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BACKGROUND: Cartilage loss is a hallmark of arthritis and follows activation of catabolic processes concomitant with a disruption of anabolic pathways like insulin-like growth factor 1 (IGF-1). We hypothesized that two natural products of South American origin, would limit cartilage degradation by respectively suppressing catabolism and activating local IGF-1 anabolic pathways. One extract, derived from cat's claw (Uncaria guianensis, vincaria), is a well-described inhibitor of NF-kappaB. The other extract, derived from the vegetable Lepidium meyenii (RNI 249), possessed an uncertain mechanism of action but with defined ethnomedical applications for fertility and vitality. METHODS: Human cartilage samples were procured from surgical specimens with consent, and were evaluated either as explants or as primary chondrocytes prepared after enzymatic digestion of cartilage matrix. Assessments included IGF-1 gene expression, IGF-1 production (ELISA), cartilage matrix degradation and nitric oxide (NO) production, under basal conditions and in the presence of IL-1beta. RESULTS: RNI 249 enhanced basal IGF-1 mRNA levels in human chondrocytes by 2.7 fold, an effect that was further enhanced to 3.8 fold by co-administration with vincaria. Enhanced basal IGF-1 production by RNI 249 alone and together with vincaria, was confirmed in both explants and in primary chondrocytes (P < 0.05). As expected, IL-1beta exposure completely silenced IGF-1 production by chondrocytes. However, in the presence of IL-1beta both RNI 249 and vincaria protected IGF-1 production in an additive manner (P < 0.01) with the combination restoring chondrocyte IGF-1 production to normal levels. Cartilage NO production was dramatically enhanced by IL-1beta. Both vincaria and RNI 249 partially attenuated NO production in an additive manner (p < 0.05). IL-1beta - induced degradation of cartilage matrix was quantified as glycosaminoglycan release. Individually RNI 249 or vincaria, prevented this catabolic action of IL-1beta. CONCLUSION: The identification of agents that activate the autocrine production of IGF-1 in cartilage, even in the face of suppressive pro-inflammatory, catabolic cytokines like IL-1beta, represents a novel therapeutic approach to cartilage biology. Chondroprotection associated with prevention of the catabolic events and the potential for sustained anabolic activity with this natural product suggests that it holds significant promise in the treatment of debilitating joint diseases. [ABSTRACT FROM AUTHOR]

Objective: To determine the Dynamin-related protein 1 (DRP1) regulation of mitochondrial fission in chondrocytes under pathological conditions, an area which is underexplored in osteoarthritis pathogenesis., Design: DRP1 protein expression was determined by immunohistochemistry (IHC) or immunofluorescence (IF) staining of cartilage sections. IL-1β-induced DRP1 mRNA expression in chondrocytes was quantified by qPCR and protein expression by immunoblotting. Mitochondrial fragmentation in chondrocytes was visualized by MitoTracker staining or IF staining of mitochondrial marker proteins or by transient expression of mitoDsRed. Mitochondrial reactive oxygen species (ROS) levels were determined by MitoSOX staining. Apoptosis was determined by lactate dehydrogenase (LDH) release assay, Caspase 3/7 activity assay, propidium iodide (PI), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and IF staining of cleaved caspase 3. Cytochrome c release was determined by confocal microscopy. Surgical destabilization of the medial meniscus (DMM) was used to induce osteoarthritis (OA) in mice., Results: Expression of DRP1 and mitochondrial damage was high in human OA cartilage and in the joints of mice subjected to DMM surgery which also showed increased chondrocytes apoptosis. IL-1β-induced mitochondrial network fragmentation and chondrocyte apoptosis via modulation of DRP1 expression and activity and induce apoptosis via Bax-mediated release of Cytochrome c. Pharmacological inhibition of DRP1 activity by Mdivi-1 blocked IL-1β-induced mitochondrial damage and apoptosis in chondrocytes. Additionally, IL-1β-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is crucial for DRP1 activation and induction of mitochondrial network fragmentation in chondrocytes as these were blocked by inhibiting ERK1/2 activation., Conclusions: These findings demonstrate that ERK1/2 is a critical player in DRP1-mediated induction of mitochondrial fission and apoptosis in IL-1β-stimulated chondrocytes., (Copyright © 2021 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: Lysosomes are the major catabolic organelle of the cell and regulate the macromolecular and organelle turnover and programmed cell death. Here, we investigated the lysosome dysfunction in cartilage and its role in chondrocytes apoptosis and the associated mechanism., Design: Lysosomal acidification in Osteoarthritis (OA) and aged cartilage was determined by LysoSensor staining. Lysosomal function in chondrocytes was blocked by siRNA mediated depletion of Lysosomal Associated Membrane Protein 2 (LAMP2) or with lysosome inhibitors. Chondrocyte apoptosis was determined by LDH release, Caspase-3/7 activation, TUNEL and PI uptake assays. Loss of mitochondrial membrane potential (MMP/ΔΨM) and mitochondrial superoxide level was determined by JC-1 and MitoSOX staining, respectively. Colocalization of mitochondria with BCL2 associated X (BAX) and Cytochrome c was determined by immunostaining. Destabilization of medial meniscus (DMM) was performed to induce OA in mice., Results: Lysosomal acidification was found to be significantly decreased in aged mouse and human and mouse OA cartilage which also showed increased chondrocyte apoptosis. Inhibition of lysosomal function resulted in increased oxidative stress, accumulation of dysfunctional mitochondria and apoptosis in chondrocytes in monolayer and in cartilage explant cultures. Depletion of LAMP2 expression or treatment of chondrocytes with lysosomal function inhibitors increased the expression and mitochondrial translocation of BAX leading to Cytochrome c release. Lysosomal dysfunction-induced apoptosis in chondrocytes was not blocked by antioxidants MitoTempo or Diphenyleneiodonium (DPI) but was abrogated by inhibiting BAX., Conclusion: Lysosomal dysfunction induce apoptosis in chondrocytes through BAX-mediated mitochondrial damage and release of Cytochrome c. Our data points to lysosomal function restoration and/or BAX inhibition in chondrocytes as a therapeutic approach for OA., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Mitochondrial function is impaired in osteoarthritis (OA) but its impact on cartilage catabolism is not fully understood. Here, we investigated the molecular mechanism of mitochondrial dysfunction-induced activation of the catabolic response in chondrocytes. Using cartilage slices from normal and OA cartilage, we showed that mitochondrial membrane potential was lower in OA cartilage, and that this was associated with increased production of mitochondrial superoxide and catabolic genes [interleukin 6 (IL-6), COX-2 (also known as PTGS2), MMP-3, -9, -13 and ADAMTS5]. Pharmacological induction of mitochondrial dysfunction in chondrocytes and cartilage explants using carbonyl cyanide 3-chlorophenylhydrazone increased mitochondrial superoxide production and the expression of IL-6, COX-2, MMP-3, -9, -13 and ADAMTS5, and cartilage matrix degradation. Mitochondrial dysfunction-induced expression of catabolic genes was dependent on the JNK (herein referring to the JNK family)/activator protein 1 (AP1) pathway but not the NFκB pathway. Scavenging of mitochondrial superoxide with MitoTEMPO, or pharmacological inhibition of JNK or cFos and cJun, blocked the mitochondrial dysfunction-induced expression of the catabolic genes in chondrocytes. We demonstrate here that mitochondrial dysfunction contributes to OA pathogenesis via JNK/AP1-mediated expression of catabolic genes. Our data shows that AP1 could be used as a therapeutic target for OA management.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Osteoarthritis (OA) is the most prevalent joint degenerative disease leading to irreversible structural and functional changes in the joint and is a major cause of disability and reduced life expectancy in ageing population. Despite the high prevalence of OA, there is no disease modifying drug available for the management of OA. Oxidative stress, a result of an imbalance between the production of reactive oxygen species (ROS) and their clearance by antioxidant defense system, is high in OA cartilage and is a major cause of chronic inflammation. Inflammatory mediators, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are highly upregulated in OA joints and induce ROS production and expression of matrix degrading proteases leading to cartilage extracellular matrix degradation and joint dysfunction. ROS and inflammation are interdependent, each being the target of other and represent ideal target/s for the treatment of OA. Plant polyphenols possess potent antioxidant and anti-inflammatory properties and can inhibit ROS production and inflammation in chondrocytes, cartilage explants and in animal models of OA. The aim of this review is to discuss the chondroprotective effects of polyphenols and modulation of different molecular pathways associated with OA pathogenesis and limitations and future prospects of polyphenols in OA treatment., (Copyright © 2020. Published by Elsevier Masson SAS.)

Ageing is a leading risk factor predisposing cartilage to osteoarthritis. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs (sncRNAs). RNA from young and old chondrocytes from macroscopically normal equine metacarpophalangeal joints was extracted and subjected to small RNA sequencing (RNA-seq). Differential expression analysis was performed in R using package DESeq2. For transfer RNA (tRNA) fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. Selected microRNA (miRNAs or miRs) and small nucleolar RNA (snoRNA) findings were validated using real-time quantitative Polymerase Chain Reaction (qRT-PCR) in an extended cohort of equine chondrocytes. tRNA fragments were further investigated in low- and high-grade OA human cartilage tissue. In total, 83 sncRNAs were differentially expressed between young and old equine chondrocytes, including miRNAs, snoRNAs, small nuclear RNAs (snRNAs), and tRNAs. qRT-PCR analysis confirmed findings. tRNA fragment analysis revealed that tRNA halves (tiRNAs), tiRNA-5035-GluCTC and tiRNA-5031-GluCTC-1 were reduced in both high grade OA human cartilage and old equine chondrocytes. For the first time, we have measured the effect of ageing on the expression of sncRNAs in equine chondrocytes. Changes were detected in a number of different sncRNA species. This study supports a role for sncRNAs in ageing cartilage and their potential involvement in age-related cartilage diseases.

