TonEBP regulates the hyperosmotic expression of aquaporin 1 and 5 in the intervertebral disc.

The central region of the intervertebral disc (IVD) is rich in proteoglycans, leading to a hyperosmotic environment, which fluctuates with daily loading. The cells of the nucleus pulposus (NP cells) have adapted to this environment via the function of tonicity enhancer binding protein (TonEBP), and NP cells have been shown to express several water channels known as aquaporins (AQP). We have previously shown that AQP1 and 5 decrease during IVD degeneration. Here, the regulation of AQP1 and 5 by hyperosmotic conditions and the role of TonEBP in this regulation was investigated. AQP1 and 5 gene expression was upregulated by hyperosmotic conditions mimicking the osmolality of the healthy IVD, which was abrogated by TonEBP knockdown. Furthermore, AQP1 and 5 immunopositivity was significantly reduced in TonEBPΔ/Δ E17.5 mice when compared with wildtype controls, indicating in vivo expression of AQP1 and 5 is controlled at least in part by TonEBP. This hyperosmotic regulation of AQP1 and 5 could help to explain the decreased AQP1 and 5 expression during degeneration, when the osmolality of the NP decreases. Together this data suggests that TonEBP-regulated osmo-adaptation may be disrupted during IVD degeneration when the expression of both AQPs is reduced.

Osmotic adaptation of nucleus pulposus cells: the role of aquaporin 1, aquaporin 4 and transient receptor potential vanilloid 4.

The microenvironment of the nucleus pulposus is hyperosmotic and fluctuates diurnally due to mechanical loading. Changes in extracellular osmolality result in cell volume alterations, responsiveness to such changes is essential for cellular homeostasis. Aquaporins allow movement of water across cell membranes and control water permeability in response to osmotic gradients. Furthermore, transient receptor potential vanilloid 4 has been shown to sense osmotic and mechanical stimuli resulting in changes to intracellular Ca2+. It has been shown previously that aquaporin 1 and 4 expression decreases during disc degeneration. Here, the expression of transient receptor potential vanilloid 4 by human nucleus pulposus cells during disc degeneration, and the roles of aquaporin 1, 4 and transient receptor potential vanilloid 4 in regulating responses to osmotic gradients was investigated. Transient receptor potential vanilloid 4 was expressed by the majority of human nucleus pulposus cells and not affected by disc degeneration. Aquaporin 4 staining co-localised with primary cilia. Nucleus pulposus cells modulated their rate of volume change, water permeability and Ca2+ influx in response to extracellular osmolality. These responses were inhibited by chemical inhibition of aquaporin 4, transient receptor potential vanilloid 4, and to a lesser extent aquaporin 1; suggesting that both aquaporins and transient receptor potential vanilloid 4 play important roles in the fundamental adaptation of nucleus pulposus cells to their osmotic environment. Co-localisation with primary cilia indicates these proteins may function synergistically to achieve adaptation, which may be lost during disc degeneration, when aquaporin 1 and 4 expression is reduced.

Potential roles of cytokines and chemokines in human intervertebral disc degeneration: interleukin-1 is a master regulator of catabolic processes.

OBJECTIVE: These studies investigated cytokine and chemokine receptor profiles in nucleus pulposus (NP) cells, and the effects of receptor stimulation on mRNA levels of extracellular matrix (ECM) components, degrading enzymes and cytokine and chemokine expression. METHOD: Immunohistochemistry (IHC) was performed to localise expression of CD4, CCR1, CXCR1 and CXCR2 in human NP tissue samples. Effects of cytokine and chemokine stimulation was performed to investigate effects related to ECM remodelling and modulation of cytokine and chemokine mRNA expression. RESULTS: IHC identified CD4, CCR1, CXCR1 and CXCR2 expression by NP cells. Differential expression profiles were observed for CD4 and CXCR2 in tissue samples from degenerate and infiltrated IVDs. In vitro stimulations of primary human NP cultures with IL-16, CCL2, CCL3, CCL7 or CXCL8 did not identify any modulatory effects on parameters associated with ECM remodelling or expression of other cytokines and chemokines. Conversely, IL-1 was seen to modulate ECM remodelling and expression of all other cytokines and chemokines investigated. CONCLUSION: This study demonstrates for the first time that NP cells express a number of cytokine and chemokine receptors and thus could respond in an autocrine or paracrine manner to cytokines and chemokines produced by NP cells, particularly during tissue degeneration. However, this study failed to demonstrate regulatory effects on ECM genes and degradative enzymes or other cytokines and chemokines for any target investigated, with the exception of IL-1. This suggests that IL-1 is a master regulator within the IVD and may exert regulatory potential over a plethora of other cytokines and chemokines.

