Agonists of peroxisome proliferators-activated receptors (PPAR) alpha, beta/delta or gamma reduce transforming growth factor (TGF)-beta-induced proteoglycans' production in chondrocytes.

OBJECTIVE: To investigate the potency of selective agonists of peroxisome proliferators-activated receptors' (PPAR) isotypes (alpha, beta/delta or gamma) to modulate the stimulating effect of transforming growth factor-beta1 (TGF-beta1) on proteoglycans' (PGs) synthesis in chondrocytes. METHOD: Rat chondrocytes embedded in alginate beads and cultured under low serum conditions were exposed to TGF-beta1 (10 ng/ml), alone or in combination with the following agonists: Wy14643 for PPARalpha, GW501516 for PPARbeta/delta, rosiglitazone (ROSI) for PPARgamma, in the presence or absence of PPAR antagonists (GW6471 for PPARalpha, GW9662 for PPARgamma). PGs' synthesis was evaluated by radiolabelled sulphate incorporation and glycosaminoglycans' (GAGs) content by Alcian blue staining of beads and colorimetric 1.9 dimethyl-methylene blue assay after beads' solubilization. Phosphorylation of Extracellular Signal-related Kinase1/2 (ERK1/2), Smad2/3 and p38-MAPK was assessed by Western Blot and production of prostaglandin E2 (PGE2) by Enzyme immuno-assay (EIA). Levels of mRNA for PPAR target genes [acyl-CoA oxidase (ACO) for PPARalpha; mitochondrial carnitin palmitoyl transferase-1 (CPT-1) for PPARbeta/delta and adiponectin for PPARgamma], aggrecan, TGF-beta1 and genes controlling GAGs' side chains' synthesis were quantified by real time polymerase chain reaction and normalized over RP29 housekeeping gene. RESULTS: ACO was selectively up-regulated by 100 microM of Wy14643, CPT-1 by 100 nM of GW501516 and adiponectin by 10 microM of ROSI without cell toxicity. TGF-beta1 increased PGs' synthesis by four-fold, GAGs' content and deposition by 3.5-fold and six-fold, respectively, while inducing aggrecan expression around 10-fold without modifying mRNA levels of GAGs' controlling enzymes. PPAR agonists inhibited the stimulating effect of TGF-beta1 by 24-44% on PGs' synthesis and over 75% on aggrecan, GAGs' content and deposition with the following rank order of potency: ROSI>GW501516> or =Wy14643. TGF-beta1-induced phosphorylation of Smad2/3 and ERK1/2 was reduced by ROSI over GW501516 but not by Wy14643 whereas stimulated PGE2 production was inhibited by Wy14643 over GW501516 but not by ROSI. The effect of PPAR agonists on PPAR target genes and TGF-beta1-induced aggrecan expression was reversed selectively by PPAR antagonists. CONCLUSION: In chondrocytes' beads, PPAR agonists reduced the stimulating effect of TGF-beta1 on PGs by inhibiting TGF-beta1-induced aggrecan expression in an isotype-selective manner. Thus, PPAR agonists could be deleterious in situation of cartilage repair although being protective in situation of cartilage degradation.

Differential distribution of adipokines between serum and synovial fluid in patients with osteoarthritis. Contribution of joint tissues to their articular production.