Objectives: Recent studies have shown that tRNA-derived RNA fragments (tRFs) are novel regulators of post-transcriptional gene expression. However, the expression profiles and their role in post-transcriptional gene regulation in chondrocytes is unknown. Here, we determined tRFs expression profile and explored tRF-3003a role in post-transcriptional gene regulation in IL-1β stimulated chondrocytes., Methods: We used qPCR arrays to determine tRNAs and tRFs expression in age- and sex-matched primary human OA chondrocytes and TC28/I2 cells stimulated with IL-1β. Chondrocytes were transfected with tRNA-Cys GCA overexpression plasmid or tRF-3003a mimic and 3'UTR luciferase reporter plasmids of mRNAs harboring predicted tRF target "seed sequence". The AGO-RNA-induced silencing complex (AGO-RISC)-dependent repressive activity of tRF-3003a was determined by siRNA-mediated knockdown of AGO2., Results: IL-1β increased the expression levels of specific tRNAs and of tRF-3003a, a type 3 tRF produced by the cleavage of tRNA-Cys GCA . tRF-3003a "seed sequence" was identified in the 3'UTR of JAK3 mRNA and tRNA-Cys GCA overexpression or transfection of a tRF-3003a mimic in chondrocytes downregulated JAK3 expression and significantly reduced the activity of the 3'UTR reporter. RIP assay showed enrichment of tRF-3003a into AGO2/RISC in IL-1β treated chondrocytes. The suppressive effect of tRF-3003a on JAK3 3'UTR reporter was abrogated with siRNA-mediated depletion of AGO2., Conclusions: We demonstrate that under pathological conditions chondrocytes display perturbations in the expression profile of specific tRNAs and tRFs. Furthermore, a specific tRF namely tRF-3003a can post-transcriptionally regulate JAK3 expression via AGO/RISC formation in chondrocytes. Identification of this novel mechanism may be of value in the design of precision therapies for OA., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: Cytokine expression is tightly regulated posttranscriptionally, but high levels of interleukin-6 (IL-6) in patients with osteoarthritis (OA) indicate that regulatory mechanisms are disrupted in this disorder. The enzyme ZCCHC6 (zinc-finger CCHC domain-containing protein 6; TUT-7) has been implicated in posttranscriptional regulation of inflammatory cytokine expression, but its role in OA pathogenesis is unknown. The present study was undertaken to investigate whether ZCCHC6 directs the expression of IL-6 and influences OA pathogenesis in vivo., Methods: Human and mouse chondrocytes were stimulated with recombinant IL-1β. Expression of ZCCHC6 in human chondrocytes was knocked down using small interfering RNAs. IL-6 transcript stability was determined by actinomycin D chase, and 3'-uridylation of microRNAs was determined by deep sequencing. Zcchc6 -/- mice were produced by gene targeting. OA was surgically induced in the knee joints of mice, and disease severity was scored using a semiquantitative grading system., Results: ZCCHC6 was markedly up-regulated in damaged cartilage from human OA patients and from wild-type mice with surgically induced OA. Overexpression of ZCCHC6 induced the expression of IL-6, and its knockdown reduced IL-6 transcript stability and IL-1β-induced IL-6 expression in chondrocytes. Reintroduction of Zcchc6 in Zcchc6 -/- mouse chondrocytes rescued the IL-1β-induced IL-6 expression. Knockdown of ZCCHC6 reduced the population of micro-RNA 26b (miR-26b) with 3'-uridylation by 60%. Zcchc6 -/- mice with surgically induced OA produced low levels of IL-6 and exhibited reduced cartilage damage and synovitis in the joints., Conclusion: These findings indicate that ZCCHC6 enhances IL-6 expression in chondrocytes through transcript stabilization and by uridylating miR-26b, which abrogates repression of IL-6. Inhibition of IL-6 expression and significantly reduced OA severity in Zcchc6 -/- mice identify ZCCHC6 as a novel therapeutic target to inhibit disease pathogenesis., (© 2018, American College of Rheumatology.)

Objective: Mitochondrial dysfunction, oxidative stress and chondrocyte death are important contributors to the development and pathogenesis of osteoarthritis (OA). In this study, we determined the expression and role of Parkin in the clearance of damaged/dysfunctional mitochondria, regulation of reactive oxygen species (ROS) levels and chondrocyte survival under pathological conditions., Methods: Human chondrocytes were from the unaffected area of knee OA cartilage (n = 12) and were stimulated with IL-1β to mimic pathological conditions. Mitochondrial membrane depolarization and ROS levels were determined using specific dyes and flow cytometry. Autophagy was determined by Western blotting for ATG5, Beclin1, immunofluorescence staining and confocal microscopy. Gene expression was determined by RT-qPCR. siRNA, wild-type and mutant Parkin plasmids were transfected using Amaxa system. Apoptosis was determined by PI staining of chondrocytes and TUNEL assay., Results: IL-1β-stimulated OA chondrocytes showed high levels of ROS generation, mitochondrial membrane damage, accumulation of damaged mitochondria and higher incidence of apoptosis. IL-1β stimulation of chondrocytes with depleted Parkin expression resulted in sustained high levels of ROS, accumulation of damaged/dysfunctional mitochondria and enhanced apoptosis. Parkin translocation to depolarized/damaged mitochondria and recruitment of p62/SQSTM1 was required for the elimination of damaged/dysfunctional mitochondria in IL-1β-stimulated OA chondrocytes. Importantly we demonstrate that Parkin elimination of depolarized/damaged mitochondria required the Parkin ubiquitin ligase activity and resulted in reduced ROS levels and inhibition of apoptosis in OA chondrocytes under pathological conditions., Conclusions: Our data demonstrates that Parkin functions to eliminate depolarized/damaged mitochondria in chondrocytes which is necessary for mitochondrial quality control, regulation of ROS levels and chondrocyte survival under pathological conditions., (Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Background/aims: Butein (2',3,4,4'-Tetrahydroxychalcone), a polyphenol produced by several plants including Butea monoserpma, has been reported to exert potent anti-inflammatory activity but the mechanism remains unknown. In the present work we investigated the mechanism of Butein-mediated suppression of IL-6 expression in normal and human osteoarthritis (OA) chondrocytes under pathological conditions., Methods: Expression level of interleukin-6 (IL-6) protein in OA cartilage was analyzed by immunohistochemistry using a validated antibody. Chondrocytes derived from normal or OA cartilage by enzymatic digestion were pretreated with Butein followed by stimulation with interleukin-1β (IL-1β) and the levels of IL-6 mRNA were quantified by TaqMan assay and the protein levels were measured by Western immunoblotting. Autophagy activation was determined by Western blotting and confocal microscopy. Autophagy was inhibited by siRNA mediated knockdown of ATG5., Results: Expression of IL-6 protein was high in the OA cartilage compared to smooth cartilage from the same patient. OA chondrocytes and cartilage explants stimulated with IL-1β showed high level expression of IL-6 mRNA and protein. Butein increased the phosphorylation of AMPKαThr-172, TSC2Ser-1387 and ULK1Ser-317 and inhibited the phosphorylation of mTORSer-2448 and its downstream target p70S6K and increased autophagy flux that correlated with the suppression of the IL-1β mediated expression of IL-6 in normal and OA chondrocytes. In OA chondrocytes with siRNA-mediated knockdown of ATG5 expression, treatment with Butein failed to activate autophagy and abrogated the suppression of IL-1β induced IL-6 expression., Conclusion: Our findings demonstrate for the first time that Butein activate autophagy in OA chondrocytes via AMPK/TSC2/ULK1/mTOR pathway. Additionally, activation of autophagy was essential to block the IL-1β-induced expression of IL-6 in OA chondrocytes. These data support further studies to evaluate the use of Butein or compounds derived from it for the management of OA., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

MicroRNAs, a group of small, noncoding RNAs that post-transcriptionally regulate gene expression, play important roles in chondrocyte function and in the development of osteoarthritis. We characterized the dynamic repertoire of the chondrocyte miRNome and miRISC-associated miRNome by deep sequencing analysis of primary human chondrocytes. IL-1β treatment showed a modest effect on the expression profile of miRNAs in normal and osteoarthritis (OA) chondrocytes. We found a number of miRNAs that showed a wide range of sequence modifications including nucleotide additions and deletions at 5' and 3' ends; and nucleotide substitutions. miR-27b-3p showed the highest expression and miR-140-3p showed the highest number of sequence variations. AGO2 RIP-Seq analysis revealed the differential recruitment of a subset of expressed miRNAs and isoforms of miRNAs (isomiRs) to the miRISC in response to IL-1β, including miR-146a-5p, miR-155-5p and miR-27b-3p. Together, these results reveal a complex repertoire of miRNAs and isomiRs in primary human chondrocytes. Here, we also show the changes in miRNA composition of the miRISC in primary human chondrocytes in response to IL-1β treatment. These findings will provide an insight to the miRNA-mediated control of gene expression in the pathogenesis of OA.

