The lonely death: chondrocyte apoptosis in TNF-induced arthritis

Inflammatory joint disease typically provokes progressive cartilage damage. The proliferative synovial inflammatory tissue directly invades the cartilage and induces the expression and activation of degrading enzymes such as matrix metalloproteases (MMPs) and aggrecanases. However, also chondrocyte apoptosis has been observed in cartilage samples of inflamed joints. It remains unclear whether this is a secondary phenomenon due to cartilage damage or a primary event initiated by the synovial inflammation. To determine the presence or absence of chondrocyte death in experimental arthritis, we longitudinally assessed proteoglycan depletion and chondrocyte apoptosis in paw sections from human tumor necrosis factor transgenic (hTNFtg) mice and wild-type littermates. Whereas, wild-type mice showed no signs of cartilage damage, hTNFtg mice exhibited progressive proteoglycan loss starting at clinical onset of arthritis. However, we already found the first apoptotic chondrocytes well before cartilage matrix breakdown occurred indicating that chondrocyte death can be induced before matrix resorption. Chondrocyte death could constantly be observed until late stages of arthritis causing a continuous increase in the number of empty cartilage lacunae. As apoptotic cells in cartilage cannot be cleared by phagocytes due to their spatial isolation in the avascular lacunae of cartilage, having no contact to professional or amateur phagocytes. The dying cells are compelled to undergo a "lonely death" inevitable ending up in secondary necrosis giving rise to perpetuation of a pro-inflammatory cascade. These data indicate that chondrocyte death may play a primary role in inflammatory arthritis fueling cartilage inflammation and damage due to secondary necrosis.     

Dectin-1 and NOD2 mediate cathepsin activation in zymosan-induced arthritis in mice

Objective  

Activation of pattern recognition receptors (PRR) may contribute to arthritis. Here, we elucidated the role of NOD2, a genetic cause of inflammatory arthritis, and several other PRR in a murine model of inflammatory arthritis.     

Bone formation on tissue-engineered cartilage constructs in vivo: effects of chondrocyte viability and mechanical loading

Interactions between bone and cartilage formation are critical during growth and fracture healing and may influence the functional integration of osteochondral repair constructs. In this study, the ability of tissue-engineered cartilage constructs to support bone formation under controlled mechanical loading conditions was evaluated using a lapine hydraulic bone chamber model. Articular chondrocytes were seeded onto polymer disks, cultured for 4 weeks in vitro, and then transferred to empty bone chambers previously implanted into rabbit femoral metaphyses. The effects of chondrocyte viability within the implanted constructs and in vivo mechanical loading on bone formation were tested in separate experiments. After 4 weeks in vivo, biopsies from the chambers consisted of a complex composite of bone, cartilage, and fibrous tissue, with bone forming in direct apposition to the cartilage constructs. Microcomputed tomography imaging of the chamber biopsies revealed that the implantation of viable constructs nearly doubled the bone volume fraction of the chamber tissue from 0.9 to 1.6% as compared with the implantation of devitalized constructs in contralateral control chambers. The application of an intermittent cyclic mechanical load was found to increase the bone volume fraction of the chamber tissue from 0.4 to 3.6% as compared with no-load control biopsies. The results of these experiments demonstrate that tissue-engineered cartilage constructs implanted into a well-vascularized bone defect will support direct appositional bone formation and that bone formation is significantly influenced by the viability of chondrocytes within the constructs and the local mechanical environment in vivo.     

Nitrogranulogen-dependent inhibition of antibody synthesis. II. In vivo studies in mice.

NTG in higher doses inhibits specific antibody responses in mice when given after but not prior to antigen. The drug also causes suppression of polyclonal humoral responses but to a lesser extent than other alkylating agents.     

Antigen-induced inhibition of human in vitro spontaneous IgE synthesis. I. Factors and kinetic studies.

