Degeneration, inflammation, regeneration, and pain/disability in dogs following destabilization or articular cartilage grooving of the stifle joint.

OBJECTIVE: The most used model for joint instability is the canine anterior cruciate ligament transection (ACLT)-model. The ACLT-model can be extended with a medial meniscectomy (MX) (i.e., ACLT-MX-model) to avoid unintentional, and with that variable, meniscal damage. The present study compares the ACLT-MX-model with the more recently introduced Groove-model on longitudinal measurements of osteophyte formation and gait as a surrogate marker of pain and disability, in addition to structural endpoint parameters. METHODS: Degenerative joint damage was induced Labrador dogs according to the ACLT-MX-model (n=7) or Groove-model (n=7). Every 4 weeks radiographs were taken to analyze osteophyte formation. Every 2 weeks gait was recorded using force-plate analysis. Joints were analyzed for features of degeneration 12 weeks after surgery. RESULTS: Both models showed similar osteophyte formation and gait changes for both experimental and contra-lateral control joints, although more pronounced for the ACLT-MX-model. This was supported by the structural endpoint measurements. Cartilage integrity, chondrocyte activity and synovial inflammation revealed similar characteristics of degenerative joint disease in both groups, again more pronounced in the ACLT-MX-model. CONCLUSIONS: The ACLT-MX-model demonstrates characteristics of joint degeneration that are related to moderate to severe osteoarthritis with clear synovial inflammatory activity. The Groove-model is a less painful and a significantly milder model of joint degeneration. The latter model might be more suitable to study subtle changes as a result of intervention than the more robust ACLT-MX-model.

The groove model of osteoarthritis applied to the ovine fetlock joint.

OBJECTIVE: Until now there have been no appropriate models for metacarpophalangeal osteoarthritis (OA), even though OA in this joint is a significant medical and economic problem in horses. A good model would be useful to evaluate progression and treatment of OA, particularly in this joint. Therefore, we translated the canine Groove model to the ovine metacarpophalangeal (fetlock) joint. METHOD: Cartilage surfaces of the metacarpal side of one fetlock joint were surgically damaged (grooved), followed by intermittent forced loading of the experimental joint. After 15 and 37 weeks, cartilage, synovial tissue and subchondral bone were analyzed by the use of macroscopy, histology, biochemistry and micro-CT. RESULTS: Technically, the model was difficult to use because cartilage surfaces were very thin. Nonetheless, all macroscopic, histologic, and biochemical cartilage parameters demonstrated adverse changes in chondrocyte activity and matrix integrity. Decreased proteoglycan content suggested slow progression of cartilage degeneration over time, while synovial inflammation diminished. Impaired subchondral bone quality and osteophyte formation were found. Although osteophyte formation was progressive, subchondral bone changes diminished over time. CONCLUSION: The canine Groove model appears to a limited extent transferable to the ovine fetlock joint. However, despite development of adverse changes consistent with early changes of OA, use of the Groove model in the ovine fetlock joint has technical limitations. Using larger animals, such as horses, may significantly improve the technical procedures and with that may provide a more reliable model of metacarpophalangeal OA that is based primarily on intrinsic cartilage damage, appropriate to evaluate the progression and treatment of OA in this particular joint.

Inhibition of COX-2 by celecoxib in the canine groove model of osteoarthritis.

OBJECTIVE: In vitro studies showed a beneficial effect of celecoxib on proteoglycan turnover and content of osteoarthritic cartilage. In the present study we evaluated whether these favourable effects of celecoxib could also be demonstrated in vivo. METHODS: In 24 Beagle dogs, osteoarthritis (OA) was induced in one knee according to the groove model. The animals were divided into three groups and received oral placebo or 100 or 200 mg celecoxib daily, starting directly after surgery. After 15 weeks joint tissue from all dogs was analysed. RESULTS: Induction of OA resulted in macroscopic and histological damage of cartilage, changes in cartilage proteoglycan turnover, loss of cartilage matrix proteoglycans and slight synovial inflammation, all characteristic of early OA. Surprisingly, none of the parameters was significantly changed upon celecoxib treatment. Synovial fluid prostaglandin E(2) levels were dose-dependently diminished by celecoxib, demonstrating that the celecoxib had reached the joint in sufficient amounts. Using an in vitro setup, canine cartilage under degenerative conditions was favourably influenced by celecoxib, demonstrating that canine cartilage is sensitive to celecoxib. CONCLUSION: The present study showed a chondroneutral effect of celecoxib on the characteristics of experimentally induced OA in vivo, in contrast to the observed beneficial effect in vitro. It could be that celecoxib had been beneficial to degenerated cartilage in vivo but that these effects were counteracted by increased loading of the affected joint and the associated progression of OA, occurring because of the well-known analgesic effects of celecoxib.