Wogonin has recently been shown to possess anti-inflammatory and chondroprotective properties and is of considerable interest due to its broad pharmacological activities. The present study highlights that Wogonin binds DNA and exerts chondroprotective effects in vitro. Wogonin showed strong binding with chondrocytes genomic DNA in vitro. The mode of binding of Wogonin to genomic-DNA was assessed by competing Wogonin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. EtBr fluorescence reduced significantly with increase in Wogonin concentration suggesting possible DNA intercalation of Wogonin. Further, in silico molecular docking of Wogonin on mammalian DNA also indicated possible intercalation of Wogonin with DNA. The denaturation and FRET studies revealed that Wogonin prevents denaturation of DNA strands and provide stability to genomic DNA against a variety of chemical denaturants. The cellular uptake study showed that Wogonin enters osteoarthritis chondrocytes and was mainly localized in the nucleus. Wogonin treatment to OA chondrocytes protects the fragmentation of genomic DNA in response to IL-1β as evaluated by DNA ladder and TUNEL assay. Treatment of chondrocytes with Wogonin resulted in significant suppression of IL-1β-mediated induction of ROS. Further, Wogonin exhibited protective potential through potent suppression of extrinsic and intrinsic apoptotic pathways and induction of anti-apoptotic proteins in IL-1β-stimulated osteoarthritis chondrocytes. Our data thus suggest that DNA intercalation by Wogonin may result in the stabilization of genomic DNA leading to protective activity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This article contains data related to the article "Wogonin, a plant derived small molecule exerts potent anti-inflammatory and chondroprotective effects through activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes" (Khan et al. 2017) [1]. The data are related to effects of Wogonin on the viability and IL-1β-stimulated activation of NF-κB and ERK1/2, JNK1/2 and p38 MAPKs in human OA chondrocytes. Gene expression data representing the chondrogenic phenotype and the efficiency of Nrf2 knockdown in monolayer culture of human OA chondrocytes were shown. Moreover, mass spectrometric calibration curve of Wogonin used to quantify the intracellular uptake were also presented. The data are presented in the form of figures and significance of these has been given in the research article (Khan et al. 2017) [1].

Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation and sustained oxidative stress. The root extract of Scutellaria baicalensis (SBE) has been used for the treatment of inflammatory and other diseases. Here, we performed activity-guided HPLC-fractionation of SBE, identified the active ingredient(s) and investigated its chondroprotective potential. We found that the Wogonin containing fraction-4 (F4) was the most potent fraction based on its ability to inhibit ROS production and the suppression of catabolic markers including IL-6, COX-2, iNOS, MMP-3, MMP-9, MMP-13 and ADAMTS-4 in IL-1β-treated OA chondrocytes. OA chondrocytes treated with F4 in the presence of IL-1β showed significantly enhanced expression of anabolic genes ACAN and COL2A1. In an in vitro model of cartilage degradation treatment with F4 inhibited s-GAG release from IL-1β-treated human cartilage explants. The inhibitory effect of F4 was not mediated through the inhibition of MAPKs and NF-κB activation but was mediated through the suppression of c-Fos/AP-1 activity at transcriptional and post transcriptional levels in OA chondrocytes. Purified Wogonin mimicked the effects of F4 in IL-1β-stimulated OA chondrocytes. Our data demonstrates that a Wogonin-rich fraction of SBE exert chondroprotective effects through the suppression of c-Fos/AP-1 expression and activity in OA chondrocytes under pathological conditions.

Osteoarthritis (OA) is the most common whole-joint disease and is characterized by progressive loss of the cartilage matrix. Matrix metallopeptidase-13 (MMP-13) is a highly active and an abundantly expressed protease in OA cartilage and chondrocytes and degrades type II collagen and proteoglycans. We investigated the mechanism of MMP-13 suppression by histone deacetylase inhibitor vorinostat (SAHA). OA chondrocytes were obtained from knee cartilage after enzymatic digestion and treated with IL-1β in the absence or presence of various histone deacetylase inhibitors. Gene expression was quantified using quantitative RT-PCR. Protein expression and chromatin modifications were determined by Western immunoblotting using specific antibodies. The effect of IL-6 on the expression of MMP-13 was determined by treating chondrocytes with recombinant IL-6 or by IL6 knockdown using IL6-specific siRNA. We found that SAHA is a potent suppressor of IL-1β-induced MMP-13, tumor necrosis factor-α, and other catabolic marker expression in OA chondrocytes. Interestingly, SAHA rescued the COL2A1 and ACAN expression in OA chondrocytes that was down-regulated by IL-1β. Of importance is our finding that IL-6-stimulated MMP-13 expression was independent of IL-1β stimulation and was blocked by SAHA, suggesting that SAHA inhibits IL-6 signaling in OA chondrocytes. Taken together, our results suggest that SAHA could be used as a therapeutic agent for the management of OA., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Enhanced and immediate expression of cyclooxygenase-2 (COX-2) mRNA is observed in IL-1β-stimulated OA chondrocytes but the synthesis of protein found significantly delayed. Here we investigated the role of stress granules (SGs), ribonucleoprotein complexes that regulate mRNA translation, in the delayed translation of COX-2 mRNAs in IL-1β-stimulated OA chondrocytes. Stimulation of human chondrocytes with IL-1β activated the stress response genes and the phosphorylation of eIF2α that triggered the assembly of SGs. Using combined immunofluorescence staining of SGs markers and COX-2 protein, RNA fluorescence in situ hybridization and RNA immunoprecipitation, the COX-2 mRNAs were found sequestered in SGs in IL-1β-stimulated OA chondrocytes. No increase in COX-2 protein expression was observed during the persistence of SGs but enhanced expression of COX-2 protein was noted upon clearance of the SGs. Inhibition of SGs clearance blocked COX-2 mRNA translation whereas blocking the assembly of SGs by TIA-1 depletion resulted in rapid and increased production of COX-2 and PGE2. Our findings show for the first time assembly of SGs and sequestration of COX-2 mRNAs in human OA chondrocytes under pathological conditions. Post-transcriptional regulation of COX-2 mRNAs translation by SGs indicates a role in IL-1β-mediated catabolic response that could be therapeutically targeted in OA.

IL-6 is an inflammatory cytokine and its overexpression plays an important role in osteoarthritis (OA) pathogenesis. Expression of IL-6 is regulated post-transcriptionally by MCPIP1. The 3' untranslated region (UTR) of MCPIP1 mRNA harbors a miR-139 'seed sequence', therefore we examined the post-transcriptional regulation of MCPIP1 by miR-139 and its impact on IL-6 expression in OA chondrocytes. Expression of miR-139 was found to be high in the damaged portion of the OA cartilage compared with unaffected cartilage from the same patient and was also induced by IL-1β in OA chondrocytes. Inhibition of miR-139 decreased the expression of IL-6 mRNA by 38% and of secreted IL-6 protein by 40%. However, overexpression of miR-139 increased the expression of IL-6 mRNA by 36% and of secreted IL-6 protein by 56%. These data correlated with altered expression profile of MCPIP1 in transfected chondrocytes. Studies with a luciferase reporter construct confirmed the interactions of miR-139 with the 'seed sequence' located in the 3' UTR of MCPIP mRNA. Furthermore, miR-139 overexpression increased the catabolic gene expression but expression of anabolic markers remained unchanged. Overexpression of miR-139 also induced apoptosis in OA chondrocytes. Importantly, we also discovered that IL-6 is a potent inducer of miR-139 expression in OA chondrocytes. These findings indicate that miR-139 functions as a post-transcriptional regulator of MCPIP1 expression and enhances IL-6 expression, which further upregulates miR-139 expression in OA chondrocytes. These results support our hypothesis that miR-139-mediated downregulation of MCPIP1 promotes IL-6 expression in OA. Therefore, targeting miR-139 could be therapeutically beneficial in the management of OA.

Objective: Enhanced expression of interleukin-6 (IL-6) plays an important role in the pathogenesis of osteoarthritis (OA). Monocyte chemoattractant protein-induced protein 1 (MCPIP-1) is a novel posttranscriptional regulator of IL-6 expression and is targeted by microRNA-9 (miR-9). We investigated the expression of MCPIP-1 in OA cartilage and explored whether targeting of MCPIP-1 by miR-9 contributes to enhanced IL-6 expression in OA., Methods: Gene and protein expression in IL-1β-stimulated human OA chondrocytes/cartilage was determined by TaqMan assay and immunoblotting, respectively. Messenger RNA (mRNA) for MCPIP-1 and IL-6 expression at the single-cell level was analyzed using RNAscope. MCPIP-1 protein interaction with IL-6 mRNA was investigated using RNA immunoprecipitation. Transient transfections were used for the small interfering RNA (siRNA)-mediated knockdown and overexpression of MCPIP-1, its RNase-defective mutant miR-9, or antagomir. The role of signaling pathways was evaluated using small-molecule inhibitors. Binding of miR-9 with the "seed sequence" in the 3'-untranslated region of MCPIP-1 mRNA was investigated using a luciferase reporter assay., Results: MCPIP-1 mRNA expression was low, but expression of miR-9 and IL-6 was high, in damaged OA cartilage. In IL-1β-stimulated OA chondrocytes, the expression of miR-9 and MCPIP-1 was mutually exclusive, and increased expression of miR-9 correlated with reduced MCPIP-1 expression and enhanced IL-6 expression. MCPIP-1 protein directly binds with IL-6 mRNA, and overexpression of wild-type MCPIP-1 destabilized the IL-6 mRNA. MCPIP-1 expression was altered by overexpression or inhibition of miR-9. Transfection with miR-9 mimics inhibited the reporter activity, and mutation of the "seed sequence" abolished the repression of reporter activity., Conclusion: These findings implicate miR-9-mediated suppression of MCPIP-1 in the pathogenesis of OA via up-regulation of IL-6 expression in IL-1β-stimulated human OA chondrocytes., (© 2015, American College of Rheumatology.)

