Klaus Kühn

N-Acetylglucosamine Prevents IL-1β-Mediated Activation of Human Chondrocytes

Glucosamine represents one of the most commonly used drugs to treat osteoarthritis. However, mechanisms of its antiarthritic activities are still poorly understood. The present study identifies a novel mechanism of glucosamine-mediated anti-inflammatory activity. It is shown that both glucosamine and N-acetylglucosamine inhibit IL-1β- and TNF-α-induced NO production in normal human articular chondrocytes. The effect of the sugars on NO production is specific, since several other monosaccharides, including glucose, glucuronic acid, and N-acetylmannosamine, do not express this activity. Furthermore, N-acetylglucosamine polymers, including the dimer and the trimer, also do not affect NO production. The observed suppression of IL-1β-induced NO production is associated with inhibition of inducible NO synthase mRNA and protein expression. In addition, N-acetylglucosamine also suppresses the production of IL-1β-induced cyclooxygenase-2 and IL-6. The constitutively expressed cyclooxygenase-1, however, was not affected by the sugar. N-acetylglucosamine-mediated inhibition of the IL-1β response of human chondrocytes was not associated with the decreased inhibition of the mitogen-activated protein kinases c-Jun N-terminal kinase, extracellular signal-related kinase, and p38, nor with activation of the transcription factor NF-κB. In conclusion, these results demonstrate that N-acetylglucosamine expresses a unique range of activities and identifies a novel mechanism for the inhibition of inflammatory processes.

The osteoprotegerin/receptor activator of nuclear factor κB/receptor activator of nuclear factor κB ligand system in cartilage

OBJECTIVE The receptor activator of nuclear factor kappaB (RANK) is a member of the tumor necrosis factor receptor family. It is activated by the secreted or cell surface-bound RANK ligand (RANKL). Osteoprotegerin (OPG) is a soluble nonsignaling receptor for RANKL and interferes with RANK activation. This receptor-ligand system regulates the differentiation of osteoclasts and dendritic cells. The present study examined human articular cartilage for the expression of these molecules and the role of RANKL in the regulation of chondrocyte function. METHODS Normal and osteoarthritic (OA) human articular cartilage was used for explant tissue culture or for isolation of chondrocytes and cell culture. Expression of RANK, RANKL, and OPG was analyzed by immunohistochemistry, Western blotting, or reverse transcription-polymerase chain reaction. Recombinant RANKL was added to cartilage or chondrocyte cultures, and gene expression, collagenase and nitric oxide production, and NF-kappaB activation were determined. RESULTS RANK, RANKL, and OPG messenger RNA (mRNA) were expressed in normal cartilage. By immunohistochemistry, RANK, RANKL, and OPG were detected in the superficial zone of normal cartilage. OA cartilage contained increased levels of OPG mRNA, and expression of the 3 proteins extended into the midzone of OA cartilage. OPG was detected by Western blotting, and was increased in response to interleukin-1beta stimulation. OPG, RANK, and RANKL protein were also detected in cultured chondrocytes. Addition of exogenous RANKL did not activate NF-kappaB, induce expression of genes encoding proinflammatory mediators in chondrocytes, or stimulate the production of collagenase and nitric oxide. CONCLUSION These results demonstrate the expression of OPG, RANK, and RANKL in cartilage. However, RANKL does not activate human articular chondrocytes.

Role of nitric oxide, reactive oxygen species, and p38 MAP kinase in the regulation of human chondrocyte apoptosis

This study addresses mechanisms by which interleukin‐1β (IL‐1β) regulates human chondrocyte apoptosis induced by a combination of the anti‐CD95 antibody CH‐11 and the proteasome inhibitor (PSI). The effect of IL‐1β on apoptosis varied among tissue samples. IL‐1β either enhanced (16/22 samples) or inhibited (6/22 samples) DNA fragmentation and caspase‐3 processing. The protective effect of IL‐1β was abrogated by the nitric oxide (NO) synthesis inhibitor N‐monomethyl‐l‐arginine (L‐NMMA) while apoptosis stimulation was not affected. The NO‐donors sodium nitroprusside (SNP) and S‐nitroso‐N‐acetyl penicillamine (SNAP) blocked DNA fragmentation, and this was associated with partial inhibition of caspase‐3 processing. Pyrrolidine dithiocarbamate (PDTC), a scavenger of reactive oxygen species (ROS) blocked apoptosis induction by CH‐11/PSI as well as the enhancement by IL‐1β. The pro‐apoptotic effects of IL‐1β were also abrogated by the p38 inhibitor SB 202190. In conclusion, IL‐1β augments CH‐11/PSI induced apoptosis in the majority of chondrocyte samples. The pro‐apoptotic effect of IL‐1β is not dependent on NO. In contrast, the anti‐apoptotic effect of IL‐1β observed in a minority of samples is partially NO‐dependent. J. Cell. Physiol. 197: 379–387, 2003© 2003 Wiley‐Liss, Inc.