Cannabinoid WIN-55,212-2 mesylate inhibits interleukin-1ß induced matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression in human chondrocytes.

OBJECTIVE: Interleukin-1ß (IL-1ß) is involved in the up-regulation of matrix metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are anti-inflammatory and reduce joint damage in animal models of arthritis. This study aimed to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2 mesylate (WIN-55) may inhibit cartilage degradation. METHODS: Effects of WIN-55 were studied on IL-1ß stimulated production of MMP-3 and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes were obtained from articular cartilage of patients undergoing total knee replacement. Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time polymerase chain reaction (PCR) was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and Enzyme Linked Immunosorbent Assay (ELISA) to measure the amount of MMP-3 and MMP-13 protein released into media. Immunocytochemistry was used to investigate the expression of cannabinoid receptors in chondrocyte cultures. RESULTS: Treatment with WIN-55 alone or in combination with IL-1ß, decreased or abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte monolayer and alginate bead cultures in both a concentration and time dependent manner. WIN-55 treatment alone, and in combination with IL-1ß, reduced MMP-3 and -13 protein production by chondrocytes cultured in alginate beads. Immunocytochemistry demonstrated the expression of cannabinoid receptors in chondrocyte cultures. CONCLUSION: Cannabinoid WIN-55 can reduce both basal and IL-1ß stimulated gene and protein expression of MMP-3 and -13. However WIN-55 also decreased basal levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by which cannabinoids may act to prevent cartilage breakdown in arthritis.

ADAMs and ADAMTSs in cancer.

ADAMs and ADAMTSs are multi-domain proteins characterised by the presence of both metalloproteinase and disintegrin-like domains. ADAM proteins are usually type 1 transmembrane proteins, and ADAMTSs are secreted from cells. The dysregulated expression of ADAMs and ADAMTSs has been reported in a wide range of human cancers, where, in many cases, they are implicated as positive regulators of cancer progression. Proteolytically active ADAMs act as ectodomain sheddases, which release extracellular regions of membrane-bound proteins (e.g., adhesion molecules, growth factors, cytokines, chemokines and receptors). Certain ADAMTSs break down extracellular matrix (ECM) proteoglycans (e.g., aggrecan, brevican and versican). Through these actions they are able to sculpt the tumour microenvironment and modulate key processes involved in cancer progression, including cell proliferation, migration and angiogenesis. Members of both groups of protein can also act to inhibit or slow cancer progression: ADAMs can interact with specific integrins to elicit inhibitory effects on cancer dissemination, and certain ADAMTSs possess antiangiogenic activity, which prevents an increase in tumour size. This review covers recent developments in the involvement of ADAM and ADAMTS proteins in human cancer.

Brevican and phosphacan expression and localization following transient middle cerebral artery occlusion in the rat.

The ECM (extracellular matrix) is a complex molecular framework that provides physical support to cells and tissues, while also providing signals for cell growth, migration, differentiation and survival. The ECM of the CNS (central nervous system) is unusual in that it is rich in CSPGs (chondroitin sulfate proteoglycans), hyaluronan and tenascins. The CSPGs are widely expressed throughout the developing and adult CNS and have a role in guiding or limiting neurite outgrowth and cell migration. Alterations in the synthesis or breakdown of the ECM may contribute to disease processes. Here, we examine changes in the brain-specific CSPGs, brevican and phosphacan, following transient middle cerebral artery occlusion, a model of stroke in the rat. We have investigated their expression at various time points as well as their spatial relationship with ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs 4). The co-localization of ADAMTS or its activity may indicate a functional role for this matrix-protease pair in degeneration/regeneration processes that occur in stroke.