OBJECTIVE: To analyze the distribution of leptin, adiponectin and resistin between paired serum and synovial fluid (SF) samples of patients with osteoarthritis (OA) and to determine the potential sources of these adipokines in the joint. The active free form of leptin was also examined by evaluating the level of the soluble leptin receptor (sOb-R). METHODS: Levels of adipokines and sOb-R were measured by a sandwich enzyme-linked immunosorbent assay in serum and SF collected from OA patients. The levels of adipokines were also determined in conditioned media from cultured joint tissues (synovium, infrapatellar fat pad, meniscus, osteophyte, cartilage and bone). RESULTS: The adipokines exhibited different patterns of distribution between the joint and the circulating compartment. Serum levels of resistin and adiponectin exceeded those in the paired SF. Conversely, leptin SF concentrations were similar or higher than those measured in serum counterparts. Leptin and adiponectin in SF may derive from each joint tissue examined, whereas resistin was not detected in conditioned media of cultured explants. Synovium and infrapatellar fat pad were the major sources of adipokines, but osteophytes released also large amounts of leptin. The sOb-R deficiency found in SF further increased the difference in the bioactive leptin levels between serum and SF. A gender-specific difference was observed with women exhibiting the highest level of free leptin in the joint. CONCLUSION: These data demonstrated that adipokines serum levels are not predictive values for SF determination. The joint cavity is a special space where each adipokine undergoes specific regulatory pathways, strengthening the hypothesis that adipokines may have local effects in the joint and may account for the high prevalence of OA in women.

[Is leptin the missing link between osteoarthritis and obesity?]. / La leptine est-elle le maillon manquant entre l'arthrose et l'obésité?

The contribution of leptin, as a possible link between osteoarthritis (OA) and obesity, was studied in cartilage and synovial fluid samples obtained from osteoarthritic patients. Its effect on cartilage was evaluated in rats after intraarticular injections of leptin. Leptin levels were measured in the synovial fluid samples by enzyme linked immunosorbent assay; leptin concentrations were correlated with the body mass index. Leptin was strongly expressed in osteophytes and OA cartilage, while, in normal cartilage, few chondrocytes produced leptin. The level of leptin expression was related to the grade of cartilage destruction and was in good relation with those of growth factors as IGF1 and TGFb. Studies in rats showed that intraarticular leptin injection stimulated anabolic functions of chondrocytes and induced the synthesis of leptin, IGF1 and TGFB in cartilage at both the chondrocytes and induced the synthesis of leptin, IGF1 and TGFB in cartilage at both the mRNA and protein levels. In conclusion, leptin may be a link between osteoarthritis and obesity, and may play a key role in cartilage metabolism. Leptin may contribute to the pathophysiology of OA.

Site specific changes in gene expression and cartilage metabolism during early experimental osteoarthritis.

OBJECTIVES: To characterize the molecular events underlying cartilage injury in the early phase of mono-iodoacetate-induced osteoarthritis (OA) in rats. METHODS: Experimental osteoarthritis was induced by intra-articular injection of 0.03mg mono-iodoacetate (MIA) in Wistar rats. Animals were killed 2, 5, 10, 15 and 20 days post-injection. Synovial tissue and standardized biopsies from different areas of knee cartilage were examined. Proteoglycan synthesis ((35)S incorporation) and gelatinase activities (zymography), semi-quantitative RT-PCR and immunohistochemistry for IL1beta, iNOS, COX2 and PPARgamma, were performed on these samples. RESULTS: Changes in proteoglycan synthesis and gelatinase activities were time and site-dependent. Proteoglycan synthesis inhibition was maximal by day 2 while the highest gelatinase activities were observed at day 5. Central part of patella and posterior plateaus and condyles, i.e. the weight-bearing cartilage areas, were the most affected. IL1beta and iNOS transcripts were induced early in cartilage at time of maximal proteoglycan synthesis inhibition, especially in weight-bearing areas. COX-2 was slightly up-regulated whereas PPARgamma gene expression remained unchanged. Gene expression profile in synovium paralleled that of cartilage, except for PPARgamma which was up-regulated at day 15 and 20. Immunostaining for IL1beta and iNOS showed that proteins were located in diseased cartilage areas at early stage of the experimental OA (day 2). At later time-points (day 20), IL1beta and iNOS were expressed in perilesional areas whereas immunostaining became below control level for COX-2 and PPARgamma. CONCLUSIONS: Time-dependent degradation of cartilage after injection of low dose of MIA (0.03mg) into rat knee joint can be related to early loss of proteoglycan anabolism, increased gelatinase activities and expression of IL1beta and downstream inflammatory genes. Increased susceptibility to MIA in weight-bearing areas of cartilage further indicate that MIA-induced experimental OA is a relevant model to study not only metabolical but also biomechanical aspects of human OA.

Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-kappa B pathway.

We recently reported that glucosamine reversed the decrease in proteoglycan synthesis and in UDP-glucuronosyltransferase I mRNA expression induced by interleukin-1 beta (IL-1 beta) [Arthritis Rheum. 44 (2001) 351-360]. In the present work, we show that glucosamine does not exert the same effects when chondrocytes were stimulated with reactive oxygen species (ROS). In order to better understand its mechanism of action, we determined if glucosamine could prevent the binding of IL-1 beta to its cellular receptors or could interfere with its signaling pathway at a post-receptor level. Addition of glucosamine to rat chondrocytes treated with IL-1 beta or with ROS decreased the activation of the nuclear factor kappa B, but not the activator protein-1. After treatment with IL-1 beta, glucosamine increased the expression of mRNA encoding the type II IL-1 beta receptor. These results emphasize the potential role of two regulating proteins of the IL-1 beta signaling pathway that could account for the beneficial effect of glucosamine in osteoarthritis.

15-Deoxy-delta12,14-PGJ2, but not troglitazone, modulates IL-1beta effects in human chondrocytes by inhibiting NF-kappaB and AP-1 activation pathways.

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to inhibit the production and the effects of proinflammatory cytokines. Since interleukin-1beta (IL-1beta) directly mediates cartilage degradation in osteoarthritis, we investigated the capability of PPARgamma ligands to modulate IL-1beta effects on human chondrocytes. RT-PCR and Western blot analysis revealed that PPARgamma expression was decreased by IL-1beta. 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), in contrast to troglitazone, was highly potent to counteract IL-1beta-induced cyclooxygenase-2 and inductible nitric oxide synthase expression, NO production and the decrease in proteoglycan synthesis. Western blot and gel-shift analyses demonstrated that 15d-PGJ2 inhibited NF-kappaB activation, while troglitazone was ineffective. Although 15d-PGJ2 attenuated activator protein-1 binding on the DNA, it potentiated c-jun migration in the nucleus. The absence or the low effect of troglitazone suggests that 15d-PGJ2 action in human chondrocytes is mainly PPARgamma-independent.

Stimulation of cyclooxygenase-2-activity by nitric oxide-derived species in rat chondrocyte: lack of contribution to loss of cartilage anabolism.

Cross-talk between inducible nitric oxide synthase (NOS II) and cyclooxygenase-2 (COX-2) was investigated in rat chondrocytes. In monolayers, interleukin-1beta (IL-1beta) induced COX-2 and NOS II expression in a dose- and time-dependent manner, to produce high prostaglandin E(2) (PGE(2)) and nitrite (NO(2)(-)) levels in an apparently coordinated fashion. COX-2 mRNA was induced earlier (30 min. versus 4 hr) and less markedly (4-fold versus 12-fold at 24 hr) than NOS II, and was poorly affected by the translational inhibitor cycloheximide (CHX). IL-1beta did not stabilize COX-2 mRNA in contrast to CHX. Indomethacin and NS-398 lacked any effect on NO(2)(-) levels whereas L-NMMA and SMT reduced PGE(2) levels at concentration inhibiting NO(2)(-) production from 50 to 90%, even when added at a time allowing a complete expression of both enzymes (8 hr). Basal COX activity was unaffected by NO donors. The SOD mimetic, CuDips inhibited COX-2 activity by more than 75% whereas catalase did not. Inhibition of COX-2 by CuDips was not sensitive to catalase, consistent with a superoxide-mediated effect. In tridimensional culture, IL-1beta inhibited radiolabelled sodium sulphate incorporation while stimulating COX-2 and NOS II activities. Cartilage injury was corrected by L-NMMA or CuDips but not by NSAIDs, consistent with a peroxynitrite-mediated effect. These results show that in chondrocytes: (i) COX2 and NOS II genes are induced sequentially and distinctly by IL-1beta; (ii) COX-1 and COX-2 activity are affected differently by NO-derived species; (iii) peroxynitrite accounts likely for stimulation of COX-2 activity and inhibition of proteoglycan synthesis induced by IL-1beta.