Objective: Hedgehog (HH) signaling has recently been associated with cartilage degradation in osteoarthritis (OA). Because interleukin-1β (IL-1β) has been implicated as a principal instigator of OA, we sought to determine whether IL-1β induces the expression of sonic HH (SHH) and its regulation by microRNAs (miRNAs) in human chondrocytes., Methods: Expression of SHH protein in human OA cartilage and in an animal model of OA was determined by immunohistochemical analysis and immunofluorescence analysis, respectively. Gene and protein expression in IL-1β- or SHH-stimulated OA chondrocytes was determined by TaqMan assays and Western blotting, respectively. The effect of overexpression of miRNA-602 (miR-602) and miR-608 or their antagomirs on SHH expression was evaluated by transient transfection of human chondrocytes and HEK 293 cells. The role of signaling pathways was evaluated using small molecule inhibitors. Binding of miRNAs with the putative seed sequence in SHH messenger RNA (mRNA) was validated using a luciferase reporter assay., Results: Expression of SHH, patched 1, Gli-1, HH-interacting protein, matrix metalloproteinase 13 (MMP-13), and Colα1(X) was high in damaged OA cartilage. In damaged cartilage and in IL-1β-stimulated OA chondrocytes, expression of SHH was inversely correlated with expression of miR-608. Cotransfection of OA chondrocytes with miR-608 or miR-602 mimic inhibited reporter activity, and mutation of the miRNA seed sequences abolished the repression of reporter activity. Overexpression of miR-602 or miR-608 inhibited the expression of SHH mRNA and protein, and this was abrogated by antagomirs. Stimulation with recombinant human SHH protein up-regulated MMP-13 expression, and inhibition of HH signaling blocked MMP-13 expression in OA chondrocytes., Conclusion: MiR-602 and miR-608 are important posttranscription regulators of SHH expression in OA chondrocytes, and their suppression by IL-1β may contribute to the enhanced expression of SHH and MMP-13 in OA., (Copyright © 2015 by the American College of Rheumatology.)

5-Hydroxymethylcytosine (5-hmC) generated by ten-eleven translocation 1-3 (TET1-3) enzymes is an epigenetic mark present in many tissues with different degrees of abundance. IL-1β and TNF-α are the two major cytokines present in arthritic joints that modulate the expression of many genes associated with cartilage degradation in osteoarthritis. In the present study, we investigated the global 5-hmC content, the effects of IL-1β and TNF-α on 5-hmC content, and the expression and activity of TETs and isocitrate dehydrogenases in primary human chondrocytes. The global 5-hmC content was found to be ∼0.1% of the total genome. There was a significant decrease in the levels of 5-hmC and the TET enzyme activity upon treatment of chondrocytes with IL-1β alone or in combination with TNF-α. We observed a dramatic (10-20-fold) decrease in the levels of TET1 mRNA expression and a small increase (2-3-fold) in TET3 expression in chondrocytes stimulated with IL-1β and TNF-α. IL-1β and TNF-α significantly suppressed the activity and expression of IDHs, which correlated with the reduced α-ketoglutarate levels. Whole genome profiling showed an erasure effect of IL-1β and TNF-α, resulting in a significant decrease in hydroxymethylation in a myriad of genes including many genes that are important in chondrocyte physiology. Our data demonstrate that DNA hydroxymethylation is modulated by pro-inflammatory cytokines via suppression of the cytosine hydroxymethylation machinery. These data point to new mechanisms of epigenetic control of gene expression by pro-inflammatory cytokines in human chondrocytes.

Objective: The C-type natriuretic peptide (CNP) signaling pathway is a major contributor to postnatal skeletal growth in humans. This study was undertaken to investigate whether CNP signaling could prevent growth delay and cartilage damage in an animal model of inflammatory arthritis., Methods: We generated transgenic mice that overexpress CNP (B6.SJL-Col2a1-NPPC) in chondrocytes. We introduced the CNP transgene into mice with experimental systemic inflammatory arthritis (K/BxN T cell receptor [TCR]) and determined the effect of CNP overexpression in chondrocytes on the severity of arthritis, cartilage damage, and linear growth. We also examined primary chondrocyte cultures for changes in gene and protein expression resulting from CNP overexpression., Results: K/BxN TCR mice exhibited linear growth delay (P < 0.01) compared to controls, and this growth delay was correlated with the severity of arthritis. Diminished chondrocyte proliferation and matrix production was also seen in K/BxN TCR mice. Compared to non-CNP-transgenic mice, K/BxN TCR mice with overexpressed CNP had milder arthritis, no growth delay, and less cartilage damage. Primary chondrocytes from mice overexpressing CNP were less sensitive to inflammatory cytokines than wild-type mouse chondrocytes., Conclusion: CNP overexpression in chondrocytes can prevent endochondral growth delay and protect against cartilage damage in a mouse model of inflammatory arthritis. Pharmacologic or biologic modulation of the CNP signaling pathway may prevent growth retardation and protect cartilage in patients with inflammatory joint diseases, such as juvenile idiopathic arthritis., (© 2014 The Authors. Arthritis & Rheumatology is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

Objective: In OA, there is enhanced expression of pro-inflammatory cytokines such as IL-1β in the affected joint. Delphinidin, an anthocyanidin found in pigmented fruits and vegetables, has been shown to possess anti-inflammatory and antioxidant properties. In the present study we determined whether delphinidin would inhibit the IL-1β-induced activation of NF-κB in human chondrocytes and determined the mechanism of its action., Methods: PGE2 levels and activation of NF-κB p65 in human OA chondrocytes were determined by ELISA-based assays. Protein expression of cyclo-oxygenase-2 (COX-2) and phosphorylation of kinases was determined by western immunoblotting. Expression level of mRNAs was determined by TaqMan assays., Results: Delphinidin inhibited IL-1β-induced expression of COX-2 and production of PGE2 in human chondrocytes. Delphinidin also inhibited IL-1β-mediated phosphorylation of IL-1 receptor-associated kinase-1(Ser376), phosphorylation of IKKα/β, expression of IKKβ, degradation of IκBα, and activation and nuclear translocation of NF-κB/p65. Phosphorylation of TGF-β-activated kinase 1 was not observed but NF-κB-inducing kinase (NIK) was phosphorylated and phosphorylation of NIK was blocked by delphinidin in IL-1β-treated human chondrocytes., Conclusion: These data identify delphinidin as a novel inhibitor of IL-1β-induced production of cartilage-degrading molecule PGE2 via inhibition of COX-2 expression and provide new insight into the mechanism of its action. Our results also identify inhibition of IRAK1(Ser376) phosphorylation by delphinidin in IL-1β-induced activation of NF-κB in human chondrocytes. Given the important role played by IL-1β-induced NF-κB activation, COX-2 expression and PGE2 production in OA, our results may have important implications for the development of novel therapeutic strategies for the prevention/treatment of OA.

Osteoarthritis (OA) is a progressive degenerative joint disease that has a major impact on joint function and quality of life. Nutraceuticals and dietary supplements derived from herbs have long been used in traditional medicine and there is considerable evidence that nutraceuticals may play an important role in inflammation and joint destruction in OA. We review the biological effects of some medicinal fruits and herbs - pomegranate, green tea, cat's claw, devil's claw, ginger, Indian olibaum, turmeric and ananas - in an attempt to understand the pivotal molecular targets involved in inflammation and the joint destruction process and to summarize their toxicities and efficacy for OA management. So far there is insufficient reliable evidence on the effectiveness of ginger, turmeric and ananas. Pomegranate and green tea only have preclinical evidence of efficacy due to the lack of clinical data. In vivo and clinical studies are required to understand their targets and efficacy in OA. Limited in vitro and in vivo evidence is available for cat's claw and Indian olibaum. More extensive studies are required before long-term controlled trials of whole cat's claw and Indian olibaum extracts, or isolated active compounds, are carried out in patients with OA to determine their long-term efficacy and safety. Devil's claw has not been rigorously tested to determine its antiarthritic potential in in vitro and in vivo models. There is strong clinical evidence of the effectiveness of devil's claw in pain reduction. However, high-quality clinical trials are needed to determine its effectiveness. No serious side effects have been reported for any fruits and herbs. Overall, these studies identify and support the use of nutraceuticals to provide symptomatic relief to patients with OA and to be used as adjunct therapy for OA management. More high-quality trials are needed to provide definitive answers to questions related to their efficacy and safety for OA prevention and/or treatment.

Background: Conventional treatments for the articular diseases are often effective for symptom relief, but can also cause significant side effects and do not slow the progression of the disease. Several natural substances have been shown to be effective at relieving the symptoms of osteoarthritis (OA), and preliminary evidence suggests that some of these compounds may exert a favorable influence on the course of the disease. The objective of this study was to investigate the anti-inflammatory/chondroprotective potential of a Herbal and amino acid mixture containing extract of the Uncaria tomentosa, Boswellia spp., Lepidium meyenii and L-Leucine on the IL-1β-induced production of nitric oxide (NO), glycosaminoglycan (GAG), matrix metalloproteinases (MMPs), aggrecan (ACAN) and type II collagen (COL2A1) in human OA chondrocytes and OA cartilage explants., Methods: Primary OA chondrocytes or OA cartilage explants were pretreated with Herbal-Leucine mixture (HLM, 1-10 μg/ml) and then stimulated with IL-1β (5 ng/ml). Effect of HLM on IL-1β-induced gene expression of iNOS, MMP-9, MMP-13, ACAN and COL2A1 was verified by real time-PCR. Estimation of NO and GAG release in culture supernatant was done using commercially available kits., Results: HLM tested in these in vitro studies was found to be an effective anti-inflammatory agent, as evidenced by strong inhibition of iNOS, MMP-9 and MMP-13 expression and NO production in IL-1β-stimulated OA chondrocytes (p < 0.05). Supporting these gene expression results, IL-1β-induced cartilage matrix breakdown, as evidenced by GAG release from cartilage explants, was also significantly blocked (p < 0.05). Moreover, in the presence of herbal-Leucine mixture (HLM) up-regulation of ACAN and COL2A1 expression in IL-1β-stimulated OA chondrocytes was also noted (p < 0.05). The inhibitory effects of HLM were mediated by inhibiting the activation of nuclear factor (NF)-kB in human OA chondrocytes in presence of IL-1β., Conclusion: Our data suggests that HLM could be chondroprotective and anti-inflammatory agent in arthritis, switching chondrocyte gene expression from catabolic direction towards anabolic and regenerative, and consequently this approach may be potentially useful as a new adjunct therapeutic/preventive agent for OA or injury recovery.

