Expression of prepro-enkephalin in human articular chondrocytes is linked to cell proliferation.

This study shows that cultured human articular chondrocytes express high levels of 1.4 kb prepro-enkephalin mRNA. Chondrocytes store met-enkephalin intracellularly and secrete this neuropeptide in mature as well as in precursor form. Gene expression is inducible by serum factors. High levels of prepro-enkephalin mRNA are detected in proliferating chondrocytes but not in confluent, contact-inhibited cells. Phorbol myristate acetate and dibutyryl cyclic AMP, but not dexamethasone, increase levels of prepro-enkephalin mRNA. Furthermore, transforming growth factor beta (TGF beta) and platelet derived growth factor (PDGF) upregulate gene expression, whereas retinoic acid, which inhibits chondrocyte proliferation, suppresses both basal and induced gene expression. Using in situ hybridization it is shown that only 1-3% of primary chondrocytes express prepro-enkephalin mRNA, whereas 52 +/- 12% of subcultured cells are strongly positive. Analysis of DNA synthesis, by autoradiography of incorporated [3H]thymidine, shows that these numbers correspond to the percentage of cells in S-phase of the cell cycle. In cultures of primary chondrocytes TGF beta promotes the formation of cartilage nodules and stimulates proliferation of adherent cells. This is associated with high levels of prepro-enkephalin mRNA in proliferating cells but not in contact-inhibited cells in cartilage nodules. In contrast, formation of cartilage nodules, proliferation and the expression of enkephalin are suppressed by interleukin-1 beta. In summary, expression of prepro-enkephalin in human articular chondrocytes is differentially controlled by cartilage regulatory factors and closely associated with cell proliferation.     

Chondrocyte response to growth factors is modulated by p38 mitogen-activated protein kinase inhibition

Inhibitors of p38 mitogen-activated protein kinase (MAPK) diminish inflammatory arthritis in experimental animals. This may be effected by diminishing the production of inflammatory mediators, but this kinase is also part of the IL-1 signal pathway in articular chondrocytes. We determined the effect of p38 MAPK inhibition on proliferative and synthetic responses of lapine chondrocytes, cartilage, and synovial fibroblasts under basal and IL-1-activated conditions.Basal and growth factor-stimulated proliferation and proteoglycan synthesis were determined in primary cultures of rabbit articular chondrocytes, first-passage synovial fibroblasts, and cartilage organ cultures. Studies were performed with or without p38 MAPK inhibitors, in IL-1-activated and control cultures. Media nitric oxide and prostaglandin E2 were assayed.p38 MAPK inhibitors blunt chondrocyte and cartilage proteoglycan synthesis in response to transforming growth factor beta; responses to insulin-like growth factor 1 (IGF-1) and fetal calf serum (FCS) are unaffected. p38 MAPK inhibitors significantly reverse inhibition of cartilage organ culture proteoglycan synthesis by IL-1. p38 MAPK inhibition potentiated basal, IGF-1-stimulated and FCS-stimulated chondrocyte proliferation, and reversed IL-1 inhibition of IGF-1-stimulated and FCS-stimulated DNA synthesis. Decreases in nitric oxide but not prostaglandin E2 synthesis in IL-1-activated chondrocytes treated with p38 MAPK inhibitors are partly responsible for this restoration of response. Synovial fibroblast proliferation is minimally affected by p38 MAPK inhibition.p38 MAPK activity modulates chondrocyte proliferation under basal and IL-1-activated conditions. Inhibition of p38 MAPK enhances the ability of growth factors to overcome the inhibitory actions of IL-1 on proliferation, and thus could facilitate restoration and repair of diseased and damaged cartilage.     

Human articular cartilage and chondrocytes produce hemopoietic colony-stimulating factors in culture in response to IL-1.

