Background: Interferon (IFN) ß displays anti-inflammatory and immunosuppressive activity and has been considered for the treatment of rheumatoid arthritis (RA). Information about the effects of this molecule on joint cells is scarce, however.
Objective: To investigate the effects of IFNß on the production of interleukin-1 receptor antagonist (IL1Ra) in human articular chondrocytes and synovial fibroblasts.
Methods: Chondrocytes and synovial fibroblasts were stimulated with IFNß alone or in combination with interleukin (IL) 1ß. IL1Ra concentrations in culture supernatants and cell lysates were determined by ELISA. Expression of mRNA encoding the secreted sIL1Ra or the intracellular icIL1Ra1 isoforms was quantified by real time reverse transcriptase-polymerase chain reaction.
Results: In chondrocytes, IFNß alone had no effect, but dose dependently enhanced the secretion of IL1Ra induced by IL1ß. Chondrocyte cell lysates contained undetectable or low levels of IL1Ra, even after stimulation with IL1ß and IFNß. Consistently, IL1ß and IFNß induced sIL1Ra mRNA expression in chondrocytes, while expression of icIL1Ra1 was not detectable. Human articular chondrocytes thus mainly produce secreted IL1Ra. In synovial fibroblasts, IFNß alone dose dependently increased IL1Ra secretion. In addition, IFNß enhanced the stimulatory effect of IL1ß on IL1Ra production. In synovial cell lysates, IFNß and IL1ß also increased IL1Ra levels. Consistently, IFNß and IL1ß induced the expression of both sIL1Ra and icIL1Ra1 mRNA in synovial fibroblasts.
Conclusion: IFNß increases IL1Ra production in joint cells, which may be beneficial in cartilage damaging diseases such as RA or osteoarthritis.
By reverse transcriptase polymerase chain reaction on messenger RNA from human polymorphonuclear cells, we have isolated a sequence identical to the cDNA coding for intracellular interleukin 1 receptor antagonist (icIL-1ra), but containing an additional in-frame 63-bp sequence located three codons downstream of the translation start of icIL-1ra. This additional sequence is inserted between the first and second exon of the intracellular form, the latter of which is colinear with part of the first exon of the secreted form of IL-1ra. The additional sequence is coded by an extra exon located 2 kb downstream the first icIL-1ra-specific exon. The complementary DNA sequence of the alternatively spliced form of icIL-1ra shows that the predicted protein differs from classical icIL-1ra in the NH2 terminus by insertion of a leaderless sequence of 21 amino acids rich in glycine and glutamic acid residues. Transcripts coding for this new form of icIL-1ra were detected in activated fibroblasts, keratinocytes, and at low levels in myelomonocytic cells. The recombinant protein expressed in COS cells had an apparent molecular mass in sodium dodecyl sulfate polyacrylamide gel electrophoresis of 25 kD compared to 22 kD of classical icIL-1ra, and was mostly intracellular. The ability of this new form of icIL-1ra to inhibit IL-1 activity, in terms of induction of E-selectin and human immunodeficiency virus replication, was comparable to that of classical icIL-1ra. We propose to refer to this new form of icIL-1ra as icIL-1ra type II.
Interleukin-1 receptor antagonist (IL-1ra) is an important modulator of IL-1 activity in a variety of tissues. IL-1ra is differentially produced by different cell types as a 22-26-kD secreted peptide (sIL-1ra) and/or a smaller 16- or 18-kD intracellular peptide (icIL-1ra). This study was undertaken to evaluate the production of IL-1ra in the human cornea. IL-1ra mRNA can be detected in early passage human corneal epithelial cells and corneal stromal fibroblasts and is significantly enhanced by IL-1. Corneal endothelial cells do not express IL-1ra mRNA. Immunohistochemical studies of cultured corneal cells and whole human cornea demonstrate IL-1ra protein production by both the epithelial and stromal cells but not the endothelial cells. Reverse transcriptase polymerase chain reaction, ELISA, and immunoprecipitation studies indicate that corneal epithelial cells are capable of producing both icIL-1ra and sIL-1ra forms of IL-1ra whereas the corneal stromal cells produce only icIL-1ra. In addition to the larger 18-kD icIL-1ra, both corneal epithelial and stromal cells are also capable of producing a smaller recently described 16-kD icIL-1ra. Thus, the differential production of IL-1ra in the human cornea is unique; whereas both epithelial and stromal cells produce icIL-1ra (type 1 and type 2), the epithelial cells appear to also produce sIL-1ra. It is proposed that these IL-1ra proteins may play an important role in regulating IL-1-induced corneal inflammation.
The interleukin 1 receptor antagonist (IL1Ra) family of molecules now includes one secreted isoform (sIL1Ra) and three intracellular isoforms (icIL1Ra1, 2, and 3). Extensive evidence indicates that the sole biological function of sIL1Ra seems to be to competitively inhibit IL1 binding to cell-surface receptors. Although intracellular IL1Ra1 may be released from keratinocytes under some conditions, the intracellular isoforms of IL1Ra may carry out additional as yet poorly defined roles inside cells. Maintenance of a balance between IL1 and IL1Ra is important in preventing the development or progression of inflammatory disease in certain organs. Both the secreted and intracellular isoforms of IL1Ra contribute to maintenance of this balance. An allelic polymorphism in intron 2 of the IL1Ra gene (IL1RN*2) predisposes to the development or severity of a variety of human diseases largely of epithelial cell origin. Both the impaired production of IL1Ra and the overproduction of IL1β are related to the presence of this allele. Restoration of the balance between IL1Ra and IL1 through a variety of approaches is a therapeutic goal in specific chronic inflammatory diseases.