Peripheral blood mononuclear cells from patients with hypersensitivity to L. perenne and D. pteronyssinus were incubated with specific antigen. They were then cultured for 7 days in the absence of antigen and the IgE contained in the supernatants was determined using an enzyme immunoassay (ELISA). Pre-incubation of the cells with antigen produced an inhibitory effect on the spontaneous IgE synthesis in most of the cases (101 of the 134 individual studied). This inhibition was more pronounced and more frequent in those cultures with an elevated spontaneous production of IgE (3,000-10,000 pg/ml). This effect depended on the dose of antigen and the duration of cell exposure. Both spontaneous IgE production kinetics and antigen-mediated inhibition were studied. A study of the IgE content of the cell pellets indicated that the antigen did not induce inhibition of the IgE release. We therefore believe that the inhibition observed must be due to some kind of IgE synthesis suppression.     

Local changes in proteoglycan synthesis during culture are different for normal and osteoarthritic cartilage.

Proteoglycan synthesis of mild-to-moderate osteoarthritic human knee cartilage was compared with that of normal cartilage of the same donor. Immediately after cartilage was obtained, the synthesis rate of proteoglycans was higher for osteoarthritic cartilage than for normal cartilage. Proteoglycan synthesis was then located, for both normal and osteoarthritic cartilage, in the middle and deep zone. However, after 4 days of culture, proteoglycan synthesis rate was higher for normal cartilage than for osteoarthritic cartilage. The reason for this transition from a lower to a higher proteoglycan synthesis rate was a strong increase in the proteoglycan synthesis in the superficial zone of normal cartilage. This was not observed for the osteoarthritic cartilage. The chondrocytes in the superficial zone of osteoarthritic cartilage, in contrast to normal cartilage, were mainly joined in cell clusters and proliferating. This may explain their inability to contribute to proteoglycan synthesis.     

Antiinflammatory effects of natural tetranortriterpenoids isolated from Carapa guianensis Aublet on zymosan-induced arthritis in mice

Objective We investigated the antiinflammatory properties of a derived fraction of tetranortriterpenoids (TNTP) obtained from the seeds of Carapa guianensis Aublet. Material and methods Zymosan-induced arthritis and pleurisy in Swiss and C57/Bl6 mice (n = 10 per group). Western blot analysis was performed to analyze nuclear factor-κB (NFκB) translocation in mice peritoneal macrophages stimulated in vitro with zymosan (500 μg/ml). ELISA was performed to evaluate cytokine levels in knee joints. Values of p ≤ 0.05 were regarded as significant. Results Zymosan intra-articular (i. a.) injection (500μg/ cavity) induced a significant increase in knee joint diameter within 6 h, peaked within 24 h and remained above control values for 20 days. Orally-given (p. o.) TNTP (100–200 mg/ kg) inhibited zymosan-induced increase in knee joint diameter and protein extravazation into synovial cavity within 6 h. TNTP (100–200 mg/kg, p. o.) also inhibited total leukocyte influx into the synovial space and tissue, as well as into the mice pleural cavity, due to neutrophil impairment 6 h after zymosan stimulation. The increase in TNF-α, IL-1β and CXCL8/IL-8 levels that were detected in knee synovial extracts obtained from zymosan-stimulated mice was also inhibited by TNTP (100 mg/kg, p. o.). Moreover, the incubation of mice peritoneal macrophages with TNTP (100 μg/ml) inhibited zymosan (500 μg/ml)-induced NFκB translocation into the nucleus 6 h after stimulation. Conclusion Taken together, these results indicate that TNTP present an important antiinflammatory effect, inhibiting zymosan-induced arthritis in mice via the impairment of TNF-α, IL-1β and CXCL8/IL-8 generation, as well as NFκB signaling pathway. Received 7 October 2005; returned for revision 16 January 2006; accepted by M. Parnham 22 May 2006     

Induction of cartilage degradation in experimental arthritis produced by allogeneic and xenogeneic proteoglycan antigens

