达水平上升,细胞培养液中白细胞介素1β和基质金属蛋白酶1的质量浓度升高,其中高能量组效果更为显著(P < 0.05)。说明关节软骨损伤后软骨细胞的功能受到影响,受损程度与创伤强度及炎性细胞因子的表达相关。     

Normal human synovial fluid and articular cartilage contain similar intact proteoglycans.

This work, directed to characterization of proteoglycans present in normal human synovial fluid by Western blotting techniques, revealed an intimate relationship of these proteoglycans to those of articular cartilage. Analyses were performed on samples subjected to digestion with chondroitinase ABC, in the presence or absence of keratanase, yielding products containing core proteins with glycosaminoglycan side chain stubs. The proteoglycan core proteins contained epitopes reactive with monoclonal antibodies that distinguish between chondroitin sulfate-4 and chondroitin sulfate-6. Additionally, these products reacted with monoclonal antibody to keratan sulfate when keratanase was omitted from the digestion. The analysis of synovial fluid revealed that the proteoglycan core proteins expressed predominantly the chondroitin sulfate-6 epitope, with expression of the chondroitin sulfate-4 epitope demonstrable only in prepubertal individuals. There was coexpression of both chondroitin sulfate epitopes in all proteoglycan core proteins of prepubertal individuals. Coexpression of chondroitin sulfate and keratan sulfate epitopes occurred in all proteoglycan core proteins. Proteoglycan core proteins had M(r) similar to those obtained from articular cartilage. Hence, in individuals free of joint disease, most proteoglycans seem to be transferred from articular cartilage to the synovial fluid without major alteration in the apparent size of the proteoglycan core protein. Only a minor set of proteoglycan core proteins had no direct articular cartilage equivalent. As this set also contained keratan sulfate, it is likely to be of articular cartilage origin, but probably modified by proteolysis.     

Independent effects of interleukin 1 on proteoglycan synthesis and proteoglycan breakdown of bovine articular cartilagein vitro

We studied the effects of human recombinant interleukin-1 on proteoglycan metabolism of bovine articular cartilage in organ culture. IL-1 was more potent in inhibiting synthesis (IC₅₀ 4 ng/mL) than in stimulating breakdown of proteoglycans (EC₅₀ 200 ng/mL). Inhibition of proteoglycan synthesis began to plateau earlier (2 days) than stimulation of proteoglycan release (4 days). Both effects could be neutralized with a polyclonal anti-IL-1 antibody; however, higher antibody titers were required to block IL-1 effects on proteoglycan synthesis than to neutralize those on proteoglycan release. Chloroquine, but not hydrocortisone, blocked IL-1-mediated proteoglycan breakdown. Both drugs, however, augmented IL-1-induced inhibition of proteoglycan synthesis. Our data suggest that the effects of IL-1 on articular cartilage proteoglycan synthesis and proteoglycan breakdown can be regulated independently.