Long-term exposure to UV irradiation and toxic chemicals is associated with chronic inflammation that contributes to skin cancer development with interleukin-1 alpha (IL-1α), constitutively produced by keratinocytes, playing a pivotal role in skin inflammation. The aim of this study was to investigate the modulation of IL-1α production in the HaCaT keratinocyte cell line. Phorbol 12-myristate 13-acetate failed to induce IL-1α in HaCaT cells, and this might be associated with the specific deficiency known to affect downstream signalling of the MEK/ERK pathway in these cells. The calcium ionophore, ionomycin, slightly enhanced the production of intracellular (icIL-1α), but this resulted in a necrotic release at higher concentrations. UV-B exposure significantly increased the production of icIL-1α in a dose-dependent manner with a maximal induction exhibited at 24 h with minimal necrotic and apoptotic effects. Validation of the HaCaT cell model indicated that the nonsteroidal anti-inflammatory drug (NSAID), ibuprofen, and the glucocorticoid, dexamethasone, inhibited icIL-1α production, and this was associated with a slight inhibition of cell viability. The UV-B-induced keratinocyte cell model provides an in vitro system that could, apart from phorbol ester-like compounds, be utilised as a screening assay in identifying skin irritants and/or therapeutic topical agents via the modulation of IL-1α production.
Leflunomide is an immunomodulatory agent used for the treatment of rheumatoid arthritis. In this study, we investigated the effect of A77 1726 – the active metabolite of leflunomide – on the production of IL-1 receptor antagonist (IL-1Ra) by human synovial fibroblasts and articular chondrocytes. Cells were incubated with A77 1726 alone or in combination with proinflammatory cytokines. IL-1Ra production was determined by ELISA. A77 1726 alone had no effect, but in the presence of IL-1β or tumour necrosis factor-α it markedly enhanced the secretion of IL-1Ra in synovial fibroblasts and chondrocytes. The effect of A77 1726 was greatest at 100 μmol/l. In synovial fibroblasts and de-differentiated chondrocytes, A77 1726 also increased IL-1β-induced IL-1Ra production in cell lysates. Freshly isolated chondrocytes contained no significant amounts of intracellular IL-1Ra. A77 1726 is a known inhibitor of pyrimidine synthesis and cyclo-oxygenase (COX)-2 activity. Addition of exogenous uridine did not significantly modify the effect of A77 1726 on IL-1Ra production, suggesting that it was not mediated by inhibition of pyrimidine synthesis. Indomethacin increased IL-1β-induced IL-1Ra secretion in synovial fibroblasts and de-differentiated chondrocytes, suggesting that inhibition of COX-2 may indeed enhance IL-1β-induced IL-1Ra production. However, the stimulatory effect of indomethacin was consistently less effective than that of A77 1726. A77 1726 increases IL-1Ra production by synovial fibroblasts and chondrocytes in the presence of proinflammatory cytokines, and thus it may possess chondroprotective effects. The effect of A77 1726 may be partially mediated by inhibition of COX-2, but other mechanisms likely concur to stimulate IL-1Ra production.
articular cartilage; IL-1 receptor antagonist; leflunomide; synovium
The regulatory mechanisms of the expression of connective tissue growth factor/CCN family member 2 (CTGF/CCN2) in human articular chondrocytes have not been clarified. We investigated the effect of prostaglandin E2 (PGE2) on CTGF/CCN2 expression in chondrocytes.
Articular cartilage samples were obtained from patients with osteoarthritis (OA) and chondrocytes were isolated and cultured in vitro. Chondrocytes were stimulated with PGE2, PGE receptor (EP)-specific agonists, or interleukin (IL)-1. CTGF expression was analyzed using quantitative polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. The inhibitory effects of EP receptor antagonists (for EP2 and EP4) against PGE2 stimulation were also investigated. Stimulation of chondrocytes with PGE2 or IL-1 significantly suppressed CTGF expression. The suppressive effect of PGE2 was reproduced by EP2/EP4 receptor agonists but not by EP1/EP3 receptor agonists, and was partially blocked by an EP4 receptor antagonist, suggesting that the EP4 receptor has a dominant role.
PGE2 may be involved in the regulation of CTGF/CCN2 expression in human articular chondrocytes via the EP4 receptor. Elucidation of EP4-mediated signaling in chondrocytes may contribute to a better understanding of the effects of PGE2 in arthritis.
Articular chondrocytes respond to osmotic stress with transient changes in cell volume and the intracellular concentration of calcium ion ([Ca2+]i). The goal of this study was to examine the hypothesis that interleukin-1 (IL-1), a pro-inflammatory cytokine associated with osteoarthritis, influences osmotically-induced Ca2+ signaling.