The hemopoietic CSF, granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), are cytokines that mediate the clonal proliferation and differentiation of progenitor cells into mature macrophages and/or granulocytes. We have employed an all-human cell culture system, specific ELISA for GM-CSF and G-CSF, and Northern analysis to investigate whether chondrocytes are a potential source of CSF in rheumatoid disease. We report that human rIL-1 stimulated in a dose-dependent manner the production of GM-CSF and G-CSF by human articular cartilage and chondrocyte monolayers in organ and cell culture, respectively. Increased levels of the CSF Ag were detected after 2 to 8 h stimulation with IL-1, and the optimum dose of IL-1 was 10 to 100 U/ml (0.06 to 0.6 nM IL-1 alpha; 0.02 to 0.2 nM IL-1 beta); neither CSF was detectable in nonstimulated cultures nor in IL-1-stimulated cultures treated with actinomycin D or cycloheximide, indicating the requirement for de novo RNA and protein synthesis. The IL-1-mediated increase in GM-CSF could also be inhibited by the corticosteroid, dexamethasone, but not by the cyclo-oxygenase inhibitor, indomethacin. Although having little effect when tested alone, TNF-alpha and lymphotoxin (TNF-beta) could synergize with IL-1 for the production of GM-CSF. Basic fibroblast growth factor, platelet-derived growth factor, and IFN-alpha and IFN-gamma each had no effect on GM-CSF levels. Results obtained by Northern analysis of chondrocyte total RNA reflected those found for the CSF Ag, namely that CSF mRNA levels were elevated in response to IL-1, but not TNF, and that there was synergy between these two cytokines. We propose that chondrocyte CSF production in response to IL-1, and the concurrent destruction of cartilage by IL-1, could provide a mechanism for the chronic nature of rheumatoid disease.     

IL-1beta synthesis by chondrocyte analyzed by 3D microscopy and flow cytometry: effect of Rhein

Several factors are known to be involved in the destruction of the articular cartilage. Interleukin-1 (IL-1) plays an important role in the pathogenesis of osteoarthritis (OA) either directly or through the stimulation of catabolic factors. The action of IL-1 on articular cartilage is multifaceted and it most likely plays an important role in the mechanism of cartilage destruction. IL-1 suppresses the synthesis of the cartilage matrix components and promotes the degradation of cartilage matrix macromolecules. Diacerein is an anthraquinone molecule that has been shown to reduce the severity of OA, both in man and in animal models. The present study was designed to evaluate in vitro effects of diacerein on IL-1beta expression in LPS or IL-1alpha stimulated chondrocytes. Intracellular IL-1beta production was analysed in articular chondrocytes cultured in monolayer or in alginate 3D-biosystems in the presence of lipopolysaccharide (LPS) or IL-1alpha, with or without diacerein. The results show that LPS and IL-1alpha increase intracellular IL-1beta and Diacerein inhibited LPS-induced and IL-1alpha induced IL-1beta production by articular chondrocytes. Moreover, the effect of mechanical stimulation was analysed. An inhibitory effect of DAR at therapeutic concentrations on IL-1beta production in articular chondrocytes is suggested.     

Proteomic analysis of the LPS-induced stress response in rat chondrocytes reveals induction of innate immune response components in articular cartilage.

Activation of toll-like receptors (TLR) in articular chondrocytes has been reported to increase the catabolic compartment, leading to matrix degradation, while the main consequence of TLR activation in monocytic cells is the expression and secretion of components of the innate immune response, particularly that of inflammatory cytokines. The objective of the work reported here was to obtain a more complete picture of the response repertoire of articular chondrocytes to TLR activation. Mass spectrometry was used to analyse the secretome of stimulated and unstimulated cells. Characterization of TLR expression in rat articular chondrocytes by RT/PCR indicated that TLR4 was the major receptor form. Exposure of these cells to lipopolysaccharide (LPS), the well-characterized TLR4 ligand, induced production not only of the matrix metalloproteinases MMP3 and 13, but also of components traditionally associated with the innate immune response, such as the complement components C1r, C3 and complement factor B, long pentraxin-3 and osteoglycin. Neither TNF-alpha nor IL-1 was detectable in culture media following exposure to LPS. One of the most prominently-induced proteins was the chitinase-like protein, Chi3L1, linking its expression to the innate immune response repertoire of articular chondrocytes. In intact femoral heads, LPS induced expression of Chi3L1 in chondrocytes close to the articular surface, suggesting that only these cells mount a stress response to LPS. Thus articular chondrocytes have a capacity to respond to TLR activation, which results in the expression of matrix metalloproteases as well as subsets of components of the innate immune response without significant increases in the production of inflammatory cytokines. This could influence the erosive processes leading to cartilage degeneration as well as the repair of damaged matrix.     