Interleukin 10 (IL-10), not IL-4 or interferon-gamma production, correlates with progression of joint destruction in rheumatoid arthritis.

OBJECTIVE: Both interleukin 4 (IL-4) and IL-10, separately and in combination, and under in vitro and in vivo conditions in animals, have been reported to inhibit characteristics of rheumatoid arthritis (RA) and experimentally induced arthritis. We investigated if IL-10 and IL-4 production, as well as interferon-gamma (IFN-gamma) production, opposing IL-4, were related to RA disease variables. A method was chosen to exclude the influence of age and disease duration. METHODS: We selected RA patients with mild and severe disease. Inclusion criteria were erythrocyte sedimentation rate (ESR) < or = 28 mm/h and > or = 50, C-reactive protein (CRP) < or = 20 and > or = 30, Thompson joint score < or = 60 and > or = 100 and radiographic joint damage score, Sharp score < or = 30 and > or = 40. Age and disease duration were restricted: 30 to 70 years and 5 to 15 years, respectively. Peripheral blood mononuclear cells were isolated and the ex vivo 48 h production of T cell IL-10, IL-4, and IFN-gamma (after CD3-CD28 stimulation) was assessed and was correlated to clinical variables. RESULTS: Only IL-10 production differed significantly between the 2 groups of RA patients, being highest in the "mild" group. Taking all patients together, a strong negative correlation was found between IL-10 production and radiographic joint damage (r = -0.53, p < 0.001) as well as progression of joint damage (r = -0.56, p < 0.0001). Similar negative correlations, although less powerful, were found between IL-10 production and ESR, CRP, and Thompson joint score. No correlation was found for IFN-gamma, IL-4, or the ratio of the 2 with disease activity variables or joint damage. CONCLUSION: The findings suggest that the high IL-10 production found in patients with RA may be protective, especially against progression of joint destruction in RA.

Articular cartilage is more susceptible to blood induced damage at young than at old age.

OBJECTIVE: It has been shown that cartilage is damaged upon intraarticular hemorrhage. We investigated differences in the susceptibility of cartilage from young adult and old animals to blood induced joint damage in a canine in vivo model. METHODS: Right knees of 6 young adult beagles (aged 2.2 +/- 0.1 yrs) and 6 old beagles (7.4 +/- 0.3 yrs) were intraarticularly injected twice in 4 days with autologous blood. Dogs were killed 4 or 16 days after the first injection and cartilage matrix proteoglycan content and synthesis and collagen damage were determined. RESULTS: Shortly after blood injection (Day 4), proteoglycan synthesis was inhibited and the proteoglycan content of the cartilage was decreased in both groups. However, the degree of the inhibition of proteoglycan synthesis was significantly greater in young adult animals than in old animals. On Day 16 proteoglycan synthesis was increased in both young adult and old cartilage, but more elevated in old cartilage. The proteoglycan content remained decreased in both young adult and old cartilage, but significantly more so in young adult cartilage than in old cartilage. CONCLUSION: Results suggest that intraarticular bleeding influences cartilage metabolism and repair, and that the cartilage of young adult animals is more susceptible to these influences than cartilage of old animals. Differences in the aging of chondrocytes and age related changes in matrix integrity may be involved. Prevention and appropriate treatment of joint bleeding is indicated and this is especially relevant for young adult cartilage.

Apocynin, a plant-derived, cartilage-saving drug, might be useful in the treatment of rheumatoid arthritis.

OBJECTIVE: To investigate whether apocynin, 1-(4-hydroxy-3-methoxyphenyl)ethanone, is able to diminish inflammation-induced cartilage destruction in rheumatoid arthritis (RA), studied in a human in vitro model. METHODS: Apocynin was added to cultures of RA peripheral blood mononuclear cells (PBMNC). Cartilage-destructive activity was determined by addition of culture supernatant to tissue samples of human articular cartilage. In addition, the proliferation of PBMNC, their production of tumour necrosis factor alpha (TN-Falpha), interleukin (IL)-1 and IL-10, and T-cell production of interferon gamma (IFN-gamma) and IL-4, as measures for T1 and T2 cell activity, were determined. RESULTS: Apocynin was able to counteract RA PBMNC-induced inhibition of cartilage matrix proteoglycan synthesis, while no effect on inflammation-enhanced proteoglycan release was found. The effect was accompanied by a decrease in IL-1 and TNF-alpha production by the MNC. No effect on T-cell proliferation was found, but the production of IFN-gamma, IL-4 and T-cell-derived IL-10 was strongly diminished. Most important, apocynin did not show any direct adverse effects on chondrocyte metabolism; on the contrary, it diminished the release of proteoglycans from the cartilage matrix. CONCLUSION: Apocynin in vitro inhibits inflammation-mediated cartilage destruction without having adverse effects on cartilage. The latter may be an advantage of apocynin over many other non-steroidal anti-inflammatory drugs. Therefore, apocynin might have an added beneficial effect in protecting RA patients from joint destruction.