Upregulation of ADAM-17 expression in active lesions in multiple sclerosis.

ADAM-17, a disintegrin and metalloproteinase, is the major proteinase responsible for the cleavage of membrane-bound tumour necrosis factor (TNF) as well as being an active sheddase of other cytokines, cytokine receptors, growth factors and adhesion molecules. TNF is a major proinflammatory cytokine that has been identified as having a pathogenic role in inflammatory diseases within the CNS including multiple sclerosis (MS). Here we report the cellular origin and distribution of ADAM-17 expression within clinically and neuropathologically confirmed MS and normal control white matter, assessed by immunohistochemistry, western blotting and PCR. ADAM-17 expression was associated with the blood vessel endothelium, activated macrophages/microglia and parenchymal astrocytes in MS white matter. Increased levels of ADAM-17 immunoreactivity were displayed in active lesions with evidence of recent myelin breakdown. Further studies into the functional role of ADAM-17 in the pathogenesis of MS and other inflammatory conditions are required.

Expression of ADAMTS-1, -4, -5 and TIMP-3 in normal and multiple sclerosis CNS white matter.

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) -1, -4 and -5 proteases have been identified in the CNS at the mRNA level. These glutamyl endopeptidases, inhibited by tissue inhibitor of metalloproteinases (TIMP)-3, are key enzymes in the degradation of the aggregating chondroitin sulphate proteoglycans (CSPGs), and may therefore play a role in CNS extracellular matrix (ECM) changes in multiple sclerosis (MS). We have investigated ADAMTS and TIMP-3 expression in normal and MS CNS white matter by real-time RT-PCR, western blotting and immunohistochemistry. We report for the first time the presence of ADAMTS-1, -4 and -5 in normal and MS white matter. Levels of ADAMTS-1 and -5 mRNA were decreased in MS compared to normal tissue, with no significant change in ADAMTS-4 mRNA levels. Protein levels of ADAMTS-4 were significantly higher in MS tissue compared to normal tissue. Immunohistochemical studies demonstrated that ADAMTS-4 was associated predominantly with astrocytes with increased expression within MS lesions. TIMP-3 mRNA was significantly decreased in MS compared to controls. These studies suggest a role for ADAMTS-4 in the pathogenesis of MS. Further studies on the activity of ADAMTS-4 will enable a better understanding of its role in the turnover of the ECM of white matter in MS.

ADAMTS-1 and -4 are up-regulated following transient middle cerebral artery occlusion in the rat and their expression is modulated by TNF in cultured astrocytes.

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) enzymes are a recently described group of metalloproteinases. The substrates degraded by ADAMTS-1, -4 and -5 suggest that they play a role in turnover of extracellular matrix in the central nervous system (CNS). ADAMTS-1 is also known to exhibit anti-angiogenic activity. Their main endogenous inhibitor is tissue inhibitor of metalloproteinases (TIMP)-3. The present study was designed to investigate ADAMTS-1, -4 and -5 and TIMP-3 expression after experimental cerebral ischaemia and to examine whether cytokines known to be up-regulated in stroke could alter their expression by astrocytes in vitro. Focal cerebral ischaemia was induced by transient middle cerebral artery occlusion in the rat using the filament method. Our results demonstrate a significant increase in expression of ADAMTS-1 and -4 in the occluded hemisphere but no significant change in TIMP-3. This was accompanied by an increase in mRNA levels for interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1ra) and tumour necrosis factor (TNF). ADAMTS-4 mRNA and protein were up-regulated by TNF in primary human astrocyte cultures. The increased ADAMTS-1 and -4 in experimental stroke, together with no change in TIMP-3, may promote ECM breakdown after stroke, enabling infiltration of inflammatory cells and contributing to brain injury. In vitro studies suggest that the in vivo modulation of ADAMTS-1 and -4 may be controlled in part by TNF.