Introduction: Epigallocatechin-3-gallate (EGCG) is a bioactive polyphenol of green tea and exerts potent anti-inflammatory effects by inhibiting signaling events and gene expression. Interleukin-1beta (IL-1β) is the principal cytokine linked to cartilage degradation in osteoarthritis (OA). The objective of this study was to evaluate the global effect of EGCG on IL-1β-induced expression of proteins associated with OA pathogenesis in human chondrocytes., Methods: Primary OA chondrocytes were pretreated with EGCG (10 to 100 uM) and then stimulated with IL-1β (5 ng/ml) for 24 hours. Culture supernatants were incubated with cytokine antibody arrays and immunoreactive proteins (80 proteins) were visualized by enhanced chemiluminiscence. Effect of EGCG on IL-1β-induced expression of 18 selected genes was verified by Real time-PCR and effect on IL-6, IL-8 and tumor necrosis factor-alpha (TNF-α) production was determined using specific ELISAs. Western immunoblotting was used to analyze the effect of EGCG on the interleukin-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF-6) proteins in IL-1β-stimulated chondrocytes. The role of nuclear factor kappa-B (NF-κB) and mitogen activated protein kinases (MAPKs) in the regulation of selected genes and the mechanism involved in EGCG mediated modulation of these genes was determined by using specific inhibitors for NF- κB (MG132) and MAPKs (p38-MAPK, SB202190; JNK-MAPK, SP600125, ERK-MAPK, PD98059)., Results: Out of 80 proteins present on the array, constitutive expression of 14% proteins was altered by EGCG treatment. No significant stimulatory effect was observed on the proteins associated with cartilage anabolic response. Stimulation with IL-1β enhanced the expression of 29 proteins. Expression of all 29 proteins up-regulated by IL-1β was found to be suppressed by EGCG. EGCG also inhibited the expression of the signaling intermediate TRAF-6 at 50 and 100 uM concentrations (P < 0.05). Our results identified several new targets of EGCG, including epithelial neutrophil activating peptide-78 (ENA-78), granulocyte macrophage colony stimulation factor (GM-CSF), growth- related oncogene (GRO), GRO-α, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), MCP-3, macrophage inflammatory protein-1beta (MIP-1β), granulocyte chemotactic protein-2 (GCP-2), MIP-3alpha, interferon-gamma-inducible protein-10 (IP-10), nucleosome assembly protein-2 (NAP-2) and leukemia inhibitory factor (LIF). The inhibitory effects of EGCG were mainly mediated by inhibiting the activation of NF-κB and c-Jun N-terminal Kinase (JNK)-MAPK in human chondrocytes., Conclusions: Our results suggest that the potential of EGCG in OA treatment/prevention may be related to its ability to globally suppress the inflammatory response in human chondrocytes. These results identify additional new targets of EGCG and advocate that EGCG may be a potent chondroprotective agent in OA.

Objective: To investigate whether advanced glycation end products (AGEs) induce the expression of IL-6 and IL-8 through the receptor for AGEs (RAGE)-activated pathways in human OA chondrocytes., Methods: OA chondrocytes were stimulated with AGE-modified BSA (AGE-BSA). Gene expression of IL-6 and IL-8 was quantified by TaqMan assays and the production was determined using ELISAs. Immunoblotting was used to analyse the activation of mitogen-activated protein kinases (MAPKs) and the degradation of IκBα. Activation of NF-κB was determined using an ELISA. Pharmacological studies to elucidate the involved pathways were executed using transfection with small interfering RNAs (siRNAs), inhibitors of MAPKs and NF-κB., Results: AGE-BSA induced the expression of IL-6 and IL-8 in OA chondrocytes, which was inhibited by pre-treatment with soluble RAGE (sRAGE) or RAGE knockdown by siRNAs. Treatment with SB202190 (p38-MAPK inhibitor) or PD98059 (ERK inhibitor) inhibited AGE-BSA-induced IL-6 and IL-8 expression. However, SP600125 (JNK inhibitor) had no effect on AGE-BSA-induced IL-6 expression but inhibited the expression of IL-8. Treatment with NF-κB inhibitors suppressed AGE-BSA-induced IL-6 and IL-8 expression., Conclusions: This is the first study to demonstrate that AGEs induce the expression of IL-6 and IL-8 in OA chondrocytes. A novel finding of our studies is that in OA chondrocytes, AGE-BSA-induced expression of IL-6, but not of IL-8, was independent of the JNK pathway. Activation of NF-κB was an absolute requirement for both IL-6 and IL-8 expression. These results demonstrate that AGE-BSA-induced expression of IL-6 and IL-8 via RAGE is mediated through different MAPK signalling pathways in OA and possibly in other degenerative diseases.

Objective: Aberrant posttranscriptional regulation of matrix metalloproteinases (MMPs) by microRNA has emerged as an important factor in human diseases. The aim of this study was to determine whether the expression of MMP-13 in human osteoarthritis (OA) chondrocytes is regulated by microRNA., Methods: Chondrocytes were stimulated with interleukin-1beta (IL-1beta) in vitro. Total RNA was prepared using TRIzol reagent. Polymerase chain reaction (PCR)-based arrays were used to determine the expression profile of 352 human microRNA. Gene expression was quantified using TaqMan assays, and microRNA targets were identified using bioinformatics. Transfection with reporter construct and microRNA mimic was used to verify suppression of target messenger RNA (mRNA). Gene expression of argonaute and Dicer was determined by reverse transcription-PCR, and expression of protein was determined by immunoblotting. The role of activated MAP kinases (MAPKs) and NF-kappaB was evaluated using specific inhibitors., Results: In IL-1beta-stimulated OA chondrocytes, 42 microRNA were down-regulated, 2 microRNA were up-regulated, and the expression of 308 microRNA remained unchanged. In silico analysis identified a sequence in the 3'-untranslated region (3'-UTR) of MMP-13 mRNA complementary to the seed sequence of microRNA-27b (miR-27b). Increased expression of MMP-13 correlated with down-regulation of miR-27b. Overexpression of miR-27b suppressed the activity of a reporter construct containing the 3'-UTR of human MMP-13 mRNA and inhibited the IL-1beta-induced expression of MMP-13 protein in chondrocytes. NF-kappaB and MAPK activation down-regulated the expression of miR-27b., Conclusion: Our data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1beta-induced activation of signal transduction pathways associated with the expression of MMP-13 down-regulated the expression of miR-27b. Thus, miR-27b may play a role in regulating the expression of MMP-13 in human chondrocytes.

Introduction: Pomegranate has been revered throughout history for its medicinal properties. p38-MAPK is a major signal-transducing pathway in osteoarthritis (OA) and its activation by interleukin-1β (IL-1β) plays a critical role in the expression and production of several mediators of cartilage catabolism in OA. In this study we determined the effect of a standardized pomegranate extract (PE) on the IL-1β-induced activation of MKK3/6, p38-MAPK isoforms and the activation of transcription factor RUNX-2 in primary human OA chondrocytes., Methods: Human chondrocytes were derived from OA cartilage by enzymatic digestion, treated with PE and then stimulated with IL-1β. Gene expression of p38-MAPK isoforms was measured by RT-PCR. Western immunoblotting was used to analyze the activation of MAPKs. Immunoprecipitation was used to determine the activation of p38-MAPK isoforms. DNA binding activity of RUNX-2 was determined using a highly sensitive and specific ELISA. Pharmacological studies to elucidate the involved pathways were executed using transfection with siRNAs., Results: Human OA chondrocytes expressed p38-MAPK isoforms p38α, -γ and -δ, but not p38β. IL-1β enhances the phosphorylation of the p38α-MAPK and p38γ-MAPK isoforms but not of p38δ-MAPK isoform in human OA chondrocytes. Activation of p38-MAPK in human OA chondrocytes was preferentially mediated via activation of MKK3. In addition, we also demonstrate that polyphenol rich PE inhibited the IL-1β-induced activation of MKK3, p38α-MAPK isoform and DNA binding activity of the transcription factor RUNX-2., Conclusions: Our results provide an important insight into the molecular basis of the reported cartilage protective and arthritis inhibitory effects of pomegranate extract. These novel pharmacological actions of PE on IL-1β stimulated human OA chondrocytes impart a new suggestion that PE or PE-derived compounds may be developed as MKK and p38-MAPK inhibitors for the treatment of OA and other degenerative/inflammatory diseases.

Background: Mast cells and basophils are multifunctional effector cells and contain plentiful secretary granules in their cytoplasm. These cell types are involved in several inflammatory and immune events and are known to produce an array of mediators including a broad spectrum of cytokines. Pomegranate fruit is rich in anthocyanins and hydrolysable tannins; a group of polyphenolic compounds shown to be potent antioxidant with anti-inflammatory activity. However, no studies have been undertaken to investigate whether a polyphenol-rich pomegranate fruit extract (POMx) inhibits the inflammatory activity of activated human mast cells and basophils. The aim of this study was to examine whether POMx modulates inflammatory reactions using human basophilic cell line KU812., Methods: KU812 cells were stimulated with phorbol-12-myristate 13-acetate plus calcium inophore A23187 (PMACI). The inhibitory effect of POMx on pro-inflammatory cytokine gene expression and production by stimulated KU812 cells was measured by quantitative RT-PCR, and cytokine-specific ELISA assays, respectively. Western blotting was used to analyze the effect of POMx on the activation of mitogen-activated protein kinases (MAPKs), and the nuclear factor (NF)-kappaB in PMACI stimulated KU812 cells. Effect on the activity of NF-kappaB was determined using Luciferase reporter assay. Significance of differences from control values were analyzed by means of standard statistical methods., Results: POMx significantly decreased PMACI stimulated inflammatory gene expression and production of interleukin (IL)-6 and IL-8 in KU812 cells. The inhibitory effect of POMx on the pro-inflammatory cytokines was MAPK subgroups c-jun N-terminal kinase (JNK)- and extracellular-regulated kinase (ERK) dependent. In addition, POMx suppressed the NF-kappaB activation induced by PMACI by inhibiting IkappaB-degradation in human basophil cells. POMx also suppressed the powerful induction of NF-kappaB promoter-mediated luciferase activity in transiently transfected KU812 cells., Conclusion: These novel pharmacological actions of POMx provide new suggestion that POMx or POMx-derived compounds may be of therapeutic use for the treatment of inflammatory diseases by suppressing mast cells/basophils activation.