Interleukin-1beta down-regulates the expression of glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis: influence of glucosamine on interleukin-1beta-mediated effects in rat chondrocytes.

OBJECTIVE: To assess the variations of galactose-beta-1,3-glucuronosyltransferase I (GlcAT-I) expression related to the decrease in proteoglycan synthesis mediated by interleukin-1beta (IL-1beta) in rat chondrocytes, and to evaluate the influence of glucosamine on the effects elicited by this proinflammatory cytokine. METHODS: Rat articular chondrocytes in primary monolayer cultures or encapsulated into alginate beads were treated with recombinant IL-1beta in the absence or presence (1.0-4.5 gm/liter) of glucosamine. Variations of GlcAT-I and expression of stromelysin 1 (matrix metalloproteinase 3 [MMP-3]) messenger RNA (mRNA) were evaluated by quantitative multistandard reverse transcriptase-polymerase chain reaction. In vitro enzymatic activity of GlcAT-I was measured by thin-layer chromatography, with radiolabeled UDP-glucuronic acid and a digalactoside derivative as substrates. Proteoglycan synthesis was determined by ex vivo incorporation of Na2-35SO4. Nitric oxide synthase and cyclooxygenase activities were monitored by the evaluation of nitrite (NO2-) and prostaglandin E2 (PGE2) produced in the culture medium, respectively. RESULTS: IL-1beta treatment resulted in a marked inhibition of GlcAT-I mRNA expression and in vitro catalytic activity, together with a decrease in proteoglycan synthesis. In addition, glucosamine was able to prevent, in a dose-dependent manner, the inhibitory effects of IL-1beta. In the same way, the amino sugar reduced NO2- and PGE2 production induced by IL-1beta. Finally, the up-regulation of stromelysin 1 (MMP-3) mRNA expression by IL-1beta was fully prevented by glucosamine. CONCLUSION: The results of this study suggest that the deleterious effect of IL-1beta on the anabolism of proteoglycan could involve the repression of GlcAT-I, a key enzyme in the biosynthesis of glycosaminoglycan. Glucosamine was highly effective in preventing these IL-1beta-mediated suppressive effects. The amino sugar also prevented the production of inflammatory mediators induced by the cytokine. This action could account for a possible beneficial effect of glucosamine on osteoarthritic articular cartilage.

Neurophysiological basis for neurogenic-mediated articular cartilage anabolism alteration.

This study was designed to investigate the pathways involved in neurogenic-mediated articular cartilage damage triggered by a nonsystemic distant subcutaneous or intra-articular inflammation. The cartilage damage was assessed 24 h after subcutaneous or intra-articular complete Freund's adjuvant (CFA) injection measuring patellar proteoglycan (PG) synthesis (ex vivo [Na(2)(35)SO(4)] incorporation) in 96 Wistar rats. Unilateral subcutaneous or intra-articular injection of CFA induced significant decrease (25-29%) in PG synthesis in both patellae. Chronic administration of capsaicin (50 mg. kg(-1). day(-1) during 4 days), which blunted the normal response of C fiber stimulation, prevented the bilateral significant decrease in cartilage synthesis. Similarly, intrathecal injection of MK-801 (10 nmol/day during 5 days), which blocked the glutamatergic synaptic transmission at the dorsal horn of signal originating in primary afferent C fibers, eliminated the CFA-induced PG synthesis decrease in both patellae. Chemical sympathectomy, induced by guanethidine (12.5 mg. kg(-1). day(-1) during 6 wk), also prevented PG synthesis alteration. Finally, compression of the spinal cord at the T3-T5 level had a similar protective effect on the reduction of [Na(2)(35)SO(4)] incorporation. It is concluded that the signal that triggers articular cartilage synthesis damage induced by a distant local inflammation 1) is transmitted through the afferent C fibers, 2) makes glutamatergic synaptic connections with the preganglionic neurons of the sympathetic system, and 3) involves spinal and supraspinal pathways.