Fluorescence ratio imaging was used to measure [Ca2+]i and cell volume in response to hypo- or hyper-osmotic stress in isolated porcine chondrocytes, with or without pre-exposure to 10 ng/ml IL-1α. Inhibitors of IL-1 (IL-1 receptor antagonist, IL-Ra), Ca2+ mobilization (thapsigargin, an inhibitor of Ca-ATPases), and cytoskeletal remodeling (Toxin B, an inhibitor of the Rho family of small GTPases) were used to determine the mechanisms involved in increased [Ca2+]i, F-actin remodeling, volume adaptation and active volume recovery.
In response to osmotic stress, chondrocytes exhibited transient increases in [Ca2+]i, generally followed by decaying oscillations. Pre-exposure to IL-1 significantly inhibited regulatory volume decrease following hypo-osmotic swelling and reduced the change in cell volume and the time to peak [Ca2+]i in response to hyper-osmotic stress, but did not affect the peak magnitudes of [Ca2+]i in those cells that did response. Co-treatment with IL-1Ra, thapsigargin, or Toxin B restored these responses to control levels. The effects were associated with alterations in F-actin organization.
IL-1 alters the normal volumetric and Ca2+ signaling response of chondrocytes to osmotic stress through mechanisms involving F-actin remodeling via small Rho GTPases. These findings provide further insights into the mechanisms by which IL-1 may interfere with normal physiologic processes in the chondrocyte, such as the adaptation or regulatory responses to mechanical and osmotic loading.
cartilage; chondrocyte; osteoarthritis; cytoskeleton; calcium channel; stretch-activated ion channel; volume regulation; cytokine
The receptor for advanced glycation end products (RAGE) binds multiple ligands, including S100 proteins, high mobility group box chromosomal protein 1 (HMGB-1), and AGEs, all of which are present in articular cartilage. Stimulation of RAGE signaling can lead to MAP kinase activation and increased NF-κB activity. The objective of the present study was to determine if chondrocytes express functional RAGE.
The presence of chondrocyte RAGE was analyzed by immunohistochemistry using normal and osteoarthritic (OA) cartilage from young and old monkeys and humans, immunoblotting of chondrocyte lysates and human cartilage extracts, and reverse transcription–polymerase chain reaction (RT-PCR) analysis of RNA from chondrocytes treated with interleukin-1 (IL-1) and fibronectin fragments. RAGE signaling was evaluated by stimulating chondrocytes with S100B and HMGB-1 and analyzing for activation of the ERK MAP kinase and NF-κB. The ability of S100B and HMGB-1 to stimulate matrix metalloproteinase 13 (MMP-13) production was also assessed. A pull-down assay using biotin-labeled S100B was used to demonstrate binding to RAGE.
RAGE was detected in sections of monkey knee cartilage and human knee and ankle cartilage. Increased immunostaining for RAGE was noted in cartilage from older adult monkeys and humans and was further increased in OA tissue. RAGE was also detected by immunoblotting and by RT-PCR, where IL-1β and fibronectin fragments were found to stimulate RAGE expression. Stimulation of chondrocytes with S100B or HMGB-1 increased phosphorylation of the ERK MAP kinase and the p65 subunit of NF-κB and increased the production of MMP-13. This signaling was inhibited in cells pretreated with soluble RAGE, and S100B was shown to bind to chondrocyte RAGE.
Articular chondrocytes express functional RAGE. The increase in RAGE noted in OA cartilage and the ability of RAGE ligands to stimulate chondrocyte MAP kinase and NF-κB activity and to stimulate MMP-13 production suggests that chondrocyte RAGE signaling could play a role in OA.
Matrix metalloproteinases are catabolic enzymes that play a key role in the articular cartilage degeneration evident in degenerative and inflammatory conditions of articular cartilage. The aim of this study is to assess the ability of pravastatin to modify matrix metalloproteinase (MMP) messenger RNA (mRNA) expression and enzyme activity in a culture of normal human chondrocytes stimulated by interleukin-1β.
Materials and methods
Normal human chondrocytes were stimulated with interleukin (IL)-1β for 6 h to induce MMP expression, simulating a catabolic state, and then treated with pravastatin (1, 5 and 10 μM) for a further 18 h before cell lysates and supernatants were harvested. Cells stimulated with IL-1β but not treated with pravastatin served as controls. Real-time polymerase chain reaction (PCR) was used to assess expression of MMP-3 and MMP-9 mRNA. MMP enzyme activity was assessed using a fluorescent MMP-specific substrate. Statistical analysis was performed using analysis of variance (ANOVA).
MMP-3 and MMP-9 mRNA expression was reduced at all concentrations tested with statistically significant trends in reduction (p = 0.002 and <0.001, respectively). Analysis of culture supernatants revealed that pravastatin treatment led to a reduction in total MMP activity but not to a statistically significant degree (p = 0.07).
Treatment with pravastatin of stimulated human chondrocytes leads to significant down-regulation of selected MMP genes and a non-significant reduction in MMP enzyme activity. Our results provide further evidence that statins may have a role to play in future treatment of disease affecting articular chondrocytes.
Articular cartilage; Statins; Matrix metalloproteinases
To elucidate the pathophysiologic links between prostaglandin E2 (PGE2) and osteoarthritis by characterizing the catabolic effects of PGE2 and its unique receptors in human adult articular chondrocytes.