Lymphocyte stimulation by CD3-CD28 enables detection of low T cell interferon-gamma and interleukin-4 production in rheumatoid arthritis.

The analysis of cytokine production is increasingly important in defining the course of an immune response and in evaluating specific therapies of immune diseases. In rheumatoid arthritis (RA), a dysregulation in T1/T2 cell balance, as defined by the production of their specific cytokines, IFN-gamma and IL-4, respectively, is suggested. A predominance of T1-cell mediated macrophage activity in the joint plays a key role in the destruction of articular cartilage and subchondral bone, whereas local T2 cell activity, mitigating disease, fails. However, analysis of the cytokines defining both T cell subsets is difficult and spontaneous production is often below detection limits. Several stimuli are therefore used to increase cytokine production. In the present study we examined whether stimulation of peripheral blood T cells in the context of mononuclear cells (PB MNC) by CD3-CD28 is a reliable method for assessing IFN-gamma and IL-4 production and is representative for the spontaneous production of these cytokines. The production of IFN-gamma and IL-4 following CD3-CD28 stimulation of RA PB MNC correlated significantly in a ratio 1 : 1 with production following ionomycin-PMA stimulation. In samples with detectable spontaneous production of IFNgamma and IL-4, production following CD3-CD28 stimulation was significantly higher than in stimulated samples with undetectable spontaneous production. Moreover, in the case of spontaneous production there was a significant positive linear correlation between the CD3-CD28 stimulated and spontaneous IFNgamma and IL-4 production, although production of both cytokines was not equally enhanced. Serial sampling did not show significant daily or weekly variation in stimulated cytokine production. The results demonstrate that a pecific T-cell stimulation by CD3-CD28 is a reliable way to enhance IFN-gamma and IL-4 production above the detection limit and so measure the T1/T2 cell balance in RA.

The immune suppressive effect of dexamethasone in rheumatoid arthritis is accompanied by upregulation of interleukin 10 and by differential changes in interferon gamma and interleukin 4 production.

OBJECTIVES: The influence of dexamethasone on interleukin 10 (IL10) production and the type 1 (T1)/type 2 (T2) T cell balance found in rheumatoid arthritis (RA) was studied. METHODS: Peripheral blood mononuclear cells (PB MNC) were isolated from 14 RA patients both before and 7 and 42 days after high dose dexamethasone pulse therapy. The ex vivo production of IL10, interferon gamma (IFN gamma) (T1 cell), and IL4 (T2 cell) by PB MNCs was assessed, along with parameters of disease activity (erythrocyte sedimentation rate, C reactive protein, Visual Analogue Scale, Thompson joint score). In addition, the in vitro effect of dexamethasone (0.02, 0.2, and 2 microM) on PB MNC IL10, IFN gamma, and IL4 production was studied. RESULTS: Dexamethasone pulse therapy resulted in a rapid and sustained decrease in RA disease activity. IL10 production increased after dexamethasone treatment and this was sustained for at least six weeks. A transient strong decrease in IFN gamma was seen shortly after corticosteroid treatment, while IL4 only decreased slightly. This led to an increased IL-4/IFN gamma ratio. In vitro, IL10 production was not detectable, IFN gamma and IL4 decreased, but the effect was more pronounced for IFN gamma than for IL4, which again resulted in an increased IL4/IFN gamma ratio. CONCLUSION: Dexamethasone therapy in RA patients leads to a rapid, clinically beneficial effect. The upregulation of IL10 production may be involved in the prolonged clinical benefit. The strong immunosuppressive effect is most evident in the decrease in IFN gamma, and is therefore accompanied by a relative shift towards T2 cell activity. In vitro evaluation showed that this shift in T cell balance was a direct effect of dexamethasone and thus independent of the hypothalamic-pituitary-adrenal axis.

Blood-induced joint damage: a human in vitro study.

