Effects of tumor necrosis factor alpha and beta on resorption of human articular cartilage and production of plasminogen activator by human articular chondrocytes

We examined the effects of recombinant human tumor necrosis factor (TNF) on human articular cartilage and chondrocytes in culture. Both TNF alpha and TNF beta stimulated cartilage matrix breakdown during prolonged culture and elevated the levels of plasminogen activator (PA) activity in both the supernatants and cell layers of cultured chondrocytes. Characterization of the PA activities by immunochemistry and by zymography following gel electrophoresis indicated that human chondrocytes produce both urokinase-type PA and tissue-type PA in response to TNF. The addition of both interleukin-1 and TNF alpha or TNF beta to chondrocyte cultures demonstrated a synergism between these cytokines in the generation of PA activity in the culture supernatants and cell layers. Our results suggest that both activated lymphocytes and monocytes may contribute to the cartilage destruction of inflammatory arthritis through their stimulation of chondrocytes with TNF beta and TNF alpha, respectively. Since PA is the only neutral proteinase reported to be elevated in TNF-stimulated chondrocyte cultures, it could have an important role in TNF-mediated cartilage destruction.     

Chondrocyte AMP‐activated protein kinase activity suppresses matrix degradation responses to proinflammatory cytokines interleukin‐1β and tumor necrosis factor α

Objective

Interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP‐activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL‐1β and TNFα.

Methods

Expression of AMPK subunits was examined, and AMPKα activity was ascertained by the phosphorylation status of AMPKα Thr¹⁷² in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL‐1β and TNFα, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPKα by small interfering RNA.

Results

Normal human knee articular chondrocytes expressed AMPKα1, α2, β1, β2, and γ1 subunits. AMPK activity was constitutively present in normal articular chondrocytes and cartilage, but decreased in OA articular chondrocytes and cartilage and in normal chondrocytes treated with IL‐1β and TNFα. Knockdown of AMPKα resulted in enhanced catabolic responses to IL‐1β and TNFα in chondrocytes. Moreover, AMPK activators suppressed cartilage/chondrocyte procatabolic responses to IL‐1β and TNFα and the capacity of TNFα and CXCL8 (IL‐8) to induce type X collagen expression.

Conclusion

Our findings indicate that AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL‐1β or TNFα. AMPK activators attenuate dephosphorylation of AMPKα and procatabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation‐induced degradation.

     

The effect of tumor necrosis factor α and γ-interferon on the resorption of human articular cartilage and on the production of prostaglandin e and of caseinase activity by human articular chondrocytes

In cultured human articular chondrocytes, addition of tumor necrosis factor α (TNFα1) stimulated caseinase activity over the range of 10⁻¹¹M to 10⁻⁷M and stimulated prostaglandin E (PGE) production over the range of 10⁻¹⁰M to 10⁻⁷M. Maximal stimulation was observed at 10⁻⁸M TNFα for both activities. Gamma-interferon (γ-IFN) had a variable effect on PGE production and no significant effect on caseinase activity in articular chondrocyte cultures over a concentration range of 0.1–1,000 units/ml. Co-incubation of TNFα with γ-IFN enhanced PGE production and decreased caseinase activity. Concentrations as low as 1 unit/ml of γ-IFN had significant effects on TNF-stimulated production of PGE and on caseinase activity. Resorption of human articular cartilage was stimulated by TNFα (10⁻⁷M) and was inhibited by γ-IFN (1,000 units/ml). It is possible that cartilage breakdown in vivo may be modulated by such interactions between cytokines.     

Production of leukemia inhibitory factor by human articular chondrocytes and cartilage in response to interleukin-1 and tumor necrosis factor α

Objective. To investigate the production of leukemia inhibitory factor (LIF) by human articular chondrocytes and cartilage. Methods. Chondrocytes and cartilage were cultured with and without added cytokines, and the conditioned media assayed for LIF by a specific radioreceptor competition assay. Results. Interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) stimulated LIF production by chondrocyte monolayers and cartilage. LIF was generally not detected in unstimulated cultures. Northern blot analysis showed increased amounts of LIF messenger RNA in IL-1– and TNFα-treated chondrocyte cultures. Conclusion. Chondrocytes, stimulated by IL-1 and/or TNFα, are potential contributors to the elevated levels of LIF observed in the synovial fluids of patients with rheumatoid arthritis and other inflammatory arthritides.     