Human adult articular chondrocytes were cultured in monolayer or alginate beads with and without PGE2 and/or agonist, antagonist of EP receptors and cytokines. Cell survival, proliferation, and total proteoglycan synthesis and accumulation were measured in alginate beads. Chondrocyte-related gene expression and PI3k/Akt signaling were assessed by real-time PCR and western blotting, respectively, using a monolayer cell culture model.
Stimulation of human articular chondrocytes with PGE2 through the EP2 receptor (i) suppresses proteoglycan accumulation and synthesis, (ii) suppresses aggrecan gene expression, (iii) does not appreciably affect expression of matrix-degrading enzymes; and (iv) decreases the collagen II:I ratio. EP2 and EP4 receptors are expressed at higher levels in knee compared to ankle cartilage, and in a grade-dependent fashion. PGE2 titration combined with IL-1 synergistically accelerates expression of pain-associated molecules such as inducible nitric oxide synthase (iNOS) and IL-6. Finally, stimulation with exogenous PGE2 or an EP2 agonist inhibits activation of Akt that is induced by insulin-like growth factor (IGF-1).
PGE2 exerts an anti-anabolic effect on human adult articular cartilage in vitro, and EP2/4 receptor antagonists may represent effective therapeutic agents for the treatment of osteoarthritis.
Accumulation of advanced glycation end products (AGEs) in joints is important in the development of cartilage destruction and damage in age-related osteoarthritis (OA). The aim of this study was to investigate the roles of peroxisome proliferator-activated receptor γ (PPARγ), toll-like receptor 4 (TLR4), and receptor for AGEs (RAGE) in AGEs-induced inflammatory signalings in human OA chondrocytes. Human articular chondrocytes were isolated and cultured. The productions of metalloproteinase-13 and interleukin-6 were quantified using the specific ELISA kits. The expressions of related signaling proteins were determined by Western blotting. Our results showed that AGEs enhanced the productions of interleukin-6 and metalloproteinase-13 and the expressions of cyclooxygenase-2 and high-mobility group protein B1 and resulted in the reduction of collagen II expression in human OA chondrocytes. AGEs could also activate nuclear factor (NF)-κB activation. Stimulation of human OA chondrocytes with AGEs significantly induced the up-regulation of TLR4 and RAGE expressions and the down-regulation of PPARγ expression in a time- and concentration-dependent manner. Neutralizing antibodies of TLR4 and RAGE effectively reversed the AGEs-induced inflammatory signalings and PPARγ down-regulation. PPARγ agonist pioglitazone could also reverse the AGEs-increased inflammatory signalings. Specific inhibitors for p38 mitogen-activated protein kinases, c-Jun N-terminal kinase and NF-κB suppressed AGEs-induced PPARγ down-regulation and reduction of collagen II expression. Taken together, these findings suggest that AGEs induce PPARγ down-regulation-mediated inflammatory signalings and reduction of collagen II expression in human OA chondrocytes via TLR4 and RAGE, which may play a crucial role in the development of osteoarthritis pathogenesis induced by AGEs accumulation.
Objective: To clarify the effect of interleukin (IL) 18 on cartilage degeneration by studying the profile of IL18 receptor (IL18R) on chondrocytes and the direct effect of IL18 on production of matrix metalloproteinases (MMPs), aggrecanases, and tissue inhibitors of metalloproteinases (TIMPs) in articular chondrocytes.
Methods: Monolayer cultured human articular chondrocytes were isolated from non-arthritic subjects and patients with rheumatoid arthritis or osteoarthritis. Gene expression of IL18, IL18Rα, IL18Rß, MMPs, and aggrecanases was detected by RT-PCR. Protein levels of IL18Rα were analysed by flow cytometry. Protein levels of IL18, MMPs, and TIMPs were measured by ELISA. Aggrecanase-2 mRNA expression was quantitatively analysed by real time RT-PCR. Protein levels of signalling molecules were assayed by western blotting.
Results: IL18 mRNA was constitutively expressed in chondrocytes, and was enhanced by IL1ß stimulation. Flow cytometric analysis showed that IL1ß, tumour necrosis factor α, and IL18 up regulated IL18Rα expression levels. The level of IL18Rß mRNA was much lower than that of IL18Rα, and was slightly up regulated by IL1ß. In chondrocytes responding to IL18, IL18 (1–100 ng/ml) slightly increased the production of MMP-1, MMP-3, and MMP-13, which was blocked by NF-κB inhibitor and p38 mitogen activated protein kinase inhibitor. IL18 up regulated mRNA expression of aggrecanase-2, but not aggrecanase-1. IL18 also slightly stimulated TIMP-1 production?through extracellular signal regulated kinase activation.
Conclusion: IL18 induces production of MMPs from chondrocytes in inflammatory arthritis. Although the direct effect of IL18 on chondrocytes may not be pivotal for the induction of cartilage degeneration, IL18 seems to play some part in the degradation of articular cartilage in arthritis.