Effects of the inhibition of cyclo-oxygenase 1 or 2 or 5-lipoxygenase on the activation of the hypothalamic-pituitary-adrenal axis induced by interleukin-1beta in the male Rat.

The limited entry of interleukin-1beta (IL-1beta) into the central nervous system has led to the hypothesis that IL-1beta acts, through IL-1beta receptors located notably on endothelial cells, on the release of prostaglandins which in turn stimulate the hypothalamic-pituitary-adrenal (HPA) axis. We used cyclo-oxygenase-1 (COX-1) and cyclo-oxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibitors, before the injection of IL-1beta, to explore the role of arachidonic acid metabolic pathways on HPA axis activation. Adult male rats were i.m injected 20 min before i.p injection of IL-1beta, with (i): a COX-1/COX-2 inhibitor (ketoprofen); (ii) a COX-2 selective inhibitor (NS 398); or (iii) a 5-LOX inhibitor (BW A4C). Following this, rats were killed 90 min after i.p. IL-1beta injection and analysis for plasma adrenocorticotropic hormone (ACTH) and corticosterone concentrations and determination of anterior pituitary pro-opio melanocortin (POMC) gene transcription was conducted. Administration of the COX-1/COX-2 inhibitor led to a complete blockage of ACTH and corticosterone secretion and POMC gene transcription. The COX-2 inhibitor led to a complete blockade of ACTH secretion and POMC gene transcription but had no effect on corticosterone secretion. The 5-LOX inhibitor had no significant effect on any parameter. These results demonstrate the crucial role of eicosanoid pathways in mediating the stimulation of the HPA axis induced by IL-1beta. Moreover, we found a clear dissociation of the effect of the blockage of COXs upon ACTH and corticosterone secretion, suggesting that IL-1beta may act at the brain as well as at the adrenal cortex to stimulate the secretion of corticosterone.

The role of 3D-microscopy in the study of chondrocyte-matrix interaction (alginate bead or sponge, rat femoral head cap, human osteoarthritic cartilage) and pharmacological application.

The potentialities of a new non-invasive optical scanning microscopy technique were evaluated through 3D analysis of chondrocyte-matrix interactions. Five different 2D or 3D culture systems were used: (1) MonoLayer (ML) of human chondrosarcoma cell line; (2) rat or human chondrocytes encapsulated in Alginate Bead (AB); (3) human chondrocytes encapsulated in Alginate Sponge (AS); (4) Rat Femoral Head Cap (RFHC); (5) slices of knee human Osteoarthritic Cartilage (HOAC). Chondrocytes ML, AB, RFHC were incubated for 24 h in vitro in the presence of recombinant human interleukin1-beta (rhIL1-beta) and the effects on cytoskeleton organisation (F-actin filament), Focal Adhesion Kinase (FAK) expression (tyrosine kinase), collagenase B expression (metalloprotease) were studied. Furthermore, the production of intracellular IL1-beta by LPS- or rhIL1-beta-stimulated chondrocytes was shown to be partly suppressed by rhein (active metabolite of diacerhein) in all culture systems. This high resolution light microscopy gave complementary information that could be important for a better understanding of the interaction of chondrocytes with the extracellular matrix in a variety of culture devices.

Evidence for the presence of peroxisome proliferator-activated receptor (PPAR) alpha and gamma and retinoid Z receptor in cartilage. PPARgamma activation modulates the effects of interleukin-1beta on rat chondrocytes.