Regulation of YKL‐40 production by human articular chondrocytes

Objective

YKL‐40 (human cartilage glycoprotein 39) is one of the most abundant proteins secreted by cultured chondrocytes. The objectives of the present study were to identify regulators of YKL‐40 production in cartilage and chondrocytes and to map the localization of YKL‐40 in chondrocytes.

Methods

Human articular chondrocytes and cartilage explants (obtained from subjects at autopsy, from a tissue bank, and from osteoarthritis [OA] patients undergoing total joint replacement surgery) were stimulated with cytokines, growth factors, and other agents. YKL‐40 expression was analyzed by Northern blot and polymerase chain reaction. YKL‐40 secretion into the media was determined by enzyme‐linked immunosorbent assay.

Results

YKL‐40 production increased to very high levels during the early phase of chondrocyte monolayer culture and in normal cartilage explant cultures as a response to tissue injury. Spontaneous YKL‐40 release was higher in OA than in normal cartilage explant cultures. In chondrocyte monolayer cultures, interleukin‐1β (IL‐1β) and transforming growth factor β (TGFβ) decreased the levels of secreted YKL‐40, and this was associated with a reduction in YKL‐40 messenger RNA levels. IL‐1β, but not TGFβ, reduced YKL‐40 production in cartilage explant cultures. Media from explants treated with cycloheximide had no detectable YKL‐40, suggesting that the released protein was newly synthesized. Immunofluorescence microscopy showed YKL‐40 staining in the Golgi system of the chondrocytes, but YKL‐40 could not be detected in the extracellular matrix.

Conclusion

The spontaneous increase in the production of YKL‐40 in the early phase of culture appears to represent a cellular response to changes in the extracellular matrix environment. This, coupled with the profound suppressive effects of IL‐1β and TGFβ on YKL‐40 production, identifies a novel regulatory pattern for this major chondrocyte‐derived protein.

     

The effect of tumor necrosis factor alpha and gamma-interferon on the resorption of human articular cartilage and on the production of prostaglandin E and of caseinase activity by human articular chondrocytes

In cultured human articular chondrocytes, addition of tumor necrosis factor alpha (TNF alpha) stimulated caseinase activity over the range of 10(-11) M to 10(-7) M and stimulated prostaglandin E (PGE) production over the range of 10(-10) M to 10(-7) M. Maximal stimulation was observed at 10(-8)M TNF alpha for both activities. Gamma-interferon (gamma-IFN) had a variable effect on PGE production and no significant effect on caseinase activity in articular chondrocyte cultures over a concentration range of 0.1-1,000 units/ml. Co-incubation of TNF alpha with gamma-IFN enhanced PGE production and decreased caseinase activity. Concentrations as low as 1 unit/ml of gamma-IFN had significant effects on TNF-stimulated production of PGE and on caseinase activity. Resorption of human articular cartilage was stimulated by TNF alpha (10(-7) M) and was inhibited by gamma-IFN (1,000 units/ml). It is possible that cartilage breakdown in vivo may be modulated by such interactions between cytokines.     

Proteomic analysis by two‐dimensional electrophoresis to identify the normal human chondrocyte proteome stimulated by tumor necrosis factor α and interleukin‐1β

Objective

To determine the intracellular proteome of normal human chondrocytes stimulated with interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) and to ascertain differences in the protein expression patterns of these 2 cytokines.

Methods

Normal human knee cartilage chondrocytes were incubated for 48 hours without stimulation or stimulated with IL‐1β (5 ng/ml) or with TNFα (10 ng/ml). For each culture condition, protein extracts from 4 normal subjects were pooled and resolved using 2‐dimensional electrophoresis. Protein spots were visualized with Sypro stain, and qualitative and quantitative analyses were performed using PDQuest software. Protein spots were then identified by mass spectrometry, using matrix‐assisted laser desorption ionization−time‐of‐flight/time‐of‐flight technology.