OBJECTIVE: To investigate mechanisms underlying cartilage damage caused by brief exposure of cartilage to blood, such as that occurring during intraarticular bleeding. METHODS: Human articular cartilage was cultured for 4 days in the presence of blood (components; 7.5-50% volume/volume). The synthesis of cartilage matrix, as determined by proteoglycan synthesis (incorporation of 35SO4(2-)), was measured directly after exposure and after a recovery period of 20 days, during which the cartilage was cultured in the absence of blood or blood components. The production of the cytokines interleukin-1 (IL-1) and tumor necrosis factor a (TNFalpha), which have a destructive effect on cartilage, was determined by enzyme-linked immunosorbent assay, and the viability of chondrocytes was determined by measuring lactate dehydrogenase release and with electron microscopy. The involvement of oxygen metabolites was evaluated by using N-acetylcysteine. RESULTS: Brief exposure to blood resulted in dose-dependent inhibition of proteoglycan synthesis. The combination of mononuclear cells and red blood cells was responsible for this effect. The effect was irreversible, independent of IL-1 and TNFalpha production, and was accompanied by chondrocyte death. These effects were partially prevented by N-acetylcysteine. CONCLUSION: Brief exposure of cartilage to blood, as occurs after a single episode or a limited number of bleeding episodes, results in lasting cartilage damage in vitro, in which cytotoxic oxygen metabolites play a role.

Blood-induced joint damage: a canine in vivo study.

OBJECTIVE: To investigate the direct and indirect (via synovial inflammation) effects of intraarticular bleeding on cartilage in vivo. METHODS: Right knees of 14 beagle dogs were injected with autologous blood on days 0 and 2. Cartilage matrix proteoglycan turnover, collagen damage, and synovial inflammation of these knees, including the cartilage-destructive properties of the synovial tissue, were determined and compared with those of the left control knees on day 4 (short-term effects; n = 7) and day 16 (long-term effects; n = 7). RESULTS: Injected knees had a diminished content of proteoglycans in the cartilage matrix, and release of proteoglycans was enhanced (days 4 and 16). The synthesis of proteoglycans was significantly inhibited on day 4 but was enhanced on day 16. On day 4 more collagen was denatured in the injected joint than in the control joint; this effect was no longer detectable on day 16. Synovial tissue showed signs of inflammation on day 4 and day 16 but had cartilage-destructive properties only on day 16. CONCLUSION: In vivo exposure of articular cartilage to blood for a relatively short time results in lasting changes in chondrocyte activity and in cartilage matrix integrity, changes that may predict lasting joint damage over time. Interestingly, the direct effect of blood on cartilage precedes the indirect effect via synovial inflammation.

Haemophilic arthropathy resembles degenerative rather than inflammatory joint disease.

AIMS: To investigate the pathogenetic mechanisms of haemophilic arthropathy (HA) by comparing end-stage arthropathy with osteoarthritis (OA; a degenerative joint disorder) and rheumatoid arthritis (RA; an inflammation-mediated joint disease). METHODS AND RESULTS: Cartilage and synovium from patients with HA (n=10), RA (n=8), OA (n=14) and normal control subjects (n=6) were examined morphologically, biochemically and histochemically. Cartilage in HA exhibited characteristics of degenerative joint disease (OA), as evidenced by morphological, histochemical (Safranin-O fast green-iron haematoxylin, Mankin grade) and biochemical (proteoglycan synthesis, glycosaminoglycan content and DNA content) changes, whereas synovium in HA showed characteristics of inflammation-mediated joint disease (RA), as evidenced by histochemical (inflammation, haematoxylin and eosin (H&E) and iron deposition, Perls' blue) and biochemical changes (interleukin (IL)-1, IL-6, tumour necrosis factor (TNF)alpha and catabolic properties). CONCLUSION: Haemophilic arthropathy shows characteristics of both inflammatory and degenerative joint disease. On the basis of these results and published information, it appears that degenerative cartilage changes have a dominant role in HA and are augmented by relatively mild inflammation of the synovium.

Experimentally induced stress in rheumatoid arthritis of recent onset: effects on peripheral blood lymphocytes.

OBJECTIVE: To examine the effects of experimentally-induced stress on the mobilization of peripheral blood lymphocytes (PBL) in patients with rheumatoid arthritis (RA) of recent onset. METHODS: Twenty-two (16 F, 6 M) patients (mean age 57.6 yrs.) and 23 (15 F, 8 M) healthy subjects (mean age 54.7 yrs.) were subjected to experimental stressors. The numbers of T-cells, B-cells, and NK-cells were determined before and after the completion of tasks inducing physical and mental effort. RESULTS: The change in PBL in response to stress was about equal for patients and healthy subjects (p > 0.75 in all PBL subsets). In patients as well as in healthy subjects, the correlations between PBL and cortisol changes in response to stress tended to be positive, while the correlations between PBL and cardiovascular changes were positive in healthy subjects, but zero or negative in patients. Moderate to high (0.32 < or = r < or = 0.55) correlations between PBL changes and pain were observed. CONCLUSION: Experimentally-induced changes in PBL (as well as cortisol) are normal in patients with early RA who are receiving long term medication, but correlations between these changes and autonomic nervous system responses are zero or negative. This apparent shift in the control of the change in PBL in response to stress is observed in particular in patients with more pain. The pathophysiological significance of these findings should be clarified in longitudinal studies.