Distinct pathways regulate facilitated glucose transport in human articular chondrocytes during anabolic and catabolic responses

Articular cartilage is an avascular, non-insulin-sensitive tissue that utilizes glucose as the main energy source, a precursor for glycosaminoglycan synthesis, and a regulator of gene expression. Facilitated glucose transport represents the first rate-limiting step in glucose metabolism. Previously, we demonstrated that glucose transport in chondrocytes is regulated by proinflammatory cytokines via upregulation of GLUT mRNA and protein expression. The objective of the present study was to determine differences in molecular mechanisms regulating glucose transport in chondrocytes stimulated with the anabolic transforming growth factor-beta1 (TGF-beta1) vs. the catabolic and proinflammatory cytokine IL-1beta. Both TGF-beta1 and IL-1beta accelerate glucose transport in chondrocytes. Although both IL-1beta and TGF-beta1 enhance glucose transport in chondrocytes to a similar magnitude, IL-1beta induces significantly higher levels of lactate. TGF-beta1-stimulated glucose transport is not associated with increased expression or membrane incorporation of GLUT1, -3, -6, -8, and -10 and depends on PKC and ERK activation. In contrast, IL-1beta-stimulated glucose transport is accompanied by increased expression and membrane incorporation of GLUT1 and -6 and depends upon activation of PKC and p38 MAP kinase. In conclusion, anabolic and catabolic stimuli regulate facilitated glucose transport in human articular chondrocytes via different effector and signaling mechanisms, and they have distinct effects on glycolysis.     

Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide.

Although IL-1 is an important modulator of chondrocyte metabolism, the postreceptor events triggered by IL-1 remain obscure. The present study shows that IL-1 induces the biosynthesis of nitric oxide (.N = O) by articular chondrocytes. Synthesis of .N = O is also induced by LPS. Other inflammatory mediators such as IFN-gamma, fibroblast growth factor, and TNF-alpha fail to provoke the production of .N = O, but they increase the potency of IL-1. A combination of IL-1, LPS, and TNF-alpha was shown to induce maximal production of 355 +/- 51 nmol/10(6) cells/72 h of nitrite (NO2-), which was measured as a stable end-product of .N = O generation. The biosynthesis of .N = O requires an induction period of approximately 6 h and continues for at least 72 h. Inhibition of .N = O production with the competitive inhibitor NG-monomethyl-L-arginine (NMA) leads to a suppression of gelatinase and PGE2 synthesis by chondrocytes activated with IL-1 alone. In contrast, NMA enhances the synthesis of both gelatinase and PGE2 after activation with a combination of IL-1, LPS, and TNF-alpha. An increase of PGE2 synthesis from 42.0 +/- 21.0 to 174.0 +/- 33.5 ng/10(6) cells/72 h resulted from the addition of NMA when these stimulatory agents were combined. Exposure of IL-1 and fibroblast growth factor-stimulated chondrocytes to authentic, exogenous .N = O led to an increase of PGE2 synthesis from 5.6 +/- 1.7 of untreated cells to 15.8 +/- 6.8 ng/10(6) of .N = O treated cells within the 1st h. This was followed by a suppression of PGE2 synthesis within the next 2 h.     

Influence of bisphosphonates on the production of pro-inflammatory cytokines by activated human articular chondrocytes

Bisphosphonates have anti-inflammatory effects in rheumatoid arthritis and chondroprotective effects in animal arthritis models but their influence on chondrocytes is not known. The aim of this study is to investigate whether bisphosphonates could influence the production of pro-inflammatory cytokines by activated chondrocytes. Therefore human articular cartilage explants were incubated at 48 h with clodronate, pamidronate or risedronate (10(-6) and 10(-8)mol/L), and dexamethasone (10(-8)mol/L). Subsequently, cultures were stimulated with IL-1, 10 ng/mL (n=6) or 1 ng/mL (n=10) for 48 h. Co-incubation was performed with or without bisphosphonates or dexamethasone. A flow cytometric microsphere-based immunoassay was used for the detection of the pro-inflammatory cytokines IL-6, IL-8, TNF-alpha, IL-1 and the regulatory cytokines IL-12p70 and IL-10 in the supernatants. Stimulation with IL-1 resulted in a dose dependent induction of IL-6 and IL-8, but no production of the other cytokines could be demonstrated. This production of IL-6 and IL-8 was neither inhibited nor enhanced by bisphosphonates. Only dexamethasone caused an inhibition of IL-6 production. In conclusion, there is no evidence on the level of articular cartilage cells that bisphosphonates would suppress or enhance IL-6 and IL-8 mediated joint destruction.     