Gene therapy used in the context of delivering a therapeutic gene(s) to chondrocytes offers a new approach for treating chondrocyte-mediated cartilage degradation associated with various human arthropathies including osteoarthritis. In this study, gene delivery to human osteoarthritis chondrocytes in monolayer culture was demonstrated using two adenoviral vectors (Ad.CMVlacZ and Ad.RSVntlacZ) carrying the Escherichia coli beta-galactosidase marker gene, and a third vector (Ad.RSV hIL-1ra) containing the cDNA for human interleukin-1 receptor antagonist. At an moi of 10(3) plaque-forming units/chondrocyte, > 90% of the infected cells stained positive for E. coli beta-galactosidase activity, indicating a high efficiency of transduction. Genetically modified chondrocytes were then transplanted onto the articular surface of osteoarthritic cartilage organ cultures with and without the underlying subchondral bone. Both in situ staining of the cartilage organ cultures for E. coli beta-galactosidase activity and examination by scanning electron microscopy indicated that the transplanted chondrocytes adhered and integrated into the articular surface and continued to express transgenic protein. Chondrocytes transduced with Ad.RSV hIL-1ra and seeded onto the surface of osteoarthritic cartilage secreted high levels of biologically active IL-1 receptor antagonist. The Ad.RSV hIL-1ra-treated cartilage samples were resistant to IL1-induced proteoglycan degradation over 10 d of sustained organ culture. These data demonstrate that transplantation of transduced chondrocytes onto the articular surface protects cartilage from IL-1-induced extracellular matrix degradation.
Because the injured joint has an actively inflammatory environment, the survival and repair potential of cartilage grafts may be influenced by inflammatory processes. Understanding the interactions of those processes with the graft may lead to concepts for pharmacologic or surgical solutions allowing improved cartilage repair.
We asked whether the maturation level of cartilaginous tissues generated in vitro by expanded human articular chondrocytes (HACs) modulate (1) the spontaneous production of cytokines and (2) the response to interleukin (IL)-1β.
Twelve pellets/donor prepared with monolayer-expanded HACs (n = 6 donors) were evaluated at six different culture times for mRNA expression (n = 72) and spontaneous baseline release of monocyte chemoattractant protein (MCP)-1, IL-8, and transforming growth factor (TGF)-β1 (n = 72). We cultured 24 pellets/donor from each of four donors for 1 or 14 days (defined as immature and mature, respectively) and exposed the pellets to IL-1β for 3 days. MCP-1, IL-8, TGF-β1, and metalloprotease (MMP)-1 and MMP-13 were quantified in pellets and culture supernatants.
By increasing culture time, the spontaneous release of IL-8 and MCP-1 decreased (12.0- and 5.5-fold, respectively), whereas that of TGF-β1 increased (5.4-fold). As compared with immature pellets, mature pellets responded to IL-1β by releasing lower amounts of MMP-1 (2.9-fold) and MMP-13 (1.7-fold) and increased levels of IL-8, MCP-1, and TGF-β1 (1.5-, 5.0-, and 7.5-fold, respectively). IL-8 and MCP-1 promptly returned to baseline on withdrawal of IL-1β.
Our observations suggest more mature cartilaginous tissues are more resistant to IL-1β exposure and can activate chemokines required to initiate tissue repair processes.
The implantation of more mature cartilaginous tissues might provide superior graft survival and improve/accelerate cartilage repair.
Elevated levels of interleukin-6 (IL-6), prostaglandin (PG)E2, PGD2 and its dehydration end product 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) have been detected in joint synovial fluids from patients with rheumatoid arthritis (RA). PGE2 directly stimulates IL-6 production in human articular chondrocytes. However, the effects of PGD2 and 15d-PGJ2 in the absence or presence of PGE2 on IL-6 synthesis in human chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestations and abnormal laboratory findings in RA patients.
Using the T/C-28a2 chondrocyte cell line as a model system, we report that exogenous PGE2 and PGD2/15d-PGJ2 exert antagonistic effects on IL-6 synthesis in human T/C-28a2 chondrocytes. Using a synthesis of sophisticated molecular biology techniques, we determined that PGE2 stimulates Toll-like receptor 4 (TLR4) synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-κB p65 subunit leading to NF-κB activation. Binding of the activated NF-κB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 chondrocytes. PGD2 or 15d-PGJ2 concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE2-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-κB-dependent IL-6 synthesis in chondrocytes.
We have delineated the signaling cascade by which PGE2 and PGD2/15d-PGJ2 exert opposing effects on IL-6 synthesis in human chondrocytes. Elucidation of the molecular pathway of IL-6 synthesis and secretion by chondrocytes will provide insights for developing strategies to reduce inflammation and pain in RA patients.
The normal structure and function of articular cartilage is the result of a precisely balanced interaction between anabolic and catabolic processes. The TGF-β family of growth factors generally exerts an anabolic or repair response; in contrast, proinflammatory cytokines such as IL-1β and TNF-α exert a strong catabolic effect. Recent evidence has shown that IL-1β, and TNF-α, and the TGF-β signaling pathways share an antagonistic relationship. The aim of this study was to determine whether the modulation of the response of articular chondrocytes to TGF-β by IL-1β or TNF-α signaling pathways occurs through regulation of activity and availability of Smad proteins.