Peroxisome proliferator-activated receptor (PPAR) alpha, PPARgamma, and retinoid acid receptor-related orphan receptor (ROR) alpha are members of the nuclear receptor superfamily of ligand-activated transcription factors. Although they play a key role in adipocyte differentiation, lipid metabolism, or glucose homeostasis regulation, recent studies suggested that they might be involved in the inflammation control and especially in the modulation of the cytokine production. This strongly suggests that these transcriptional factors could modulate the deleterious effects of interleukin-1 (IL-1) on cartilage. However, to date, their presence in cartilage has never been investigated. By quantitative reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry analysis, we demonstrated, for the first time, the presence of PPARalpha, PPARgamma, and RORalpha in rat cartilage, at both mRNA and protein levels. Comparatively, the PPARalpha mRNA content in cartilage was much lower than in the liver but not significantly different to that of the adipose tissue. PPARgamma mRNA expression in cartilage was weak, when compared with adipose tissue, but similar to that found in the liver. RORalpha mRNA levels were similar in the three tissues. mRNA expression of the three nuclear receptors was very differently modulated by IL-1 or mono-iodoacetate treatments. This indicates that they should be unequally involved in the effects of IL-1 on chondrocyte, which is in accordance with results obtained in other cell types. Indeed, we showed that 15d-PGJ2 mainly, but also the drug troglitazone, that are ligands of PPARgamma could significantly counteract the decrease in proteoglycan synthesis and NO production induced by IL-1. By contrast, PPARalpha ligands such as Wy-14,643 or clofibrate had no effect on this process. Therefore, the presence of PPARgamma in chondrocytes opens up new perspectives to modulate the effects of cytokines on cartilage by the use of specific ligands. The function of the two other transcription factors, PPARalpha and RORalpha identified in chondrocytes remains to be explored.

[Non-steroidal anti-inflammatory drugs with selectivity for cyclooxygenase-2 in Alzheimer's disease. Rationale and perspectives]. / Anti-inflammatoires non stéroïdiens sélectifs de la cyclooxygénase-2 pour la maladie d'Alzheimer. Justification et perspectives.

POSSIBLE INFLAMMATORY MECHANISMS: Alzheimer's disease (AD) is a degenerative disease of the brain including possibly inflammatory mechanisms, as illustrated by the presence of activated microglial cells in the periphery of senile plaques and neurofibrillary tangles and the subsequent release of proinflammatory mediators with neurotoxic potency. RATIONALE FOR NSAID USE: Although not firmly demonstrated, the rationale for the prescription of non steroidal anti-inflammatory drugs (NSAIDS) as neuroprotective agents in AD lies on epidemiological data having shown a reduced risk of developing AD in patients on long-term therapy with NSAIDs (non selective cyclo-oxygenase [COX] inhibitors). RATIONALE FOR THE USE OF SELECTIVE COX-2 INHIBITORS: The rationale for the prescription of selective COX-2 inhibitors as neuroprotective drugs in AD lies on: Epidemiological data having shown a reduced risk of developing AD in patients treated with anti-inflammatory doses of classical NSAIDs (inhibition of COX-1 and COX-2) but not with antithrombotic doses of aspirin (selective inhibition of COX-1), Cellular experiments, Demonstration of a better gastro-intestinal (GI) safety profile with selective COX-2 inhibitors than with classical NSAIDs in short-term studies, allowing a possible long-term use in AD. BEFORE PRESCRIBING: COX-2 may have an ambivalent functionality in the brain since the basal production of prostaglandins through COX-2 may participate in neuronal homeostasis whereas the expression of COX-2 is associated with brain development. Classical NSAIDs are ineffective in reducing the formation of senile plaque and neurofibrillary tangles in AD, which is consistent with an ability to reduce inflammation associated with activation of microglia but illustrates their failure to suppress the degenerative process. Prophylactic use of selective COX-2 NSAIDs can be considered on the basis of their good GI safety after 6 months of marketing in United States but need to be confirmed for a longer time. CURRENT TRIALS: Clinical studies focusing on both the prevention and the slowing down of early AD are under way with two recently launched selective COX-2 inhibitors, celecoxib and rofecoxib.

Pefloxacin-induced achilles tendon toxicity in rodents: biochemical changes in proteoglycan synthesis and oxidative damage to collagen.