Differential expression of ADAMTS-1, -4, -5 and TIMP-3 in rat spinal cord at different stages of acute experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of inflammatory demyelination, a pathological event common to multiple sclerosis (MS). During CNS inflammation there are alterations in the extracellular matrix (ECM). A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS)-1, -4 and -5 are proteases present in the CNS, which are able to cleave the aggregating chondroitin sulphate proteoglycans, aggrecan, phosphacan, neurocan and brevican. It is therefore important to investigate changes in their expression in different stages of EAE induction. We have investigated expression of ADAMTS-1, -4, -5 and tissue inhibitor of metalloproteinase (TIMP)-3, by real-time RT-PCR. We have also examined protein expression of ADAMTS-1, -4 and -5 by western blotting and immunocytochemistry in spinal cord from animals at different stages of disease progression. Our study demonstrated a decrease in ADAMTS-4 mRNA and protein expression. TIMP-3 was decreased at the mRNA level although protein levels were increased in diseased animals compared to controls. Our study identifies changes in ADAMTS expression during the course of CNS inflammation which may contribute to ECM degradation and disease progression.

Acute effect of high-calcium milk with or without additional magnesium, or calcium phosphate on parathyroid hormone and biochemical markers of bone resorption.

OBJECTIVE: To assess whether there are any differences in the postprandial physiological responses to apple drink (control), calcium phosphate (tricalcium phosphate, TCP) and high-calcium skim milk (HCSM) with or without additional magnesium in postmenopausal women. DESIGN: Randomized, controlled, cross-over. Measurements after overnight fast before each drink, and subsequently every hour for 8 h postprandially. SETTING: Human Nutrition Studies Laboratory, Milk and Health Research Centre, Massey University, Palmerston North, New Zealand. SUBJECTS: Twenty-one healthy postmenopausal women. INTERVENTION: Four drinks, each 400 ml. (1) Apple drink (25% fruit juice). (2) TCP dispersed in water containing 1200 mg Ca. (3) HCSM containing 1200 mg Ca and 65.5 mg Mg. (4) HCSM containing 1200 mg Ca and 172 mg Mg. RESULTS: There was no difference in baseline serum calcium, PTH or C-telopeptide levels between drinks. There were no overall differences in serum calcium after apple or after either milk, but after TCP serum calcium increased from a baseline value of 2.12+/-0.08 to a mean peak of 2.21+/-0.12 mmol/l (s.d.) (P=0.0001) after 2 h. There were no significant differences in serum PTH after either apple or HCSM+Mg. In contrast, after TCP, serum PTH decreased from 2.76+/-0.69 to a mean nadir of 2.23+/-0.65 pmol/l (P=0.0001) after 1 h, and after HCSM, it decreased from 2.71+/-0.78 to a mean nadir of 2.51+/-0.87 pmol/l (P=0.007) after 2 h. Serum C-telopeptides decreased after each drink, reaching nadirs after 5 h. At this time the serum values for each of the high calcium drinks were not different from each other, but were significantly less than for apple (P=0.001 for each), being 0.22+/-0.09 ng/ml for apple, 0.15+/-0.08 for TCP, 0.14+/-0.07 for HCSM and 0.16+/-0.07 for HCSM+Mg. CONCLUSION: Despite differences in serum calcium and PTH responses to the three high-calcium drinks that we tested, there was no distinguishable difference in serum C-telopeptides between high calcium drinks.

Astrocyte and endothelial cell expression of ADAM 17 (TACE) in adult human CNS.

ADAM 17, also known as TACE, is an important sheddase for a number of proteins, including tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-alpha (TGF-alpha), L-selectin, p75, and p55 TNF receptors, and interleukin-1 receptor II (IL-1RII). The presence of ADAM 17 mRNA in adult mouse and rat CNS was recently reported (Karkkainen et al. Mol Cell Neurosci 15:547-560, 2000). However, the cellular origin of ADAM 17 remains unknown. In this study, we have used an anti-ADAM 17 antibody in an immunohistochemical study of its distribution in human adult CNS tissue. Cells with astrocytic and endothelial morphology were ADAM 17-positive. This finding was further confirmed using double immunofluorescence with antibodies against GFAP and von Willebrand factor, which label astrocytes and endothelial cells, respectively. This study demonstrates that ADAM 17 is expressed by astrocytes and endothelial cells in normal brain tissue and may have a role in normal brain function.

