Interferon-gamma protects against the development of structural damage in experimental arthritis by regulating polymorphonuclear neutrophil influx into diseased joints.

OBJECTIVE: Local interaction between soluble mediators within the inflamed synovium is a key factor that governs the pathologic outcome of inflammatory arthritides. Our aim was to investigate the interplay between the Th1 lymphokine interferon-gamma (IFNgamma) and pivotal cytokines that drive rheumatoid arthritis (RA) pathology (interleukin-1beta [IL-1beta] and tumor necrosis factor alpha [TNFalpha]) in modulating inflammation and arthritis in vitro and in vivo. METHODS: Monarticular antigen-induced arthritis (AIA) was initiated in IFNgamma-deficient (IFNgamma(-/-)) mice and age-matched wild-type (IFNgamma(+/+)) mice. Joint swelling was measured and histologic analysis was performed in order to assess changes in both inflammatory and degenerative parameters in vivo. In vitro, the influence of IFNgamma in regulating IL-1beta- and TNFalpha-driven CXCL8 and CCL2 production was quantified by enzyme-linked immunosorbent assay. RESULTS: In murine AIA, both inflammatory and degenerative arthritis parameters were significantly exacerbated in the absence of IFNgamma. IFNgamma appeared to be a crucial factor in regulating CXCR2+ neutrophil influx in the joint. In in vitro studies using RA fibroblast-like synoviocytes, IFNgamma modulated both IL-1beta- and TNFalpha-driven chemokine synthesis, resulting in the down-regulation of CXCL8 production. CONCLUSION: IFNgamma exerts antiinflammatory, chondroprotective, and antiosteoclastogenic effects in murine AIA through a mechanism that involves the regulation of chemokine synthesis and local neutrophil recruitment. These studies suggest a potential therapeutic role of modulating IFNgamma signaling in the treatment of inflammatory arthritides.

Inhibition of aggrecan turnover in short-term explant cultures of bovine tendon.

The large aggregating proteoglycan, aggrecan, better known for its physiological role in articular cartilage where it serves to facilitate resistance of compressive forces during joint articulation, is also present within the distinct functional regions of tendon (i.e., compressed/fibrocartilaginous and tensional). Previous studies demonstrate that an increased turnover of aggrecan occurs in tendon, which is mediated principally by the 'aggrecanases' and, as such, these proteinases may play an important role in the normal functioning of the tissue. In the present study, utilising bovine tendon explant culture systems, we demonstrated that aggrecanase-mediated tendon aggrecan turnover may be modulated by generic metalloproteinase inhibitors (i.e., the aggrecanase inhibitor, actinonin and the broad-spectrum MMP inhibitor, marimistat). As expected, no MMP-generated aggrecan catabolites were detected in the culture system, suggesting that tendon aggrecanases may be inhibited by marimistat. Furthermore, immunohistochemical analyses revealed that aggrecan metabolites are present in the endotenon, surrounding the collagen fibre bundles, suggesting that aggrecan may provide functions of water imbibement and resistance of reversible and repeated compressive loads manifest between the collagen fibres; these functions, in turn, may be associated with increased aggrecan turnover in this tissue. Thus, inhibition of tendon aggrecanases and consequently aggrecan turnover in this tissue, may be related to some of the deleterious effects observed in the tendons of patients undergoing drug therapy with broad-spectrum MMP inhibitors for cancer and arthritis.

Catabolism of aggrecan proteoglycan aggregate components in short-term explant cultures of tendon.

Hyaluronan (HA) and link protein are essential components of the aggrecan proteoglycan aggregate, whereby HA binds multiple aggrecan monomers, an interaction which is stabilised by link protein. In this study, we have examined the turnover of the aggregate components, HA, link protein, and the N-terminal G1 domain of aggrecan, in explant cultures of tissue from compressed and tensional regions of young and mature bovine tendons. Western blot analyses revealed the release of highly processed link protein and G1-containing metabolites, in the absence of catabolic agents, indicating an increased turnover of these components in tendon. In addition, significant levels of HA were released from the tissue matrix into the media compartment. Furthermore, RT-PCR analysis showed that the mRNA expression of link protein and enzymes/proteins associated with HA metabolism may be modulated in the distinct functional regions of tendon with development. Perturbation of normal aggrecan aggregate metabolism may lead to tissue dysfunction.