Mutual antagonism of rheumatoid arthritis and hay fever; a role for type 1/type 2 T cell balance.

OBJECTIVES: The balance between interferon gamma (IFN gamma) and interleukin 4 (IL4) producing T cells (T1 and T2 cells) seems to be of importance in many (auto)immune disorders. In general, T1 cell activity is important in cellular immunity whereas T2 cell activity plays a part in humoral responses. T1 cell activity predominates in joints of patients with rheumatoid arthritis (RA) whereas T2 cell activity is characteristic of atopic syndromes. This study investigated whether the prevalence of hay fever in RA is low and if severity of RA (T1 cell activity) can be influenced by the concomitant occurrence of a T2 cell mediated disease (hay fever). METHODS: The prevalence of hay fever was assessed in 643 consecutive (RA and non-RA) patients seen in our outpatient clinic and confirmed by skin test and specific IgE. Of this group the 12 RA patients with hay fever were compared with RA patients without hay fever (matched for age, sex, and disease duration). RESULTS: The prevalence of hay fever in RA patients is lower than in non-RA patients (4% versus 8%), and yields a relative risk for RA patients to develop hay fever of 0.48. RA patients with hay fever showed a lower disease activity (erythrocyte sedimentation rate, C reactive protein, Thompson joint score, and radiographic joint damage (Sharp) score) than RA patients without hay fever. The clinical data were related to peripheral blood T1/T2 cell balance: a lower IFN gamma/IL4 ratio was observed for RA patients with hay fever, indicating a comparatively increased T2 cell activity in RA patients with hay fever. CONCLUSION: These results argue in favour of the exploration of treatments aimed at regulation of a possible imbalance in T1/T2 cell activity in RA.

Iron deposits and catabolic properties of synovial tissue from patients with haemophilia.

Haemophilic arthropathy is characterised by iron deposits in synovial tissues. We investigated the suggestion that iron plays an important role in synovial changes. We obtained synovial tissue from six patients with haemophilia during arthroplasty, finding that brown haemosideritic tissue was often adjacent to tissue with a macroscopically normal appearance in the same joint. Samples from both types of synovial tissue were analysed histologically and biochemically to determine catabolic activity. Macroscopically haemosideritic synovium showed a significantly higher inflammatory activity than that with a normal appearance. Cultures of abnormal synovial tissue gave a significantly enhanced production of IL-1, IL-6 and TNF alpha compared with cultures of synovial tissue with a normal appearance. In addition, the supernatant fluids from the cultures showed greater catabolic activity from haemosideritic tissue, as determined by the inhibition of the synthesis of articular cartilage matrix. We conclude that in patients with haemophilic arthropathy, local synovial iron deposits are associated with increased catabolic activity.

Cartilage damage as a result of hemarthrosis in a human in vitro model.

OBJECTIVE: To investigate the direct effect of blood and blood components on human cartilage in vitro. METHODS: Healthy human articular cartilage tissue was obtained post mortem and cultured according to standard procedures. The harmful effects of whole blood and various isolated blood components as well as the reversibility of these effects were assessed by means of proteoglycan synthesis and proteoglycan release. RESULTS: Whole blood anticoagulated with heparin, coagulated blood, mononuclear cells (MNC), erythrocytes [red blood cells (RBC)] and plasma, in this order of potency, decreased proteoglycan synthesis in a dose dependent manner. The effect of the combination of MNC and RBC in concentrations equivalent to those in whole blood was significantly greater than the effect of each of these isolated components alone and did not differ from that of whole blood. Moreover, cartilage exposed for 4 days to this combination exhibited irreversible inhibition of proteoglycan synthesis. The effect was similar to that of whole blood, the opposite of that of the individual components or other combinations. CONCLUSION: Results suggest a direct irreversible harmful effect of whole blood on cartilage, whereby MNC and RBC together are the main factors. Taking into account that the concentration of blood during hemarthrosis approaches 100% and the natural evacuation time of blood from a joint is about 4 days, our results suggest that prompt evacuation of intraarticular blood or prevention of intraarticular bleeding might be crucial in preventing cartilage damage.