Blood pressure responses to high-calcium skim milk and potassium-enriched high-calcium skim milk.

OBJECTIVE: This study was designed to evaluate the effect of high-calcium skim milk or potassium-enriched high-calcium skim milk on blood pressure compared with nonenriched skim milk. DESIGN: This was a randomized double-blind controlled trial. Each milk intervention lasted for 4 weeks, with a minimum of 4 weeks of wash-out between interventions. METHODS: We recruited 38 healthy people, aged over 40 years, to take part in a double-blind, randomized, controlled cross-over study. We asked them to replace their usual liquid milk with two servings per day of skim milk (control), high-calcium skim milk or potassium-enriched high-calcium skim milk. We measured office blood pressures (seated and standing) at the start and after 2 and 4 weeks of milk intervention and we measured daytime ambulatory blood pressures at the start and after 4 weeks of milk intervention. Each milk intervention was interspaced by a 4-week interval. RESULTS: Office systolic blood pressure (standing) decreased from 127 +/- 16 to 124 +/- 16 mmHg (P<0.05) after 4 weeks of skim milk and from 130 +/- 18 to 126 +/- 17 mmHg (P<0.05) after 4 weeks of high calcium skim milk. After 4 weeks of consuming the potassium-enriched high-calcium milk, systolic blood pressure decreased from 125 +/- 18 to 117 +/- 16 mmHg (P<0.001) seated, and from 130 +/- 16 to 122 +/- 15 mmHg (P<0.001) standing. There were no significant changes in office diastolic blood pressure after any milk. There was no change in ambulatory blood pressure after either skim milk or high-calcium skim milk. After 4 weeks of potassium-enriched high-calcium milk, ambulatory daytime systolic blood pressure decreased from 138 +/- 13 to 135 +/- 11 mm Hg (P<0.05) and daytime diastolic blood pressure decreased from 80 +/- 8 to 78 +/- 9 mmHg (P<0.05). CONCLUSIONS: High-calcium milk enriched with potassium has a small hypotensive effect in healthy people aged over 40 years.

Evidence that the inhibition of cartilage proteoglycan breakdown by mannosamine is not mediated via inhibition of glycosylphosphatidylinositol anchor formation.

The effect of mannosamine, an inhibitor of glycosylphosphatidylinositol (GPI) anchor formation, on chondrocyte-mediated cartilage proteoglycan breakdown was investigated using cartilage explant cultures. Mannosamine inhibited interleukin 1alpha-, tumour necrosis factor alpha- and retinoic acid-stimulated proteoglycan release from bovine nasal and articular cartilage, and retinoic acid-stimulated proteoglycan release from human cartilage. Its effects on two GPI-anchored proteins [the urokinase receptor, which binds urokinase-type plasminogen activator (uPA) to cell surfaces, and alkaline phosphatase] were also studied using bovine chondrocytes. Enzyme histochemistry and zymography demonstrated cell-associated uPA-like serine proteinase activity and PA activity respectively which was not reduced by treatment of chondrocytes with mannosamine at concentrations effective at inhibiting cartilage proteoglycan breakdown. Similarly, the activity of cell-associated alkaline phosphatase was not reduced, except at mannosamine concentrations much higher than those used to inhibit proteoglycan breakdown. These results demonstrate that inhibition of proteoglycan breakdown by mannosamine is too potent to be explained by an effect on GPI-anchor formation.

Metalloproteinases and tissue inhibitor of metalloproteinase 1 (TIMP-1) in endometrial flushings from pre- and post-menopausal women and from women with endometrial adenocarcinoma.