Regulation of CD95 (Fas/APO-1)–induced apoptosis in human chondrocytes

OBJECTIVE To examine the role of nuclear factor kappaB (NF-kappaB) and caspases 3, 8, and 9 in CD95-mediated apoptosis of normal chondrocytes. METHODS First-passage chondrocytes from normal human knee cartilage were stimulated with CD95 antibody, and cell death was determined by annexin V binding and by an enzyme-linked immunosorbent assay. Activation of caspases 3, 8, and 9 was measured by Western blotting, and their role in death signaling was evaluated using caspase-specific small peptide inhibitors. The influence of NF-kappaB was determined by electrophoretic mobility shift assay (EMSA) and proteasome inhibition-dependent blocking of the degradation of inhibitor of NF-kappaB. RESULTS Low levels of NF-kappaB activity were detected by EMSA in unstimulated chondrocytes. NF-kappaB activity was increased in response to agonistic CD95 antibody. CD95 antibody-induced apoptosis was potentiated by the proteasome inhibitors MG-132 and PS1, and this was associated with a reduced nuclear translocation of NF-kappaB. Proteasome inhibitors also caused the induction of DNA fragmentation by tumor necrosis factor alpha. Procaspase 3 processing was enhanced by the proteasome inhibitor MG-132. Procaspase 8 was undetectable by immunoblotting in whole cell lysates of chondrocytes, but caspase 8 messenger RNA was detected by reverse transcription-polymerase chain reaction. Furthermore, apoptosis induced by CD95 stimulation and proteasome inhibitors was blocked by the caspase 8-specific inhibitor Ac-IETD-CHO. Processing of procaspase 9 was not observed, and inhibition of CD95-dependent cell death by the caspase 9 inhibitor Ac-LEHD-CHO was not significant. CONCLUSION These results suggest that CD95-dependent cell death is enhanced by NF-kappaB inhibition at and/or downstream of caspase 8 activation and that caspase 9 activation is not involved in CD95-mediated apoptosis in chondrocytes.

CELL DENSITY MODULATES APOPTOSIS IN HUMAN ARTICULAR CHONDROCYTES

This study addresses the effects of cell density and serum on CD95 (APO‐1/Fas) and CD95L (Fas Ligand) expression and on the induction of CD95‐dependent apoptosis in human articular chondrocytes from normal knees. Subsets of articular chondrocytes in first passage monolayer culture expressed CD95 and CD95L on the cell surface. The expression of both molecules was influenced by cell density: 22.3% of chondrocytes plated at subconfluent density expressed CD95L while expression in confluent cultures was reduced to 8.2%. CD95 expression was 32.1% under subconfluent and 12.2% under confluent conditions. Induction of specific apoptosis by agonistic antibody to CD95 was 15 times higher in confluent cultures than in subconfluent cultures despite higher levels of CD95 and CD95L expression in subconfluent cells, suggesting that protective antiapoptotic mechanisms were activated in low‐density cultures. In subconfluent cultures, serum withdrawal had no effect on the sensitivity of the cells toward CD95 antibody‐induced apoptosis. However, in confluent cultures, serum withdrawal led to a significant reduction of CD95‐dependent apoptosis. Together, these findings demonstrate that cell density is an important modulator of CD95/CD95L expression and susceptibility to CD95‐mediated apoptosis in cultured human chondrocytes. J. Cell. Physiol. 180:439–447, 1999.

Mechanisms of sodium nitroprusside-induced death in human chondrocytes

The nitric oxide (NO) donor sodium nitroprusside (SNP) has been used to study NO-dependent cell death in human chondrocytes. This study compares SNP-induced chondrocyte death and SNP-activated signaling mechanisms with apoptosis induced by CD95 activation. Sodium nitroprusside increased cell death dose-dependently. Compared to CD95 stimulation, SNP induced only low levels of internucleosomal DNA fragmentation as measured by cell-death enzyme-linked immunosorbent assay (ELISA). However, SNP caused substantial nuclear DNA cleavage, as evidenced by terminal deoxynucleotidyltransferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL). Caspase-3 processing in response to SNP was not detected. The pancaspase inhibitor Z-VAD.FMK partially abrogated the TUNEL signal but did not block cell death or internucleosomal DNA fragmentation. The caspase-3-specific inhibitor Ac-DEVD-CHO did not inhibit the SNP-induced TUNEL signal or internucleosomal DNA fragmentation. DNA degradation was not blocked by the p38 inhibitor SB 202190 but by the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine. The results of this study support the hypothesis that the phenotype and mechanisms of SNP-induced chondrocyte death are distinct from apoptosis induction via CD95.