Despite a relatively low incidence of serious side effects, fluoroquinolones and the fluoroquinolone pefloxacin have been reported to occasionally promote tendinopathy that might result in the complication of spontaneous rupture of tendons. In the present study, we investigated in rodents the intrinsic deleterious effect of pefloxacin (400 mg/kg of body weight) on Achilles tendon proteoglycans and collagen. Proteoglycan synthesis was determined by measurement of in vivo and ex vivo radiosulfate incorporation in mice. Collagen oxidative modifications were measured by carbonyl derivative detection by Western blotting. An experimental model of tendinous ischemia (2 h) and reperfusion (3 days) was achieved in rats. Biphasic changes in proteoglycan synthesis were observed after a single administration of pefloxacin, consisting of an early inhibition followed by a repair-like phase. The depletion phase was accompanied by a marked decrease in the endogenous serum sulfate level and a concomitant increase in the level of sulfate excretion in urine. Studies of ex vivo proteoglycan synthesis confirmed the in vivo results that were obtained. The decrease in proteoglycan anabolism seemed to be a direct effect of pefloxacin on tissue metabolism rather than a consequence of the low concentration of sulfate. Pefloxacin treatment for several days induced oxidative damage of type I collagen, with the alterations being identical to those observed in the experimental tendinous ischemia and reperfusion model. Oxidative damage was prevented by coadministration of N-acetylcysteine (150 mg/kg) to the mice. These results provide the first experimental evidence of a pefloxacin-induced oxidative stress in the Achilles tendon that altered proteoglycan anabolism and oxidized collagen.

Proteoglycan and collagen biochemical variations during fluoroquinolone-induced chondrotoxicity in mice.

Although fluoroquinolone antibacterials have a broad therapeutic use, with a relatively low incidence of severe side effects, they have been reported to induce lesions in the cartilage of growing animals by a mechanism that remains unclear. This study was undertaken to determine the potentially deleterious effect of a high dose of pefloxacin (400 mg/kg of body weight) on two main constituents of cartilage in mice, i.e., proteoglycans and collagen. Variations in levels of proteoglycan anabolism measured by in vivo [(35)S]sulfate incorporation into cartilage and oxidative modifications of collagen assessed by detection of carbonyl derivatives were monitored after administration of pefloxacin. Treatment of mice with 1 day of pefloxacin treatment significantly decreased the rate of biosynthesis of proteoglycan for the first 24 h. However, no difference was observed after 48 h. The decrease in proteoglycan synthesis was accompanied by a marked drop in serum sulfate concentration and a concomitant increase in urinary sulfate excretion. The decrease in proteoglycan synthesis, also observed ex vivo, may suggest a direct effect of pefloxacin on this process, rather than it being a consequence of a low concentration of sulfate. On the other hand, treatment with pefloxacin for 10 days induced oxidative damage to collagen. In conclusion, this study demonstrates, for the first time, that pefloxacin administration to mice leads to modifications in the metabolism and integrity of extracellular proteins, such as collagen and proteoglycans, which may account for the side effects observed. These results offer new insights to explain quinolone-induced disorders in growing articular cartilage.

Cartilage protection by nitric oxide synthase inhibitors after intraarticular injection of interleukin-1beta in rats.