Results

We identified 37 spots by mass spectrometry (MS) or by MS/MS, corresponding to 35 different proteins. In IL‐1β–stimulated chondrocytes, IL‐1β was found to modulate 22 proteins, as compared with unstimulated chondrocytes. All of these proteins except connective tissue growth factor (CCND2) were up‐regulated. Proteins involved in cellular metabolism and energy (23%) that were up‐regulated or induced by IL‐1β included nicotinamide phosphoribosyltransferase, long‐chain fatty acid–coenzyme A ligase 4, δ‐aminolevulinic acid dehydratase, triosephosphate isomerase, and an isoform of glyceraldehyde‐3‐phosphate dehydrogenase. In TNFα‐stimulated chondrocytes, TNFα was found to modulate 20 proteins, as compared with unstimulated chondrocytes. All of these except chitinase 3–like 1 (cartilage glycoprotein 39), proteasome activator complex subunit 2, and G3PDH, were up‐regulated. Eighteen proteins were differently modulated by IL‐1β and TNFα. Of these, 45% were related to metabolism.

Conclusion

IL‐1β and TNFα induce different profiles of intracellular protein expression in healthy human chondrocytes. Most of the proteins that are differently regulated are proteins that are implicated in the generation of cellular energy and in glycolysis.

     

Responses of normal and rheumatic human articular chondrocytes cultured under various experimental conditions in agarose

The purpose of the present study was to test if agarose could support the maintenance of normal and arthritic human chondrocytes in culture, and under which experimental conditions they could be successfully grown. Cultures of chondrocytes isolated from articular cartilage from patients with rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA), and healthy controls were assessed by light microscopy, alcian blue staining, formazan uptake and incorporation of radiosulfate into the extracellular matrix. The results showed that both normal and arthritic chondrocytes proliferated, and synthesized proteoglycan (PG) in agarose in short term and long term culture. Proliferation and PG synthesis occurred at a slower rate in chondrocytes from adult rheumatic patients than from healthy controls. Supplements to the medium influenced chondrocyte proliferation, PG synthesis and release into the medium. Serum from RA patients stimulated chondrocyte responses more than normal human serum (NHS), and NHS promoted PG synthesis more than fetal calf serum (FCS). Exposure to inflammatory synovial fluid (SF) enhanced PG synthesis of healthy chondrocytes, but suppressed it in arthritic chondrocytes. We conclude that species-specific serum is optimal for chondrocyte cultures, and that disease related culture conditions change the chondrocyte response. As metabolic responses of human chondrocytes are maintained in agarose, this culture system appears as a suitable in vitro tool for further studies of human joint disease.     

Synthesis and release of phospholipase A2 by unstimulated human articular chondrocytes

High activity of proinflammatory, type II phospholipase A2 (PLA2) was found in synovial fluids (SF) in inflammatory arthritis. In search for the sources of this PLA2, we cultured human articular chondrocytes and cartilage explants from healthy, osteoarthritic and rheumatoid joints. All cultures, unstimulated by cytokines, released PLA2 extracellularly. Cultures obtained from the deep layers of the cartilage released more PLA2 than those obtained from the superficial layers. Deep layer explants released 0.38 to 18.16 pmol/min/mg protein PLA2/day, whereas superficial layer explants released 0.39-3.18 pmol/min/mg/day. Chondrocyte cell cultures continuously released PLA2, in the first day 909-46347 pmol/min/(10)6 cells and after 9-26 days of culture 166-2115 pmol/min/10(6) cells. PLA2 released from chondrocytes was calcium dependent and had optimum activity at pH 7.5. Cycloheximide markedly inhibited its release. Chondrocyte cultures also released muramidase (LZM) but there was no correlation between PLA2 and LZM release. It may be concluded that cytokine unstimulated human articular chondrocytes synthesize and release PLA2 extracellularly which is similar to that found in the SF. Thus, chondrocytes may possibly serve as one of the sources of intraarticular PLA2.     

Inhibition of interleukin‐1β‐stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage

Objective

To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.