A single intra-articular injection of proteoglycan (PG) antigens induced sterile inflammatory response and cartilage degradation in preimmunized rabbits and dogs. Both cell-mediated and humoral immune reactions could be detected against PG antigens in these animals, but there was no response against collagen Type II. The lymphocytes isolated from the inflamed synovial layer proved to be predominantly of T cell type. The cartilage degradation was indicated by the accumulation of IgG and complement in the superficial layer of the articular cartilage. By the diminished number of chondrocytes and by the increased binding of specific antibodies in the ground substance. The results suggest that PG antigens trigger local immune reactions which become self-sustaining by enzymatic exposure of antigenic sites. The anti-PG antibodies are cytotoxic to articular chondrocytes and thus can block the continuous neogenesis of matrix components which may lead ultimately to deterioration of the cartilage. This type of experimental arthritis appears to be a model closely related to rheumatoid arthritis.     

Apoptotic chondrocyte death in cell-matrix biocomposites used in autologous chondrocyte transplantation.

Tissue engineering may be a promising approach for the treatment of focal articular cartilage defects. Programmed cell death (apoptosis) plays an important role in multiple degenerative processes of cartilage (e.g. osteoarthritis). It is known that matrix provides a trophic signal for the cells and an altered matrix may influence the availability of factors that regulate apoptosis. In this study we investigate the viability of chondrocytes seeded on a Chondrogide scaffold (Geistlich Biomaterials, CH), which we use in matrix-induced autologous chondrocyte transplantation (MACT). By now, we have studied material from 29 patients treated for localized articular cartilage defects in the knee. Our results indicate that light microscopy (Mayer's hematoxylin-eosin, Masson-Goldner, Trypan-blue and TUNEL method) and electron microscopy can be used to investigate for apoptotic cells grown on a Chondrogide resorbable scaffold. Neither the handling of the cell-matrix biocomposite nor the procedures for fixation could destroy the scaffold or the cell sheet adhering firmly to the matrix. Apoptotic cells were revealed in all samples and with all techniques used. Mayer's hematoxylin-eosin and Masson-Goldner staining show cells with a condensed, pycnotic nucleus and shrunken cytoplasm. In electron microscopy we observed cells with chromatin condensation and volume shrinkage consistent with apoptosis. The results of the Trypan-blue staining show a mean viability of 92.1 +/- 9.8% (range 57-100%). The TUNEL method revealed 44.6 +/- 20.4% positive cells. Our results indicate that apoptosis plays an important role in chondrocytes grown on a scaffold. An optimal scaffold will determine the growth, morphology and phenotype of the chondrocytes by its physical and chemical characteristics.     

A dominant interference collagen X mutation disrupts hypertrophic chondrocyte pericellular matrix and glycosaminoglycan and proteoglycan distribution in transgenic mice.

Collagen X transgenic (Tg) mice displayed skeleto-hematopoietic defects in tissues derived by endochondral skeletogenesis.(1) Here we demonstrate that co-expression of the transgene product containing truncated chicken collagen X with full-length mouse collagen X in a cell-free translation system yielded chicken-mouse hybrid trimers and truncated chicken homotrimers; this indicated that the mutant could assemble with endogenous collagen X and thus had potential for dominant interference. Moreover, species-specific collagen X antibodies co-localized the transgene product with endogenous collagen X to hypertrophic cartilage in growth plates and ossification centers; proliferative chondrocytes also stained diffusely. Electron microscopy revealed a disrupted hexagonal lattice network in the hypertrophic chondrocyte pericellular matrix in Tg growth plates, as well as altered mineral deposition. Ruthenium hexamine trichloride-positive aggregates, likely glycosaminoglycans (GAGs)/proteoglycans (PGs), were also dispersed throughout the chondro-osseous junction. These defects likely resulted from transgene co-localization and dominant interference with endogenous collagen X. Moreover, altered GAG/PG distribution in growth plates of both collagen X Tg and null mice was confirmed by a paucity of staining for hyaluronan and heparan sulfate PG. A provocative hypothesis links the disruption of the collagen X pericellular network and GAG/PG decompartmentalization to the potential locus for hematopoietic failure in the collagen X mice.     