Introduction: The major risk factor for osteoarthritis (OA) is aging, but the mechanisms underlying this risk are only partly understood. Age-related accumulation of advanced glycation end products (AGEs) can activate chondrocytes and induce the production of proinflammatory cytokines and matrix metalloproteinases (MMPs). In the present study, we examined the effect of epigallocatechin-3-gallate (EGCG) on AGE-modified-BSA (AGE-BSA)-induced activation and production of TNFalpha and MMP-13 in human OA chondrocytes., Methods: Human chondrocytes were derived from OA cartilage by enzymatic digestion and stimulated with in vitro-generated AGE-BSA. Gene expression of TNFalpha and MMP-13 was measured by quantitative RT-PCR. TNFalpha protein in culture medium was determined using cytokine-specific ELISA. Western immunoblotting was used to analyze the MMP-13 production in the culture medium, phosphorylation of mitogen-activated protein kinases (MAPKs), and the activation of NF-kappaB. DNA binding activity of NF-kappaB p65 was determined using a highly sensitive and specific ELISA. IkappaB kinase (IKK) activity was determined using an in vitro kinase activity assay. MMP-13 activity in the culture medium was assayed by gelatin zymography., Results: EGCG significantly decreased AGE-stimulated gene expression and production of TNFalpha and MMP-13 in human chondrocytes. The inhibitory effect of EGCG on the AGE-BSA-induced expression of TNFalpha and MMP-13 was mediated at least in part via suppression of p38-MAPK and JNK activation. In addition, EGCG inhibited the phosphorylating activity of IKKbeta kinase in an in vitro activity assay and EGCG inhibited the AGE-mediated activation and DNA binding activity of NF-kappaB by suppressing the degradation of its inhibitory protein IkappaBalpha in the cytoplasm., Conclusions: These novel pharmacological actions of EGCG on AGE-BSA-stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG-derived compounds may inhibit cartilage degradation by suppressing AGE-mediated activation and the catabolic response in human chondrocytes.

Several recent studies have documented that supplementation with pomegranate fruit extract inhibits inflammatory symptoms in vivo. However, the molecular basis of the observed effects has not been fully revealed. Although previous studies have documented the inhibition of nitric oxide and cyclooxygenase (COX) activity in vitro by plant and fruit extracts added directly into the culture medium but whether concentrations of bioactive compounds sufficient enough to exert such inhibitory effects in vivo can be achieved through oral consumption has not been reported. In the present study we determined the effect of rabbit plasma obtained after ingestion of a polyphenol rich extract of pomegranate fruit (PFE) on COX enzyme activity ex vivo and the IL-1beta-induced production of NO and PGE2 in chondrocytes in vitro. Plasma samples collected before and 2 hr after supplementation with PFE were tested. Plasma samples collected after oral ingestion of PFE were found to inhibit the IL-1beta-induced PGE2 and NO production in chondrocytes. These same plasma samples also inhibited both COX-1 and COX-2 enzyme activity ex vivo but the effect was more pronounced on the enzyme activity of COX-2 enzyme. Taken together these results provide additional evidence of the bioavailability and bioactivity of compounds present in pomegranate fruit after oral ingestion. Furthermore, these studies suggest that PFE-derived bioavailable compounds may exert an anti-inflammatory effect by inhibiting the inflammatory cytokine-induced production of PGE2 and NO in vivo.

Objective: Expression of matrix metalloproteinase 9 (MMP-9) is up-regulated in osteoarthritis (OA) and usually presents as multiple bands when synovial fluid (SF) from OA patients is analyzed by zymography. Among these bands is an approximately 125-130-kd band for high molecular weight (HMW) gelatinase, which has not been characterized. This study was undertaken to characterize the HMW MMP activity in OA SF., Methods: MMP activity in OA SF was determined by gelatin zymography. Recombinant MMPs were used to identify MMP activity on the zymogram. Western immunoblotting, immunoprecipitation, and immunodepletion analyses were performed using antibodies specific for human MMP-9 and human neutrophil gelatinase-associated lipocalin (NGAL). Human cartilage matrix degradation was determined by dimethylmethylene blue assay., Results: Zymographic analysis showed that the HMW gelatinase in OA SF comigrated with a purified NGAL-MMP-9 complex. Results of Western immunoblotting showed that the HMW gelatinase was also recognized by antibodies specific for human NGAL or human MMP-9. These same antibodies also immunoprecipitated the HMW gelatinase activity from OA SF. The NGAL-MMP-9 complex was reconstituted in vitro in gelatinase buffer. In the presence of NGAL, MMP-9 activity was stabilized; in the absence of NGAL, rapid loss of MMP-9 activity occurred. MMP-9-mediated release of cartilage matrix proteoglycans was significantly higher in the presence of NGAL (P < 0.05)., Conclusion: Our findings demonstrate that the HMW gelatinase activity in OA SF represents a complex of NGAL and MMP-9. The ability of NGAL to protect MMP-9 activity is relevant to cartilage matrix degradation in OA and may represent an important mechanism by which NGAL may contribute to the loss of cartilage matrix proteins in OA.

Background: The Amazonian medicinal plant Sangre de grado (Croton palanostigma) has traditional applications for the treatment of wound healing and inflammation. We sought to characterize two extracts (progrado and zangrado) in terms of safety and oligomeric proanthocyanidin chain length. Additionally progrado was evaluated for antioxidant activity and possible chondroprotective actions., Methods: Acute oral safety and toxicity was tested in rats according under OECD protocol number 420. The profile of proanthocyanidin oligomers was determined by HPLC and progrado's antioxidant activity quantified by the ORAC, NORAC and HORAC assays. Human cartilage explants, obtained from surgical specimens, were used to assess chondroproteciton with activity related to direct inhibitory effects on human matrix metalloproteinase (MMP, gelatinolytic) activity using synovial fluid and chondrocytes activated with IL-1beta (10 ng/ml). Additionally, progrado (2-10 mug/ml) was tested for its ability to maintain optimal IGF-1 transcription and translation in cartilage explants and cultured chondrocytes., Results: Both progrado and zangrado at doses up to 2000 mg/kg (po) displayed no evidence of toxicity. Oligomeric proanthocyanidin content was high for both progrado (158 mg/kg) and zangrado (124 mg/kg), with zangrado almost entirely composed of short oligomers (<6 mer), whereas the majority of oligomers in progrado exceeded 10 mers. Progrado was a remarkably potent antioxidant in the standardized tests ORAC, NORAC and HORAC. Progrado was exceptionally effective in reducing both basal and IL-1beta induced glycosaminoglycan release from human cartilage explants at concentrations that also directly blocked the gelatinolytic activity of MMP-2 and MMP-9. Progrado prevented IL-1beta induced suppression of IGF-1 production from human cartilage explants as well as stimulating basal IGF-1 production (P < 0.05). Comparable changes in IGF-1 gene expression were noted in cultured human chondrocytes., Conclusion: Progrado has a promising safety profile, significant chondroprotective and antioxidant actions, directly inhibits MMP activity and promotes the production of the cartilage repair factor, IGF-1. This suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation.

Osteoarthritis (OA) of the knee and hip is a debilitating disease affecting more women than men and the risk of developing OA increases precipitously with aging. Rheumatoid arthritis (RA), the most common form of inflammatory joint diseases, is a disease of unknown etiology and affects approximately 1% of the population worldwide, and unlike OA, generally involves many joints because of the systemic nature of the disease. Non-steroidal anti-inflammatory drugs (NSAIDs) are the first drugs of choice for the symptomatic treatment of both OA and RA. Because of the risks associated with the use of NSAIDs and other limitations, the use of alternative therapies, such as acupuncture and medicinal herbs, is on the rise and according to reports approximately 60-90% of dissatisfied arthritis patients are likely to seek the option of complementary and alternative medicine (CAM). This paper reviews the efficacy of some of the common herbs that have a history of human use and their anti-inflammatory or antiarthritic properties have been evaluated in animal models of inflammatory arthritis, in studies employing well defined and widely accepted in vitro models that use human chondrocytes/cartilage explants or in clinical trials. Available data suggests that the extracts of most of these herbs or compounds derived from them may provide a safe and effective adjunctive therapeutic approach for the treatment of OA and RA. This, in turn, argues for trials to establish efficacy and optimum dosage of these compounds for treating human inflammatory and degenerative joint diseases.

Interleukin (IL)-1beta induces the expression of matrix metalloproteinases (MMPs) implicated in cartilage resorption and joint degradation in osteoarthritis (OA). Pomegranate fruit extract (PFE) was recently shown to exert anti-inflammatory effects in different disease models. However, no studies have been undertaken to investigate whether PFE constituents protect articular cartilage. In the present studies, OA chondrocytes or cartilage explants were pretreated with PFE and then stimulated with IL-1beta at different time points in vitro. The amounts of proteoglycan released were measured by a colorimetric assay. The expression of MMPs, phosphorylation of the inhibitor of kappaBalpha (IkappaBalpha) and mitogen-activated protein kinases (MAPKs) was determined by Western immunoblotting. Expression of mRNA was quantified by real-time PCR. MAPK enzyme activity was assayed by in vitro kinase assay. Activation of nuclear factor-kappaB (NF-kappaB) was determined by electrophoretic mobility shift assay. PFE inhibited the IL-1beta-induced proteoglycan breakdown in cartilage explants in vitro. At the cellular level, PFE (6.25-25 mg/L) inhibited the IL-1beta-induced expression of MMP-1, -3, and -13 protein in the medium (P < 0.05) and this was associated with the inhibition of mRNA expression. IL-1beta-induced phosphorylation of p38-MAPK, but not that of c-Jun-N-terminal kinase or extracellular regulated kinase, was most susceptible to inhibition by low doses of PFE, and the addition of PFE blocked the activity of p38-MAPK in a kinase activity assay. PFE also inhibited the IL-1beta-induced phosphorylation of IkappaBalpha and the DNA binding activity of the transcription factor NF-kappaB in OA chondrocytes. Taken together, these novel results indicate that PFE or compounds derived from it may inhibit cartilage degradation in OA and may also be a useful nutritive supplement for maintaining joint integrity and function.