Biological basis for the benefit of nutraceutical supplementation in arthritis.

Arthritis is a common disease in which the end-point results in joint replacement surgery. This article reviews the use of nutraceuticals as alternative treatments for pathological manifestations of arthritic disease. The efficacy of fish oils (e.g. cod liver oil) in the diet has been demonstrated in several clinical trials, animal feeding experiments and in vitro models that mimic cartilage destruction in arthritic disease. In addition, there is some evidence for beneficial effects of other nutraceuticals, such as green tea, herbal extracts, chondroitin sulphate and glucosamine. However, in most cases, there is little scientific evidence at the cellular and molecular levels to explain their mechanisms of action.

Immunolocalisation and expression of proteoglycan 4 (cartilage superficial zone proteoglycan) in tendon.

Cartilage superficial zone protein/proteoglycan (SZP) or proteoglycan 4 (PRG4), has been demonstrated to have the potential for several distinct biological functions including cytoprotection, lubrication and matrix binding. In the present study, we have examined both the immunolocalisation and the mRNA expression pattern of PRG4 in tissue harvested from the compressed and tensional regions of young and mature bovine tendons. Immunohistochemical analyses, utilizing monoclonal antibody 3-A-4 which recognizes a conformational-dependent epitope on native PRG4, demonstrated that PRG4 is present predominantly at the surface of fibrocartilaginous regions of tendon, with the intensity of immunoreactivity in this region increasing with age. RT-PCR analyses revealed that the expression of PRG4 mRNA can be modulated by exposure to cytokines and growth factors. In addition, analyses of human pathological tendon revealed that PRG4 may also be expressed as an alternatively spliced form lacking exons which encode part of the N-terminal matrix-binding and cell-proliferative domain; however, it remains to be determined whether such splice variants are a feature of human tendon, regardless of disease state. Taken together, these data indicate that PRG4 may play an important cytoprotective role by preventing cellular adhesion to the tendon surface as well as providing lubrication during normal tendon function, in a manner complimentary to cartilage PRG4. Structural modifications to SZP, together with a reduction in synthesis during tendon inflammation with injury and disease may account for the formation of tendon adhesions and contribute to the overall dysfunction of the tissue.

Effects of n-3 fatty acids on cartilage metabolism.

Although the clinical benefits of dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) has been recognised for a number of years, the molecular mechanisms by which particular PUFA affect metabolism of cells within the synovial joint tissues are not understood. This study set out to investigate how n-3 PUFA and other classes of fatty acids affect both degradative and inflammatory aspects of metabolism of articular cartilage chondrocytes using an in vitro model of cartilage degradation. Using well-established culture models, cartilage explants from normal bovine and human osteoarthritic cartilage were supplemented with either n-3 or n-6 PUFA, and cultures were subsequently treated with interleukin 1 to initiate catabolic processes that mimic cartilage degradation in arthritis. Results show that supplementation specifically with n-3 PUFA, but not n-6 PUFA, causes a decrease in both degradative and inflammatory aspects of chondrocyte metabolism, whilst having no effect on the normal tissue homeostasis. Collectively, our data provide evidence supporting dietary supplementation of n-3 PUFA, which in turn may have a beneficial effect of slowing and reducing inflammation in the pathogenesis of degenerative joint diseases in man.

Pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n-3 fatty acids.

OBJECTIVE: To determine if n-3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n-6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage. METHODS: The metabolic profile of human OA cartilage was determined at the time of harvest and after 24-hour exposure to n-3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin-1 (IL-1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs]). RESULTS: Supplementation with n-3 PUFA (but not other fatty acids) reduced, in a dose-dependent manner, the endogenous and IL-1-induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS-4, MMP-13, and MMP-3 (but not TIMP-1, -2, or -3) was also specifically abolished with n-3 PUFA supplementation. In addition, n-3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5-lipoxygenase, 5-lipoxygenase-activating protein, tumor necrosis factor alpha, IL-1alpha, and IL-1beta) without affecting the expression of message for several other proteins involved in normal tissue homeostasis. CONCLUSION: These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n-3 PUFA, but not to other classes of fatty acids.