The presence of metalloproteinase activity in endometrial flushings obtained from premenopausal women, during the proliferative and secretory phases of the menstrual cycle, control post-menopausal women and women with post-menopausal bleeding (PMB) with or without adenocarcinoma was analysed by zymography. In addition, quantitative measurements of matrix metalloproteinase 2 (MMP-2), MMP-3, MMP-9 and tissue inhibitor of metalloproteinase 1 (TIMP-1) in the flushings were obtained by ELISA. The zymography results showed eight bands of activity, with molecular weights ranging from 51 to 208 kDa in the flushings from pre-menopausal women and post-menopausal women, particularly those with adenocarcinoma. Both zymography and ELISA showed that MMP-2 and MMP-9 were the major metalloproteinases found in the flushings and only low concentrations of MMP-3 were found. Concentrations of MMP-2 in pre-menopausal women were higher in flushings obtained during the secretory phase of the menstrual cycle than those obtained in the proliferative phase (P < 0.05), suggesting that it may play a role in embryo implantation. Concentrations of MMP-2 (P < 0.001), MMP-9 (P < 0.05) and TIMP-1 (P < 0.001) in the flushings from post-menopausal control women were lower than those from pre-menopausal women. Concentrations of MMP-2 (P < 0.05) and TIMP-1 (P < 0.05) were higher in flushings from women with PMB without carcinoma compared with post-menopausal controls and concentrations of MMP-9 (P < 0.01) and TIMP-1 (P < 0.05) in flushings from women with adenocarcinoma were higher than in post-menopausal controls. Among subjects with PMB, concentrations of MMP-9 in women with adenocarcinoma were higher than those without carcinoma (P < 0.05). Our results show that concentrations of MMP-2, MMP-9 and TIMP-1, but not MMP-3, are associated with endometrial activity and, therefore, may have a role in the breakdown of endometrial tissue. In addition, the increased concentrations of MMP-9 in flushings of women with adenocarcinoma indicate that this particular proteinase is associated with the presence of endometrial neoplastic cells.

The effect of suramin on the resorption of bovine nasal cartilage.

Suramin is an anti-neoplastic drug. Its actions include the inhibition of binding of urokinase-type plasminogen activator (uPA) to its receptor, an event which may prevent cartilage breakdown. The aim of this work was to determine the effect of suramin on cartilage resorption. Cartilage expiants, stimulated with interleukin-1alpha, tumour necrosis factor-alpha or retinoic acid were incubated with suramin. Release of incorporated (35)S-sulphate from pre-labelled expiants was used as a measure of proteoglycan breakdown and toluidine blue staining was used to visualise proteoglycan loss.Suramin inhibited the resorption of cytokine and retinoic acid-stimulated bovine nasal cartilage at concentrations between 100-1000 microM. These findings were confirmed by histochemistry. Though reversibility studies indicated that suramin toxicity could not be excluded above 100 muM, retention of suramin in the expiants may have contributed to this. There was no significant effect on lactate production up to 500 muM. The observed inhibition of cartilage resorption may reflect actions of suramin on the PA/plasmin system or on cytokine action.

Cognitive and physical measures in rehabilitation of patients with lupus.

During the past 2 years, four papers describing new research studies and three papers reviewing the literature were published. These seven papers address longstanding issues, including 1) the presence or absence of cognitive impairment in systemic lupus erythematosus (SLE), 2) the prevalence of such impairment within SLE and related conditions (eg, antiphospholipid antibody syndrome, active vs inactive SLE), 3) the specific pattern of cognitive functions impaired and the possibility of a "lupus-specific" pattern, 4) the etiopathogenesis of cognitive impairments and the potential role of confounding factors such as corticosteroid use or depression, and 5) longitudinal studies examining the course of SLE-related cognitive impairment. The review period also includes two studies using both static and functional neuroimaging to identify the potential relation between neuroanatomic or neurophysiologic abnormalities and cognitive impairment in patients with SLE. No papers specific to Sjögren's syndrome were identified.

A serine proteinase inactivator inhibits chondrocyte-mediated cartilage proteoglycan breakdown occurring in response to proinflammatory cytokines.