The inducible IkappaB kinase (IKKi/IKKepsilon) is a recently described serine-threonine kinase that activates the transcription factors NFkappaB, interferon regulatory factor-3 (IRF3) and CCAAA/enhancer-binding protein (C/EBPdelta). Several inflammatory agents have been shown to induce the expression of the IKKi gene in macrophages and other cell types but the mechanism is unknown. We have found that the IKKi expression was constitutive in human chondrocytes from OA cartilage and a human chondrocytic cell line C28/I2 but was up-regulated by the inflammatory cytokines TNFalpha or IL-1betain an NFkappaB-dependent manner. To understand the constitutive and inducible expression of the IKKi gene we localized the transcription start site (TSS), cloned and sequenced a 2 kb genomic DNA fragment 5' of the TSS and characterized the putative promoter region (PPR), and identified the motifs therein that are required for basal and cytokine-induced IKKi gene promoter activity. We found that IKKi core promoter was TATA-less and by using PCR generated deletion mutants of the PPR we found that the cis-elements responsible for basal transcriptional activity were located between -51 and -100 bp upstream of the TSS while the cytokine response elements were located distally between -501 and -1000 bp upstream of the TSS. The DNA region containing the cytokine response elements had two kappaB sites as the most relevant regulatory motifs. The results of site-directed mutagenesis revealed that the kappaB site located between -833 and -847 bp upstream of the TSS was biologically functional and required for cytokine-induced IKKi promoter activity in human chondrocytes and HeLa cells. The silence of the other kappaB site (-816/-802) was positional, rather than sequence-specific. Over-expression of NFkappaB p65 mimics the TNFalpha-induced activation of the IKKi promoter. Also the gel shift assay suggested that NFkappaB p65 is responsible for activation of the IKKi promoter. These data for the first time characterize the promoter region and provide further insights into the transcriptional regulation of IKKi in human chondrocytes and other cell types.

Objective: The proinflammatory cytokine interleukin-1beta (IL-1beta) induces the production of high levels of nitric oxide (NO) in human chondrocytes. Green tea (Camellia sinensis) polyphenols are potent antiinflammatory agents and have been shown to inhibit NO production in tumor cell lines. In the present study, we examined the effect of epigallocatechin-3-gallate (EGCG), a green tea polyphenol, on IL-1beta-induced production of NO in primary human osteoarthritis (OA) chondrocytes., Methods: Human chondrocytes were derived from OA cartilage and were treated with EGCG (100 microM) and IL-1beta (2 ng/ml) for different periods, and inducible nitric oxide synthase (iNOS) messenger RNA and protein expression was determined by real-time quantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively. Production of NO was determined as nitrite in culture supernatant. Activation and translocation of nuclear factor kappaB (NF-kappaB), levels of inhibitor of nuclear factor kappaB (IkappaB), and NF-kappaB DNA binding activity were determined by Western blotting and a highly sensitive and specific enzyme-linked immunosorbent assay. Activity of IkappaB kinase was determined using in vitro kinase assay., Results: Human chondrocytes cotreated with EGCG produced significantly less NO compared with chondrocytes stimulated with IL-1beta alone (P < 0.005). The inhibition of NO production correlated with the suppression of induction and expression of NF-kappaB-dependent gene iNOS. EGCG inhibited the activation and translocation of NF-kappaB to the nucleus by suppressing the degradation of its inhibitory protein IkappaBalpha in the cytoplasm., Conclusion: Our results indicate that EGCG inhibits the IL-1beta-induced production of NO in human chondrocytes by interfering with the activation of NF-kappaB through a novel mechanism. Our data further suggest that EGCG may be a therapeutically effective inhibitor of IL-1beta-induced inflammatory effects that are dependent on NF-kappaB activation in human OA chondrocytes.

CD6, a 130-kDa surface glycoprotein, is expressed primarily on cells of T lineage. A co-stimulatory role for CD6 in mature T cells has been shown, but the function of CD6 during thymocyte development is unknown. Since CD6 ligands are expressed on thymic epithelium, their interactions with CD6 could be important in thymic selection. In this report we show that CD6 is developmentally regulated in human and mouse thymocytes, and further demonstrate that increase in the level of CD6 expression correlates with expression of the selection marker CD69. We also show that activation via CD2 induces CD6 expression on mature human thymocytes and on a subset of immature human thymocytes that are resistant to apoptosis. In human and mouse thymocytes that express heterogeneous TCR, CD6 increases occur as double-positive thymocytes are selected to a single-positive stage. In contrast, in thymocytes from TCR transgenic mice, CD6 is barely increased following selection, suggesting that as functional avidity increases, requirements for CD6-dependent co-stimulation decrease. Taken together, these results indicate that during thymic development CD6-dependent signals may contribute both to thymocyte survival, and to the overall functional avidity of selection in both man and mouse.

Objective: To determine the expression profile of protein kinase (PK) and protein tyrosine kinase (PTK) genes in human primary osteoarthritis (OA) chondrocytes and to compare it with that of immortalized human chondrocytes T/C 28a4 with a view to learning whether T/C 28a4 cells can be used for elucidating signal transduction pathways in human chondrocytes., Design: We used the Atlas Human cDNA Array and a method based on PCR with degenerate primers to analyse the expression profile of protein kinase genes in primary human OA chondrocytes and compared it with that of immortalized human chondrocyte cell line T/C 28a4 using RT-PCR and Western blotting., Results: A total of 21 PTK genes were identified and several of these have never been shown to be expressed in human OA chondrocytes. Comparative expression analysis of some selected kinase genes showed that the mRNA expression pattern of many protein kinase genes in OA chondrocytes was identical to that of T/C 28a4 cells. However, there were differences in the level of protein expression of selected protein kinases in these cells. For example, mRNA expression of the novel kinase HCK was detected in OA chondrocytes and in the cell lines analysed but by Western blotting HCK protein was not detected in OA chondrocytes. In these studies, we also identified a novel mutant form of the discoidin domain receptor 2 (DDR2) transcript from chondrocyte-like cell line HTB-94., Conclusions: Our results provide novel information about protein kinase gene expression in OA chondrocytes and indicate that the transformed chondrocyte cell line T/C 28a4 may be suitable for elucidating signal transduction pathways in chondrocytes and to investigate how they regulate chondrocyte function in inflammatory and degenerative joint diseases., (Copyright 2001 OsteoArthritis Research Society International.)

Objective: Collagen induced arthritis (CIA) in mice is mediated by synergistic T cell and humoral immune responses specific for type II collagen (CII). We have previously shown that in arthritic joints of BUB mice (TCR Vbetaa, H-2q) the TCR repertoire is enrichedfor Vbeta10 expressing T cells, and that immunization with a Vbeta10 peptide (Vbeta10p) prevents the phenotypic expression of disease. The objective of the present study was to understand how immunization with a synthetic TCR Vbeta peptide affected the development of the pathogenic CII-specific immune response in BUB mice., Methods: Arthritic and protected animals were tested for Vbeta10p- and CII-specific cytokine production by a highly specific and sensitive ELISA spot assay, andfor CII-specific antibody production by standard ELISA. In adoptive transfer experiments, Vbeta10p-specific LN cells (INF-gamma producing) were injected into naive mice prior to immunization with type-II collagen/CFA., Results: Immune cells from arthritic animals produced IFN-gamma and IL-2, without IL-4 and IL-5 in response to CII and an immunodominant epitope, A2, derivedfrom CII. Serum from these mice contained anti-CII antibodies of both IgGI and IgG2a subtypes. Our results show for thefirst time that immunization with Vbeta10p resulted in Vbeta10p-specific IFN-gamma and IL-2 production that was restricted to the CD4+ T cell subset. Emergence of this Vbeta10p-specific immune response was associated with a dramatic decrease in the frequency of CII and A2-specific, cytokine producing T cells in arthritis protected mice. Protective immunity was cell mediated and could be adoptively transferred. In contrast, the protective immunization had only a marginal effect on the anti-CII antibody response indicating that the CII specific humoral immune response was not significantly affected., Conclusion: Immunization with TCR Vbeta10p leads to expansion of a population of Vbeta10p- specific CD4+ Tcells. This anti-TCR Vbeta10p specific type 1 cytokine producing immune response was protective in adoptive transfer studies and appears to inhibit the expansion of the pathogenic anti-CII cellular immunity. Additionally, the anti-TCR Vbeta10p-specific cellular immune response was mediated by CD4+ T cells and these T cells did not produce IL-4 or IL-5. Thus, our results suggest that protection against CIA in mice immunized with synthetic TCR Vbeta10p was achieved by a specific down-regulation of the CII-specific Thl type cellular immune response and not via immune deviation.