Immobilization aggravates cartilage damage during antigen-induced arthritis in mice. Attachment of polymorphonuclear leukocytes to articular cartilage.

The early and late effects of short-term immobilization on arthritic joints have been studied. Knee joints of mice in which an antigen-induced unilateral arthritis was elicited were immobilized in extension for 3, 5, and 7 days. After 5 and 7 days' immobilization, arthritis was significantly more severe. More leukocytes infiltrated the periarticular tissues and more cellular exudate was found in the joint space. A striking observation was that large numbers of polymorphonuclear leukocytes (PMN) attached to the surface of the cartilage, a phenomenon not found in mobile arthritic joints. Electron-microscopy confirmed PMN adhesion and showed severe ruffling of the cartilage surface under immobilized conditions. Further examination of factors determining PMN sticking revealed that attachment is rapid when the cartilage surface is already damaged, and that retained immune complexes and complement play a pivotal role. The late effects of immobilization were studied after a remobilization period of 2 weeks. Enhanced matrix depletion and chondrocyte death persisted in arthritic joints that were previously immobilized for 5 and 7 days, and the latter also showed significantly increased osteophyte formation. Although these results are speculative for the human situation, this study indicates that treatment of arthritic joints by complete rest should be applied with caution.     

Role of chondrocyte death and hypocellularity in ageing human articular cartilage and the pathogenesis of osteoarthritis

Apoptotic death of articular chondrocytes has been implicated in the pathogenesis of human osteoarthritis. Although nitric oxide and Fas ligand have been shown to be inducers of chondrocyte apoptosis in vitro and in vivo, the contribution of other triggers such as hypoxia, matrix acidosis, abnormal shear stress and catabolic cytokines like interleukin-1beta and tumour necrosis factor alpha has not been examined. It is also not known if growth factors such as insulin like growth factor 1 or anabolic cytokines prevent apoptosis. The intracellular mechanism of effecting apoptotic death depend on whether damage to the mitochondrion or receptor ligation is the primary apoptotic stimulus, since these activate different initiator caspases which then deliver the apoptotic signal to common downstream effector caspases and other proteases. The hypothesis proposed here suggests that by using chondrocytes derived from control and osteoarthritis joints and established human chondrocyte cell lines, it is possible to investigate the relative contributions of major cell death inducing mechanisms and correspondingly which initiating caspase is activated. This understanding is essential for developing appropriately targeted anti-protease therapies for the inhibition of chondrocyte apoptosis in the rational treatment of osteoarthritis.     

自噬与软骨细胞生存及软骨损伤

背景：自噬是细胞通过溶酶体途径处理内源性底物的过程,它普遍存在于机体细胞中,又被看作是细胞Ⅱ型程序性死亡,自噬可能是正常软骨细胞的一种保护或平衡机制。 目的：就自噬与软骨及其损伤相关性的最新研究进展进行讨论,旨在对自噬在软骨及其损伤修复中的作用有一个更好的理解。 方法：应用计算机检索中国知网、万方数据库及PubMed数据库最近20年有关自噬与软骨损伤方面的文献,中文检索词为“自噬、软骨、软骨细胞”,英文检索词为“autophagy、cartilage、chondrocytes、beclin1、LC3”。 结果与结论：软骨细胞能感受关节内微环境变化而做出应答,以调整细胞基质代谢,维持关节软骨生物学功能,而软骨细胞所处低氧环境是引起细胞自噬的重要因素。自噬是正常软骨细胞的一种平衡或者保护机制,虽然自噬与软骨及其损伤相关性在近些年的研究中取得了长足的进步,但不得不承认其仍处于初级阶段,在分子水平上一些Atg的发现加深了对自噬的认识,但在软骨中如何诱导自噬途径,自噬信号是如何传导的,对软骨细胞生存会产生怎样的影响等方面的了解还不够丰富,有待广大学者进一步研究。     