Human articular chondrocytes isolated from knee joints from patients with osteoarthritis (OA) or normal bovine chondrocytes were cultured in suspension in polyHEMA-coated dishes with either 10% FBS media or serum-deprived media six hours before treatment with IL-1β alone, TNF-α alone or IL-1β followed by TGF-β. Nuclear extracts were examined by electrophoretic mobility shift assays (EMSA) for NF-κB and Smad3/4 DNA binding. Nuclear extracts were also subjected to the TranSignal Protein/DNA array (Panomics, Redwood City, CA) enabling the simultaneous semiquantitative assessment of DNA-binding activity of 54 different transcription factors. Nuclear phospho-Smad2/3 and total Smad7 protein expression in whole cell lysates were studied by Western blot. Cytoplasmic Smad7, COL2A1, aggrecan and SOX9 mRNA expression was measured by real-time PCR.
The DNA-binding activity of Smad3/4 in the TranSignal Protein/DNA array was down-regulated by TNF-α (46%) or IL-1β treatment (42%). EMSA analysis showed a consistent reduction in Smad 3/4 DNA-binding activity in human articular chondrocytes treated with IL-1β or TNF-α. TGF-β-induced Smad3/4 DNA-binding activity and Smad2/3 phosphorylation were also reduced following pre-treatment with IL-1β in human OA and bovine chondrocytes. Real-Time PCR and Western blot analysis showed that IL-1β partially reversed the TGF-β stimulation of Smad7 mRNA and protein levels in TGF-β-treated human OA cells. In contrast, TGF-β-stimulated COL2A1, aggrecan, and SOX9 mRNA levels were abrogated by IL-1β.
IL-1β or TNF-α exerted a suppressive effect on Smad3/4 DNA-binding activity in human articular chondrocytes, as well, as on TGF-β-induced stimulation of Smad3/4 DNA-binding activity and Smad 2/3 phosphorylation in human OA and bovine articular chondrocytes. IL-1β partially reversed the increase in TGF-β-stimulated Smad7 mRNA or protein levels suggesting that Smad7 may not be involved in the suppression of TGF-β signaling induced by IL-1β or TNF-α in articular chondrocytes. The balance between the IL-1β or TNF-α and the TGF-β signaling pathways is crucial for maintenance of articular cartilage homeostasis and its disruption likely plays a substantial role in the pathogenesis of OA.
Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA.
Primary human articular chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity.
Chondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5 (ADAMTS5), as well as down-regulation of aggrecan after FGF-2 stimulation. Flow cytometry, qPCR and immunoblotting analyses suggested that FGFR1 and FGFR3 were the major FGFR isoforms expressed in human articular chondrocytes. FGFR1 was activated more potently than FGFR3 upon FGF-2 stimulation. In osteoarthritic chondrocytes, FGFR3 was significantly down regulated (P < 0.05) with a concomitant increase in the FGFR1 to FGFR3 expression ratio (P < 0.05), compared to normal chondrocytes. Our results also demonstrate that FGFR3 was negatively regulated by FGF-2 at the transcriptional level through the FGFR1-ERK (extracellular signal-regulated kinase) signaling pathway in human articular chondrocytes.
FGFR1 is the major mediator with the degenerative potential in the presence of FGF-2 in human adult articular chondrocytes. FGFR1 activation by FGF-2 promotes catabolism and impedes anabolism. Disruption of the balance between FGFR1 and FGFR3 signaling ratio may contribute to the pathophysiology of OA.
The mitogen-activated protein kinase (MAPK) pathway is stimulated in differentiated chondrocytes and is an important signaling cascade for chondrocyte differentiation and survival. Pro-inflammatory cytokines such as interleukin 1β (IL-1β) play important roles in the pathogenesis of osteoarthritis (OA) and rheumatoid arthritis (RA). In this study, we investigated whether curcumin and resveratrol can synergistically inhibit the catabolic effects of IL-1β, specifically the inhibition of the MAPK and subsequent apoptosis in human articular chondrocytes. Chondrocytes were either left untreated or treated with 10 ng/ml IL-1β or 1 μM U0126, a specific inhibitor of MAPK pathway alone for the indicated time periods or pre-treated with 10 μM curcumin, 10 μM resveratrol or 10 μM resveratrol and 10 μM curcumin for 4 h followed by co-treatment with 10 ng/ml IL-1β or 1 μM U0126 and 10 μM resveratrol, 10 μM curcumin or 10 μM resveratrol and 10 μM curcumin for the indicated time periods. Cultures were evaluated by immunoblotting and transmission electron microscopy. Incubation of chondrocytes with IL-1β resulted in induction of apoptosis, downregulation of β1-integrins and the extracellular signal-regulated kinase 1/2 (Erk1/2). Interestingly, U0126 induced apoptosis and blocked the above-mentioned proteins in a similar way to IL-1β. Furthermore, curcumin and resveratrol inhibited IL-1β- or U0126-induced apoptosis and downregulation of β1-integrins and Erk1/2 in human articular chondrocytes. These results suggest that combining these two natural compounds activates MEK/Erk signaling, a pathway that is involved in the maintenance of chondrocyte differentiation and survival.