Tumour necrosis factor alpha (TNF-alpha) is a cytokine with pleiotropic effects on cells ranging from proliferation to apoptosis. These biological effects of TNF-alpha are believed to be elicited by the induction or enhancement of the expression of TNF-alpha responsive genes in the target cells. TNF-alpha is pro-inflammatory and a principal mediator in the pathogenesis of arthritis. The activation of an inflammatory cascade by TNF-alpha in arthritis results in the degradation of cartilage, joint destruction and loss of function. Because TNF-alpha is an important mediator in the pathogenesis of arthritis, the present study addresses the identification of novel TNF-alpha responsive genes in HTB-94 cell line which is of human origin and maintains a chondrocytic phenotype. The three identified cDNAs were previously not known to be induced or upregulated by TNF-alpha in chondrocytes or cells of chondrocytic lineage. One of the identified cDNAs had sequence similarity to human hydroxyl lyase mRNA (PLOD), an enzyme involved in collagen biosynthesis and its metabolism; the second cDNA had sequence similarity to the human cytoplasmic anti-proteinase-2 mRNA (CAP-2), a member of a group of proteins shown to be associated with protecting cells from TNF-alpha-induced apoptosis; and the third cDNA had sequence similarity to a dual specificity kinase, TTK, which is associated with cell proliferation. Relative gene expression level analysis by PCR and by Northern blotting revealed that treatment with TNF-alpha enhanced the expression of PLOD, CAP2 and TTK transcripts which confirmed the results obtained with display gels. Furthermore, TTK mRNA expression was also induced in human articular chondrocytes treated with TNF-alpha but not in untreated chondrocytes. Our results suggest that these genes may play a role in chondrocytic responses to TNF-alpha-mediated stimuli affecting the cartilage homeostasis., (Copyright 2000 Academic Press.)

Identification of common dietary substances capable of affording protection or modulating the onset and severity of arthritis may have important human health implications. An antioxidant-rich polyphenolic fraction isolated from green tea (green tea polyphenols, GTPs) has been shown to possess anti-inflammatory and anticarcinogenic properties in experimental animals. In this study we determined the effect of oral consumption of GTP on collagen-induced arthritis in mice. In three independent experiments mice given GTP in water exhibited significantly reduced incidence of arthritis (33% to 50%) as compared with mice not given GTP in water (84% to 100%). The arthritis index also was significantly lower in GTP-fed animals. Western blot analysis showed a marked reduction in the expression of inflammatory mediators such as cyclooxygenase 2, IFN-gamma, and tumor necrosis factor alpha in arthritic joints of GTP-fed mice. Histologic and immunohistochemical analysis of the arthritic joints in GTP-fed mice demonstrated only marginal joint infiltration by IFN-gamma and tumor necrosis factor alpha-producing cells as opposed to massive cellular infiltration and fully developed pannus in arthritic joints of non-GTP-fed mice. The neutral endopeptidase activity was approximately 7-fold higher in arthritic joints of non-GTP-fed mice in comparison to nonarthritic joints of unimmunized mice whereas it was only 2-fold higher in the arthritic joints of GTP-fed mice. Additionally, total IgG and type II collagen-specific IgG levels were lower in serum and arthritic joints of GTP-fed mice. Taken together our studies suggest that a polyphenolic fraction from green tea that is rich in antioxidants may be useful in the prevention of onset and severity of arthritis.

Alloreactive T lymphocytes can respond to foreign MHC complexed with foreign peptides through the direct pathway of allorecognition and can additionally recognize allopeptides expressed in the context of recipient (self) MHC through the indirect pathway. To better elucidate how indirect pathway-responsive CD4(+) T cells mediate allograft rejection, we isolated and characterized a TH1 T cell line from BALB/c recipients of B10.A skin that responds to a defined immunodominant, self-restricted allopeptide, I-Abetak58-71. When transferred into BALB/c severe combined immunodeficiency recipients of B10.A skin allografts, this cell line specifically induced a form of skin graft rejection characterized by the presence of TH1 cytokines, macrophage infiltration, and extensive fibrosis. Recall immune responses and immunofluorescence of the rejecting skin revealed only the presence of the peptide-specific T cells within the recipient animals, with no evidence of a direct pathway alloresponse. These studies demonstrate that T cells reactive to a single self-restricted allopeptide can mediate a form of allogeneic skin graft rejection that exhibits characteristics of a chronic, fibrosing process.

Collagen-induced arthritis (CIA) in susceptible strains of mice is an animal model of T cell-mediated inflammatory polyarthritis. Analysis of T cell receptor (TCR) V beta gene usage in cells isolated from arthritic joints of BUB/BnJ (BUB) mice (H-2q, TCR V beta a) showed that TCR V beta chain gene usage was limited to TCR V beta 3 and V beta 10 gene families. All of the BUB mice immunized with a mixture of TCR V beta 3 and TCR V beta 10 peptides, but not with control TCR V beta 14 peptide, were refractory to the induction of CIA. Immunization with TCR V beta 3 and V beta 10 peptides completely blocked the development of clinical and subclinical inflammation, formation of pannus and synovial hyperplasia, and the erosion of cartilage and bone. Further studies revealed that preimmunization of BUB mice with V beta 10 peptide alone was sufficient to render the mice resistant to CIA. Analysis of TCR V beta chain gene expression in lymph node cells from arthritic and arthritis-protected mice showed the expression of TCR V beta 10 subfamily in all of the arthritic mice, but not in arthritis-protected mice. Immunization with TCR V beta peptides did not diminish the humoral responses to chicken type-II collagen and also elicited significant levels of anti-V beta 3 and anti-V beta 10 peptide antibodies. Antibodies cross-reactive with mouse chicken type-II collagen were detected in both the arthritic and arthritis-protected mice. Adoptive transfer of serum from arthritis-protected BUB mice significantly delayed the onset (P < 0.005) of arthritis in recipient BUB mice. In contrast, mice injected with serum from arthritic mice had early onset of arthritis. These results demonstrate that immunization of BUB mice with TCR V beta chain peptides elicited antibodies reactive with the self-TCR and prevented the induction of collagen-induced arthritis by eliminating or downregulating pathogenic T cells and consequently blocking the development of humoral immune response. These findings may have clinical applications in treating human autoimmune diseases characterized by common TCR gene usage.

The proteoglycans synthesized by human osteoarthritic femoral head cartilage and nonarthritic articular cartilage age-matched to the osteoarthritic cartilage specimens was studied in explant cultures and in chondrocytes generated by explant outgrowth from the cartilages. Twenty-four hours after explanation, both nonarthritic articular cartilage and osteoarthritic cartilage synthesized principally one large proteoglycan core protein that migrated on 3-5% acrylamide gels with an apparent molecular mass (M(r)) of approximately 520 kDa after enzymatic digestion with chondroitinase ABC and keratanase. The proteoglycan was found in both the explant itself and in the medium compartment of the culture as well. This proteoglycan contained chondroitin-6-sulfate, keratan sulfate and the hyaluronan binding region as evidenced by immunoblotting with murine anti-proteoglycan monoclonal antibodies indicating that the proteoglycan was aggrecan. To a much lesser extent two additional proteoglycan core proteins were also found in the explant but were not seen in the culture medium compartment. These proteoglycans possessed apparent M(r)'s of approximately 480 kDa and approximately 390 kDa on 3-5% acrylamide gels after chondroitinase ABC and keratanase digestion. The medium compartment contained principally the approximately 520 kDa proteoglycan core protein. In osteoarthritic cartilage explants, the pattern of newly synthesized proteoglycans recovered from the tissue as assessed on 3-16% polyacrylamide gradient gels remained relatively the same from day 1 after explantation up to 36 days of culture. By contrast, the proteoglycans recovered from the culture medium contained chondroitin sulfate and keratan sulfate after 1, 7, and 21 days in culture but by 36 days appeared to contain only chondroitin sulfate. Chondrocytes generated from osteoarthritic cartilage and age-matched nonarthritic articular cartilage synthesized different patterns of large (greater than 200 kDa) proteoglycan. Whereas chondrocytes derived from osteoarthritic cartilage continued to synthesize principally the approximately 520 kDa proteoglycan core protein, the chondrocytes derived from nonarthritic cartilage synthesized in addition to this proteoglycan, abundant amounts of the other two proteoglycan core proteins as well.

Objective: To investigate the occurrence and clinical correlation of the arg519-to-cys mutation in the type II procollagen gene in patients with osteoarthritis (OA)., Methods: Sixty-six subjects from 7 families with a strong family history of generalized OA and 13 patients with erosive OA were evaluated clinically and radiologically. Blood samples from 58 subjects in the familial OA group and from all 13 patients with erosive OA were obtained for DNA analysis. Exon 31 of COL2A1, which spans residue 519, was amplified by polymerase chain reaction., Results: The arg519-to-cys mutation was detected in 2 of the 7 families with generalized OA. In these 2 families, the mutation was present in the 2 probands and in 19 other clinically affected family members, as well as in 3 (so-far) clinically unaffected family members (ages 25, 14, and 11 years). It was absent in 18 clinically unaffected members tested. The mutation was associated with a distinctive pattern of early-onset, aggressive, generalized OA with a mild spinal chondrodysplasia. Inheritance was autosomal dominant. No mutation was found in any of the patients with erosive OA., Conclusion: The arg519-to-cys mutation defines a new pathogenic factor in generalized OA with characteristic clinical and radiologic features. The demonstration of a mutation in 3 of 8 families with OA studied thus far suggests a significant incidence of genetically related clinical OA.

We have used PCR to study the expression of T-cell antigen receptor beta RNA containing particular variable region (V) elements from transcripts directly in the cells isolated from joints and lymph nodes of B10.Q mice (H-2q) immunized with chicken type II collagen. Our data show that the T cells present in arthritic joints expressed only a few V beta transcripts--V beta 2, -6, -7, -8.2, -9, -10, and -15. V beta 6 and -8.2 were expressed predominantly (six out of seven animals) while others were expressed at a relatively low level in different animals. In lymph node cells, transcripts for V beta 6, -8.2, and -9 were detected in four out of seven animals. The data indicate that in collagen-induced arthritis there is a restrictive usage of TCR V beta elements and that V beta 6 and -8.2 are probably used preferentially.