Qualitative changes in human osteoarthritic hip cartilage proteoglycan synthesis during long-term explant culture

Cartilage explants from 14 human osteoarthritic (OA) femoral heads synthesize 4 subpopulations of proteoglycan (PG) based on hydrodynamic size on Sepharose CL-2B (Kav: I, 0.05; II, 0.28; III, 0.68; IV, 0.9-1.0). A detailed analysis of newly synthesized PG monomer from each PG subpopulation was made during protracted (20 days vs. 1 day) explant culture of the cartilage specimens. Subpopulations I and II--high-density PG each eluted off Sepharose CL-2B as a unimodal peak, Kav, 0.2-0.25. High-density PG from subpopulation II appeared as a broad polydisperse symmetrical peak. Subpopulation IV eluted as 2 peaks; a minor peak of large size, and a major peak, Kav, 0.9. Large pore composite polyacrylamide gel electrophoresis (CAPAGE) of intact PG monomer (fraction D1D1) resolved at least 2 discrete PG subpopulations as constituents of both subpopulations I and II. Subpopulations III and IV PG monomer consisted of several heterogeneous subpopulations. The size of the high-density PG from subpopulations I and II decreased with time-in-culture. No significant differences were found in the average glycosaminoglycan (GAG) chain size (Kav, 0.65 on Sepharose CL-6B) or in susceptibility to chondroitinase ABC or AC-II (both 80%) in subpopulations I, II and III high density PG as a function of time-in-culture. The average GAG chain length of subpopulation IV high density PG (Kav, 0.75-0.9) was significantly shorter than the high-density PG of other subpopulations. Variations in the average GAG length and chondroitinase susceptibility did not appear to underly the smaller size of subpopulations I and II high-density PG with culture age. By contrast, keratanase susceptibility of subpopulations I and II high-density PG increased as a function of culture time.     

Increased turnover of proteoglycan aggregates in tendon vs. cartilage

Introduction Although the function of proteoglycans (PGs) within the tendon extracellular matrix are not fully understood, changes in their turnover have been associated with tendinopathies ( Riley et al. 1994 ). In contrast to cartilage, aggrecanases are constitutively expressed and active in tendon ( Rees et al. 2000 ), indicative of a high rate of aggrecan turnover. Clinical trials investigating the use of active site MMP inhibitors have been confounded by side effects, which involve tendonitis and ‘musculoskeletal syndrome’. Such side effects may relate to nonspecific inhibition of tendon aggrecanases required to maintain normal metabolic homeostasis. The purpose of this study, therefore, was to compare the rate of turnover of tendon and cartilage PGs derived from the same joint and to determine the effect of MMP inhibitors (actinonin and marimastat) on aggrecan catabolism. Materials and methods Deep digital flexor tendon explants from compressed and tensional regions were dissected from young and mature bovine. Explants were precultured and then cultured for a further 4 days with or without marimastat (0–2 µm ) or actinonin (0–200 µm ). PG and lactate quantification, Western blot analysis of degradation products and RT‐PCR analyses were performed. In a separate experiment for measurement of PG turnover, explants were set up as described above and then pulse chase labelled with [³⁵S] sulfate. The rate of turnover of ³⁵S‐labelled PGs from the matrix of tendon (and articular cartilage obtained from the same animal) was subsequently calculated from the amount of ³⁵S‐labelled macromolecules appearing in the medium each day and that remaining in the matrix of explants at the termination of culture. Results PG turnover (presumably predominantly aggrecan) was markedly higher in tendon vs. cartilage. This difference was apparent in tendons from all regions and ages. Both marimastat and actinonin inhibited aggrecanase‐mediated PG catabolism in both tendon and cartilage explants. As expected, mRNA expression for the aggrecanases, MMPs and TIMPs was unaffected by the addition of these inhibitors to the culture medium. Discussion Aggrecan turnover in tendon is higher than that of articular cartilage, which may be attributed to distinct physiological properties of this PG in tendon. Importantly, immunohistochemical staining for aggrecan in tendon indicates its presence in between collagen fibres and fibril bundles ( Vogel et al. 1999 ), and thus aggrecan aggregates may dissipate resultant compressive loads by resisting the flow of water in these locations. In addition, aggrecan may facilitate the sliding of fibrils during the small amount of elongation of the tendon whilst under tension. Thus, the half‐life of tendon aggrecan is significantly reduced because it constantly participates in repeated resistance to compression. Our data also demonstrates that both marimastat and actinonin can inhibit aggrecanase‐mediated PG catabolism in tendon cultures. This suggests that the occurrence of ‘musculoskeletal syndrome’ in clinical trial patients may be due to the fact that these inhibitors affect the activity of aggrecanases in tendon, thus preventing them from playing their normal role in tendon aggrecan turnover and consequently perturbing normal physiological function.     