Chondrocyte; MAPK pathway; Integrins; OsteoArthritis; IL-1β; Curcumin; Resveratrol
In inflammatory diseases such as rheumatoid arthritis, functions of chondrocytes including synthesis of matrix proteins and proteinases are altered through interactions with cells of the infiltrating pannus. One of the major secreted products of mononuclear inflammatory cells is IL-1. In this study we found that recombinant human IL-1 beta suppressed synthesis of cartilage-specific type II collagen by cultured human costal chondrocytes associated with decreased steady state levels of alpha 1 (II) and alpha 1(IX) procollagen mRNAs. In contrast, IL-1 increased synthesis of types I and III collagens and levels of alpha 1(I), alpha 2(I), and alpha 1(III) procollagen mRNAs, as we described previously using human articular chondrocytes and synovial fibroblasts. This stimulatory effect of IL-1 was observed only when IL-1-stimulated PGE2 synthesis was blocked by the cyclooxygenase inhibitor indomethacin. The suppression of type II collagen mRNA levels by IL-1 alone was not due to IL-1-stimulated PGE2, since addition of indomethacin did not reverse, but actually potentiated, this inhibition. Continuous exposure of freshly isolated chondrocytes from day 2 of culture to approximately half-maximal concentrations of IL-1 (2.5 pM) completely suppressed levels of type II collagen mRNA and increased levels of types I and III collagen mRNAs, thereby reversing the ratio of alpha 1(II)/alpha 1(I) procollagen mRNAs from greater than 6.0 to less than 1.0 by day 7. IL-1, therefore, can modify, at a pretranslational level, the relative amounts of the different types of collagen synthesized in cartilage and thereby could be responsible for the inappropriate repair of cartilage matrix in inflammatory conditions.
Monocyte influx and activation in synovial joints are important in the pathogenesis of both degenerative and inflammatory arthropathies. In this study, we demonstrate the potential of articular cartilage to directly modulate these events. IL-1-stimulated human articular chondrocytes transcribed 0.7-kb monocyte chemoattractant protein-1 (MCP-1) mRNA. In situ hybridization of cartilage organ cultures revealed MCP-1 transcripts in chondrocytes in the superficial tangential zone within 2 h of stimulation with IL-1. Chondrocytes in deeper layers responded by 4 h and reached maximum MCP-1 mRNA levels by 8-12 h. IL-1-stimulated cartilage organ and chondrocyte monolayer cultures released functional monocyte chemotactic activity. This was neutralized by a monoclonal antibody specific for MCP-1, and was associated with the synthesis and secretion of immunoreactive 13-kD and 15-kD isoforms of MCP-1. Regulators and signal transduction pathways involved with the expression of the MCP-1 gene in chondrocytes were analyzed. Steady-state mRNA levels were increased by the known chondrocyte activators IL-1, tumor necrosis factor alpha, LPS, platelet-derived growth factor, and transforming growth factor beta. In addition, leukemia inhibitory factor induced MCP-1 gene expression and protein synthesis, identifying this cytokine as a new regulator of chondrocyte function. Dexamethasone blunted the induction of MCP-1 gene expression by IL-1 and by activators of protein kinase A as well as protein kinase C signal transduction pathways. In contrast, retinoic acid strongly increased phorbol myristate acetate-induced MCP-1 expression and potentiated the effects of IL-1 and LPS. In conclusion, chondrocytes express MCP-1 in response to factors that are present in cartilage or synovium. This provides a mechanism by which cartilage can play an active role in the initiation and progression of arthritis.
The signaling protein p38 mitogen-activated protein kinase is required for inflammatory signaling in chondrocytes that regulates MMP production. We sought to determine the role of specific p38 isoforms in chondrocyte catabolic signaling in response to IL-1β and fibronectin fragments.
Human articular chondrocytes isolated from normal ankle cartilage from tissue donors or from osteoarthritic knee cartilage obtained during knee replacement were stimulated with IL-1β or fibronectin fragment (Fn-f), with or without pretreatment with p38 inhibitors (SB203580 or BIRB796) or growth factors (IGF-1 and OP-1). p38 isoform phosphorylation was measured by antibody array and immunoblotting. MMP-13 expression was measured by real-time PCR, ELISA, and immunoblotting. Chondrocytes were transfected with plasmids expressing constitutively active (CA) p38γ or with adenovirus expressing dominant negative (DN) p38γ.
Stimulation of chondrocytes with either IL-1β or Fn-f led to enhanced phosphorylation of p38α and p38γ, with little phosphorylation of p38β or p38δ isoforms. p38α localized to the nucleus and p38γ to the cytosol. Inhibition of both p38α and p38γ with BIRB796 resulted in less inhibition of MMP-13 production in response to IL-1β or FN-f than did inhibition of only p38α with SB203580. Transfection with CAp38γ resulted in decreased MMP-13 production while transduction with DNp38γ resulted in increased MMP-13 production. IGF-1 and OP-1 pretreatment inhibited p38α phosphorylation but not p38γ phosphorylation.
p38γ is activated by catabolic stimulation of human articular chondrocytes, but interestingly suppresses MMP-13 production. Treatments that increase p38γ activation may be of therapeutic benefit in reducing chondrocyte production of MMP-13.
chondrocyte; articular cartilage; integrins; cytokines; cell signaling; matrix metalloproteinase
Extracellular inorganic pyrophosphate (ePPi) plays a key role in the regulation of normal and pathologic mineralization. The purpose of this work was to evaluate the role of P1 and P2 purine receptors in modulating ePPi production by articular chondrocytes.