Neutrophil migration induced by IL-8-activated mast cells is mediated by CINC-1

The aim of this study was to characterize the mediators released by mast cells responsible for IL-8-induced neutrophil migration. It was observed that IL-8 induces a dose-dependent neutrophil migration into peritoneal cavity of rats, but not into air-pouch cavity in which resident mast cells are not present. The transference of peritoneal mast cells to the air-pouch renders this cavity responsive to IL-8. The neutrophil migration induced by IL-8 into the peritoneal cavity was not observed when the peritoneal-resident mast cells were depleted by compound 48/80 or distilled water treatment. Confirming the importance of mast cells, IL-8-stimulated mast cells supernatant induced significant neutrophil migration when injected into peritoneal and air-pouch cavities. The IL-8-induced neutrophil migration was observed not to be dependent on LTB(4), prostaglandins or TNF-alpha, since MK886, indomethacin or thalidomide were unable to block the IL-8-induced neutrophil accumulation 'in vivo' or the release of neutrophil chemotactic factor "in vitro" by IL-8-stimulated mast cells. However, dexamethasone, an inhibitor of the synthesis of pro-inflammatory cytokines, blocked the neutrophil migration induced by IL-8 "in vivo" and also inhibited the release of the neutrophil chemotactic factor by IL-8-stimulated mast cells. Moreover, the incubation of IL-8-stimulated mast cells supernatant with antibody against cytokine-induced neutrophil chemoattractant 1 (CINC-1), but not against TNF-alpha or IL-1beta, inhibited its neutrophil chemotactic activity. Furthermore, we found a significant amount of CINC-1 in this supernatant. In conclusion, we demonstrated that the neutrophil migration induced by IL-8 is dependent on CINC-1 release from mast cells.     

Characterization of granulocyte chemotactic activity from human cytokine-stimulated chondrocytes as interleukin 8

Human articular chondrocytes, when stimulated with interleukin 1 beta (IL 1 beta), tumor necrosis factor-alpha (TNF-alpha), or with the double stranded RNA poly (rI).poly (rC), produce a chemotactic activity for granulocytes. The induction with IL 1 beta could be abolished by an antibody to IL 1 beta but not by an antibody to interleukin 6 (IL 6), indicating that the latter is not a mediator for the production of chemotactic activity. The inducers had no direct chemotactic effect on granulocytes. The granulocyte chemotactic factor from chondrocytes was characterized with a specific antibody against leukocyte-derived interleukin 8 (IL 8). The specificity of this antibody was demonstrated by immunochemical and biological criteria such that it could immunoprecipitate only the 6-7 kDa IL 8 protein from fibroblasts, and that it did not neutralize a structurally related monocyte chemotactic protein. This antibody against IL 8 completely neutralized the granulocyte chemotactic activity from stimulated chondrocytes. This demonstrates the identity of chondrocyte IL 8 with leukocyte- and fibroblast-derived IL 8. Our data show that leukocyte chemotaxis into the inflamed joint can be mediated by IL 8, induced in both synovial fibroblasts and chondrocytes by the inflammatory cytokines IL 1 and TNF-alpha.     

Differential effects of ebselen on neutrophil recruitment,chemokine, and inflammatory mediator expression in a rat model of lipopolysaccharide-induced pulmonary inflammation

We postulated that the seleno-organic compound ebselen would attenuate neutrophil recruitment and activation after aerosolized challenge with endotoxin (LPS) through its effect as an antioxidant and inhibitor of gene activation. Rats were given ebselen (1-100 mg/kg i.p.) followed by aerosolized LPS exposure (0.3 mg/ml for 30 min). Airway inflammatory indices were measured 4 h postchallenge. Bronchoalveolar lavage (BAL) fluid cellularity and myeloperoxidase activity were used as a measure of neutrophil recruitment and activation. RT-PCR analysis was performed in lung tissue to assess gene expression of TNF-alpha, cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage-inflammatory protein-2 (MIP-2), ICAM-1, IL-10, and inducible NO synthase. Protein levels in lung and BAL were also determined by ELISA. Ebselen pretreatment inhibited neutrophil influx and activation as assessed by BAL fluid cellularity and myeloperoxidase activity in cell-free BAL and BAL cell homogenates. This protective effect was accompanied by a significant reduction in lung and BAL fluid TNF-alpha and IL-1 beta protein and/or mRNA levels. Ebselen pretreatment also prevented lung ICAM-1 mRNA up-regulation in response to airway challenge with LPS. This was not a global effect of ebselen on LPS-induced gene expression, because the rise in lung and BAL CINC-1 and MIP-2 protein levels were unaffected as were lung mRNA expressions for CINC-1, MIP-2, IL-10, and inducible NO synthase. These data suggest that the anti-inflammatory properties of ebselen are achieved through an inhibition of lung ICAM-1 expression possibly through an inhibition of TNF-alpha and IL-1 beta, which are potent neutrophil recruiting mediators and effective inducers of ICAM-1 expression.     