Methods

IL‐1β was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800‐kd HA to assess its effect on IL‐1β‐induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti‐CD44 antibody were performed to investigate the mechanism of action of HA.

Results

Treatment and pretreatment with 800‐kd HA at 1 mg/ml resulted in significant suppression of IL‐1β‐stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA‐binding blocking experiments using anti‐CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti‐CD44 antibody, which suppressed IL‐1β‐stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL‐1β in normal and OA cartilage.

Conclusion

This study demonstrates that HA effectively inhibits IL‐1β‐stimulated production of MMP‐1, MMP‐3, and MMP‐13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL‐1β may involve direct interaction between HA and CD44 on chondrocytes.

     

Regulation of YKL-40 production by human articular chondrocytes

OBJECTIVE: YKL-40 (human cartilage glycoprotein 39) is one of the most abundant proteins secreted by cultured chondrocytes. The objectives of the present study were to identify regulators of YKL-40 production in cartilage and chondrocytes and to map the localization of YKL-40 in chondrocytes. METHODS: Human articular chondrocytes and cartilage explants (obtained from subjects at autopsy, from a tissue bank, and from osteoarthritis [OA] patients undergoing total joint replacement surgery) were stimulated with cytokines, growth factors, and other agents. YKL-40 expression was analyzed by Northern blot and polymerase chain reaction. YKL-40 secretion into the media was determined by enzyme-linked immunosorbent assay. RESULTS: YKL-40 production increased to very high levels during the early phase of chondrocyte monolayer culture and in normal cartilage explant cultures as a response to tissue injury. Spontaneous YKL-40 release was higher in OA than in normal cartilage explant cultures. In chondrocyte monolayer cultures, interleukin-1beta (IL-1beta) and transforming growth factor beta (TGFbeta) decreased the levels of secreted YKL-40, and this was associated with a reduction in YKL-40 messenger RNA levels. IL-1beta, but not TGFbeta, reduced YKL-40 production in cartilage explant cultures. Media from explants treated with cycloheximide had no detectable YKL-40, suggesting that the released protein was newly synthesized. Immunofluorescence microscopy showed YKL-40 staining in the Golgi system of the chondrocytes, but YKL-40 could not be detected in the extracellular matrix. CONCLUSION: The spontaneous increase in the production of YKL-40 in the early phase of culture appears to represent a cellular response to changes in the extracellular matrix environment. This, coupled with the profound suppressive effects of IL-1beta and TGFbeta on YKL-40 production, identifies a novel regulatory pattern for this major chondrocyte-derived protein.     

Tumor necrosis factor α activation of the apoptotic cascade in murine articular chondrocytes is associated with the induction of metalloproteinases and specific pro‐resorptive factors

Objective

Tumor necrosis factor α (TNFα) blockade provides substantive reduction of the symptoms of rheumatoid arthritis (RA). While the biologic actions of TNFα have been well characterized in immune and synovial cells, which are known to be major contributors to the progression of cartilage destruction in RA, the current studies were designed to assess the direct effects of TNFα on chondrocytes.

Methods

We examined the expression of several groupings of messenger RNA (mRNA) that define key biologic pathways that have previously been associated with either the general actions of TNFα or cartilage destruction, in murine articular chondrocytes isolated from wild‐type mice and TNFα receptor–null (p55/p75^−/−) mice.

Results

TNFα induced the expression of multiple mRNA that facilitate apoptosis and lead to apoptosis‐induced cell death. The induction of apoptosis was accompanied by the increased expression of several factors involved in the regulation of skeletal tissue proteolysis and resorption. Quantitative increases from 2‐fold to >10‐fold were seen for inducible nitric oxide synthase, matrix metalloproteinase 3, macrophage colony‐stimulating factor, and osteoprotegerin mRNA expression. The dependence of the induction of these mRNA on TNFα was confirmed by comparison with the effects of TNFα on chondrocytes isolated from receptor‐null mice.

Conclusion

These findings demonstrate that TNFα alters the expression of a complex array of genes within murine chondrocytes that contribute to the destruction of joint surfaces, independent of its actions on synovial and immune cells. Further studies are needed to clarify the biologic actions of TNFα in human cartilage cells.