Anti-aging effects of high molecular weight proteoglycan from salmon nasal cartilage in hairless mice

Proteoglycans comprise a family of complex macromolecules consisting of a core protein with covalently attached glycosaminoglycan (GAG) chains. The skin anti-aging effects of oral administration of proteoglycan fractions with different molecular weights from salmon nasal cartilage were investigated in a hairless mouse model of skin aging; aging was caused by repeated ultraviolet B (UVB) irradiation. Three proteoglycan fractions of different molecular weights were prepared from salmon nasal cartilage water extract by ion-exchange column chromatography and gel filtration column chromatography. Physiological and histological analysis of the skin indicated that oral administration of high molecular weight proteoglycan inhibited UVB-induced skin aging, defined as increased erythema, increased transepidermal water loss (TEWL), decreased hydration, and epidermal and dermal hypertrophies. The serum and dorsal skin inflammatory cytokine levels indicated that high molecular weight proteoglycan acts on gut immunity and improves skin by inhibiting surplus inflammatory cytokines produced by UVB irradiation. These results suggest that high molecular weight proteoglycan from salmon nasal cartilage is effective in preventing skin aging.     

Co-culture with infrapatellar fat pad differentially stimulates proteoglycan synthesis and accumulation in cartilage and meniscus tissues

Purpose: Although osteoarthritis is widely viewed as a disease of the whole joint, relatively few studies have focused on interactions among joint tissues in joint homeostasis and degeneration. In particular, few studies have examined the effects of the infrapatellar fat pad (IFP) on cartilaginous tissues. The aim of this study was to test the hypothesis that co-culture with healthy IFP would induce degradation of cartilage and meniscus tissues. Materials and Methods: Bovine articular cartilage, meniscus, and IFP were cultured isolated or as cartilage-fat or meniscus-fat co-cultures for up to 14 days. Conditioned media were assayed for sulfated glycosaminoglycan (sGAG) content, nitrite content, and matrix metalloproteinase (MMP) activity, and explants were assayed for sGAG and DNA contents. Results: Co-cultures exhibited increased cumulative sGAG release and sGAG release rates for both cartilage and meniscus, and the cartilage (but not meniscus) exhibited a substantial synergistic effect of co-culture (sGAG release in co-culture was significantly greater than the summed release from isolated cartilage and fat). Fat co-culture did not significantly alter the sGAG content of either cartilage or meniscus explants, indicating that IFP co-culture stimulated net sGAG production by cartilage. Nitrite release was increased relative to isolated tissue controls in co-cultured meniscus, but not the cartilage, with no synergistic effect of co-culture. Interestingly, MMP-2 production was decreased by co-culture for both cartilage and meniscus. Conclusions: This study demonstrates that healthy IFP may modulate joint homeostasis by stimulating sGAG production in cartilage. Counter to our hypothesis, healthy IFP did not promote degradation of either cartilage or meniscus tissues.