Porcine cartilage explants and chondrocyte monolayers were cultured in the presence of P1 agonists, or a P2 agonist or antagonist and inhibitors of P2 signaling. Ambient media ePPi concentrations were measured after 48 to 96 hours.
The P1 agonists NECA and CGS 21680 significantly decreased ePPi concentrations surrounding chondrocytes and cartilage explants. The P2 agonist, ADP, increased ePPi levels, and the P2 antagonist, suramin, decreased ePPi concentrations. Thapsigargin and BAPTA, which dampen Ca2+-related P2 signaling, suppressed the response to ADP.
Purine receptors are important regulators of ePPi production by chondrocytes. P1 receptor stimulation diminishes and P2 receptor stimulation enhances ePPi production. Alterations in receptor signaling or aberrations of extracellular purine nucleotide metabolism resulting in abnormal quantities or proportions of P1 and P2 receptor ligands could foster changes in ePPi production that in turn affect mineralization. We propose a homeostatic role for extracellular purine nucleotides and purine receptors in stabilizing ePPi concentrations.
ATP; articular cartilage; chondrocytes; biomineralization; purine receptors
S100A4 has been shown to be increased in OA cartilage and to stimulate chondrocytes to produce MMP-13 through activation of the receptor for advanced glycation end products (RAGE). The aim of this study was to understand the mechanism of S100A4 secretion by chondrocytes.
Human articular chondrocytes isolated from ankle cartilage were stimulated with 10ng/ml of IL-1β, IL-6, IL-7 or IL-8. Cells were pretreated with either a JAK-3 inhibitor, brefeldin-A, or cycloheximide. Immunoblotting with phospho-specific antibodies was used to determine the activation of signaling proteins. Secretion of S100A4 was measured in the conditioned media by immunoblotting and MMP-13 was measured by ELISA.
Chondrocyte secretion of S100A4 was observed after treatment with IL-6 or IL-8 but was much greater in cultures treated with equal amounts of IL-7 and was not observed with IL-1β. IL-7 activated the JAK/STAT pathway with increased phosphorylation of JAK-3 and STAT-3 leading to increased production of S100A4 and MMP-13. Overexpression of a dominant negative RAGE construct inhibited IL-7 mediated production of MMP-13. Pretreatment of chondrocytes with a JAK-3 inhibitor or with cycloheximide blocked the IL-7 mediated secretion of S100A4. However, pretreatment of chondrocytes with brefeldin-A did not.
IL-7 stimulates chondrocyte secretion of S100A4 via activation of JAK/STAT signaling and then S100A4 acts in an autocrine manner to stimulate MMP-13 production via RAGE. Since both IL-7 and S100A4 are upregulated in OA cartilage and can stimulate MMP-13 production by chondrocytes, this signaling pathway could contribute to cartilage destruction during the development of OA.
Interleukin 1 receptor antagonist (IL-1Ra) levels are elevated in the blood of patients with a variety of infectious, immune, or traumatic conditions. To examine whether IL1Ra is produced by liver cells with characteristics resembling an acute-phase protein, human primary hepatocytes isolated from liver biopsies and HepG2 hepatoma cells were stimulated with IL-1beta, IL-6, and TNFalpha. IL-1Ra was present in the supernatants of both cells, with production significantly enhanced by IL-1beta, and by the combination of IL-1beta and IL-6. The term IL-1Ra refers to two different proteins encoded by the same gene, but generated by alternative splicing of two different first exons. One isoform is secreted (17-kD sIL-1Ra), and the other isoform remains in the cytoplasm (18-kD icIL-1Ra). By Western blot analysis, the supernatants of human hepatoma (HepG2) cells contained only sIL-1Ra, whereas the lysates contained a novel smaller molecular mass isoform of 16 kD. RT-PCR and ribonuclease protection assay with RNA from HepG2 cells showed that only sIL-1Ra mRNA was expressed, and confirmed the inducing effect of IL-1beta and IL-6. Transfection studies were performed using constructs containing the promoters of either sIL-1Ra or icIL-1Ra coupled to the luciferase reporter gene. The sIL-1Ra promoter was active in HepG2 cells stimulated by IL-1beta and/or IL-6, whereas the icIL-1Ra promoter was inactive. Mutation of binding sites for transcription factors NF-kappaB and/or C/EBP within the proximal sIL-1Ra promoter led to significant decreases in response to IL-1beta and IL-6 in comparison to the wild-type promoter. Electromobility gel shift assays confirmed the presence of NF-kappaB and C/EBP binding sites within the sIL-1Ra promoter, and indicated a significant increase in the binding activities of nuclear proteins from HepG2 cells treated with IL-1beta and IL-6. In summary, sIL-1Ra, but not icIL-1Ra, is produced by hepatocytes, and is regulated by proinflammatory cytokines as an acute-phase protein. In addition, NF-kappaB and C/EBP family members are likely to play important roles in the full expression of IL-1Ra by hepatocytes during inflammatory conditions.