Dynamic compression inhibits the synthesis of nitric oxide and PGE(2) by IL-1beta-stimulated chondrocytes cultured in agarose constructs

Both mechanical loading and interleukin-1beta (IL-1beta) are known to regulate metabolic processes in articular cartilage through pathways mediated by nitric oxide ((*)NO) and PGE(2). This study uses a well-characterized model system involving isolated chondrocytes cultured in agarose constructs to test the hypothesis that dynamic compression alters the synthesis of (*)NO and PGE(2) by IL-1beta-stimulated articular chondrocytes. The data presented demonstrate for the first time that dynamic compression counteracts the effects of IL-1beta on articular chondrocytes by suppressing both (*)NO and PGE(2) synthesis. Inhibitor experiments indicated that the dynamic compression-induced inhibition of PGE(2) synthesis and stimulation of proteoglycan synthesis were (*)NO mediated, while compression-induced stimulation of cell proliferation was (*)NO independent. The inhibition of (*)NO and PGE(2) by dynamic compression is a finding of major significance that could contribute to the development of novel strategies for the treatment of cartilage-degenerative disorders.     

Cell hypertrophy and type X collagen synthesis in cultured articular chondrocytes

Articular cartilage is a permanent tissue whose cells do not normally take part in the endochondral ossification process. To determine whether articular chondrocytes possess the potential to express traits associated with this process such as cell hypertrophy and type X collagen, chondrocytes were isolated from adult chicken tibial articular cartilage and maintained in long-term suspension cultures. As a positive control in these experiments, we used parallel cultures of chondrocytes from the caudal portion of chick embryo sternum. Both articular and sternal chondrocytes readily proliferated and progressively increased in size with time in culture. Many had undergone hypertrophy by 4-5 weeks. Analysis of medium-released collagenous proteins revealed that both articular and sternal chondrocytes initiated type X collagen synthesis between 3 and 4 weeks of culture; synthesis of this macromolecule increased with further growth. Immunofluorescence analysis of 5-week-old cultures showed that about 15% of articular chondrocytes and 30% of sternal chondrocytes produced type X collagen; strikingly, there appeared to be no obvious relationship between type X collagen production and cell size. The results of this study show that articular chondrocytes from adult chicken tibia possess the ability to express traits associated with endochondral ossification when exposed to a permissive environment. They suggest also that the process of cell hypertrophy and initiation of type X collagen synthesis are independently regulated both in articular and sternal chondrocytes.     

Pycnogenol attenuates the inflammatory and nitrosative stress on joint inflammation induced by urate crystals

Acute gouty arthritis results from monosodium urate (MSU) crystal deposition in joint tissues. Deposited MSU crystals induce an acute inflammatory response which leads to damage of joint tissue. Pycnogenol (PYC), an extract from the bark of Pinus maritime, has documented antiinflammatory and antioxidant properties. The present study aimed to investigate whether PYC had protective effects on MSU-induced inflammatory and nitrosative stress in joint tissues both in vitro and in vivo. MSU crystals upregulated cyclooxygenase 2 (COX-2), interleukin 8 (IL-8) and inducible nitric oxide synthase (iNOS) gene expression in human articular chondrocytes, but only COX-2 and IL-8 in synovial fibroblasts. PYC inhibited the up-regulation of COX-2, and IL-8 in both articular chondrocytes and synovial fibroblasts. PYC attenuated MSU crystal induced iNOS gene expression and NO production in chondrocytes. Activation of NF-κB and SAPK/JNK, ERK1/2 and p38 MAP kinases by MSU crystals in articular chondrocytes and synovial fibroblasts in vitro was attenuated by treatment with PYC. The acute inflammatory cell infiltration and increased expression of COX-2 and iNOS in synovial tissue and articular cartilage following intra-articular injection of MSU crystals in a rat model was inhibited by coadministration of PYC. Collectively, this study demonstrates that PYC may be of value in treatment of MSU crystal-induced arthritis through its anti-inflammatory and anti-nitrosative activities.