The role played by serine proteinases with trypsin-like specificity in chondrocyte-mediated cartilage proteoglycan breakdown was investigated by use of a selective proteinase inactivator, 7-amino-4-chloro-3-(-3-isothiureidopropoxy)isocoumarin, in explant culture systems. This compound was a rapid inactivator of urokinase-type plasminogen activator. It potently inhibited interleukin 1- and tumor necrosis factor-stimulated proteoglycan release from both nasal and articular cartilage. Its less potent inhibition of basal and retinoic acid-stimulated release appeared to be due to cytotoxic effects. The functional half-life of the inactivator in culture medium was 95 min, and its concentration in cartilage was 2.5-fold higher than in the surrounding medium. Following spontaneous hydrolysis the breakdown products of the inactivator were unable to inhibit proteoglycan release. Trypsin-like activity was demonstrated by enzyme histochemistry to be chondrocyte-associated and inhibited by the serine proteinase inactivator. Cell-associated and secreted plasminogen activator activity was detected by zymography. These results suggest the involvement of a serine proteinase(s) with trypsin-like specificity, possibly urokinase-type plasminogen activator, in chondrocyte-mediated cartilage proteoglycan breakdown occurring as a result of stimulation with proinflammatory cytokines. Basal proteoglycan breakdown may occur via a different pathway. Our findings point to a pathological role for serine proteinase(s) in the development of cartilage diseases such as arthritis, possibly in a cascade which results in the activation of the enzyme(s) directly responsible for proteoglycan breakdown. It remains to be shown whether the target serine proteinase is urokinase-type plasminogen activator.

Effects of transforming growth factor beta on the production of prostaglandin E and caseinase activity of unstimulated and interleukin 1-stimulated human articular chondrocytes in culture.

Transforming growth factor beta (TGF beta) has previously been shown to have actions on chondrocytes and cartilage both in vitro and in vivo which suggest a role in connective tissue repair. In particular, some of its actions have been shown to be antagonistic to those of interleukin 1 (IL-1). In this study, the effects of TGF beta on prostaglandin E (PGE) production and caseinase activity, in the presence and absence of IL-1, in human articular chondrocytes were investigated. TGF beta 1 and TGF beta 2 were shown to modulate IL-1 beta-stimulated PGE production and caseinase activity. Both TGF beta 1 and beta 2 inhibited IL-1 beta-stimulated PGE production in the absence of serum and augmented it in the presence of serum. TGF beta 1 and TGF beta 2 inhibited IL-1-stimulated caseinase activity with and without serum. In general, the TGF beta s had little or no effect on basal PGE or caseinase levels. TGF beta s may be important modulators of chondrocyte metabolism, their effects on PGE production may depend on cytokine interactions; furthermore, their effects on caseinase activity may help prevent cartilage breakdown.

Plasminogen activators and their inhibitors in synovial fluids from normal, osteoarthritis, and rheumatoid arthritis knees.

OBJECTIVES: To establish baseline concentrations of plasminogen activators and their inhibitors in normal knee synovial fluids, and to compare them with well characterised osteoarthritis (OA) and rheumatoid arthritis (RA) knee fluids. METHODS: A total of 26 normal subjects, 71 patients with OA, and 17 patients with RA underwent knee aspiration. Patients with OA were subclassified according to presence of nodal generalised OA (NGOA) and synovial fluid calcium pyrophosphate crystals. Clinical assessment of inflammation (graded 0-6) was undertaken in OA and RA patients. Plasminogen activator (PA), plasminogen activator inhibitor (PAI), and urokinase-type PA receptor (uPAR) antigen concentrations were determined by enzyme linked immunosorbent assay. The species of PAs present were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. RESULTS: Concentrations of all antigens (uPA, tissue-type PA (tPA), uPAR, and PAI-1), were significantly greater in RA than OA; those in OA were significantly greater than normal. The concentrations showed no direct association with clinically assessed inflammation of the knee. In normal fluids, no associations with age were observed. Antigen concentrations (uPA, tPA, and uPAR) in NGOA differed from those in other subclasses of OA, but the species of PA present did not appear to vary between disease groups. The predominant PA appeared to have identity with uPA. CONCLUSION: Because of the greater concentrations of these antigens in OA compared with normal fluids, OA cannot be used as a surrogate normal control in studies of the PA/PAI system. Alteration of the PA/PAI system was confirmed in RA and OA knee fluids, with greater changes evident in RA. The finding of different concentrations of PA antigens in NGOA compared with other OA fluids further supports a different pathogenic mechanism in this subset.