OBJECTIVE: To evaluate the effect of nitric oxide synthase (NOS) inhibitors on proteoglycan synthesis following intraarticular administration of interleukin-1beta (IL-1beta) in rats. METHODS: Recombinant human IL-1beta and NOS inhibitors with different selectivity for inducible NOS (N-monomethyl-L-arginine [L-NMA], N-iminoethyl-L-ornithine [L-NIO], and S-methylisothiourea [SMT]) were simultaneously administered in rats by a single intraarticular injection in each knee. L-NMA was also infused for 72 hours using an Alzet mini osmotic pump implanted into the peritoneal cavity 24 hours before IL-1beta challenge. NO production was determined as nitrate and nitrite, either in synovial fluid or ex vivo in supernatants of synovium and patellae. Proteoglycan synthesis was measured by ex vivo incorporation of 35SO4(2-) into patellar cartilage. RESULTS: IL-1beta induced a time-dependent increase in NO production in synovial fluid. Synovium and patellae released large amounts of nitrate and nitrite under ex vivo conditions, indicating that both tissues are effective sources of NO within the joint. This production of NO was accompanied by a delayed inhibition of proteoglycan synthesis. The intraarticular administration of L-NMA and L-NIO reduced NO release in synovial fluid and resulted in a partial recovery of proteoglycan synthesis. Under our experimental conditions, SMT failed to reduce NO synthesis and to restore proteoglycan synthesis. The protection of cartilage was improved by the systemic and sustained delivery of L-NMA. However, the complete inhibition of NO production in synovial fluid was not sufficient to fully restore cartilage anabolism. CONCLUSION: Our findings show that in rats: 1) NO may be an early mediator of the effect of IL-1beta on cartilage, 2) NO inhibition may have therapeutic relevance, although it is not sufficient to fully reverse the deleterious effects of IL-1beta, 3) among NOS inhibitors tested, only amino acid derivatives are effective, 4) protection can be achieved by local administration of NOS inhibitors, and 5) systemic and sustained delivery of the NOS inhibitor with the highest efficacy after intraarticular injection provides the most benefit.

Evidence for neurogenic transmission inducing degenerative cartilage damage distant from local inflammation.

OBJECTIVE: To investigate involvement of the nervous system in ipsilateral and contralateral joint inflammation. METHODS: Freund's complete adjuvant (CFA; 1 mg or 1 microg) was injected unilaterally and the messages (a) from the hind paw to the ipsilateral and contralateral knees and (b) from one knee to the contralateral knee were analyzed. The degenerative impact of the local injury on distant cartilage was assessed using patellar proteoglycan synthesis as an indicator. Neurogenic mechanisms were blocked either by spinal cord compression or by injection of neurokinin 1 (NK-1) antagonist, or they were mimicked by intraarticular injection of substance P. The data were compared with those gathered in a model of systemic inflammation, characterized by fever and serum interleukin-6 production. RESULTS: After unilateral subcutaneous injection of CFA, proteoglycan anabolism decreased bilaterally. Spinal cord compression and administration of the NK-1 antagonist inhibited the response in the contralateral limb. Following 1 mg CFA subcutaneous injection, the ipsilateral response implicated both neurogenic and systemic mechanisms, whereas the nervous system alone was implicated after 1 microg subcutaneous CFA injection. The 1 microg CFA intraarticular injection induced a degenerative contralateral signal, which was abolished by spinal cord compression and by pretreatment with the NK-1 antagonist. Intraarticular injection of 1 microg CFA also induced an ipsilateral increase of anabolism, which was enhanced by spinal cord compression. Similar results were obtained after intraarticular injections of substance P. These effects were not reproduced with turpentine treatment, a systemic model, in which spinal cord compression had no effect. CONCLUSION: A unilateral inflammation can induce, by neurogenic mechanisms, distal bilateral degeneration of articular cartilage, implicating involvement of neuropeptides.

Biotelemetry: an original approach to experimental models of inflammation.

Biotelemetry is a new biological technology which evaluates continuous spontaneous locomotor activity and body temperature in rodents. The telemetry system comprises a transmitter implanted in the peritoneal cavity of the rodent, and a receiver placed beneath the animal's cage. The receiver detects the radio waves and the activity of the rodents as counts which are registered in the computer system, and the adapter detects the calibrated body temperature. First, we showed that biotelemetric studies of different species (rats, guinea pigs, mice and gerbils) provide substantial information about their circadian rhythms. Second, using the most common examples employed in pharmacology of inflammation (hyperthermia, arthritis, ischemia-reperfusion and so on) biotelemetry has helped us to clarify the pathophysiological significance of the parameters of temperature and mobility in several experimental models in rodents.

Modulation of IL-1 effects on cartilage by NO synthase inhibitors: pharmacological studies in rats.

Objective To compare the ability of L-arginine (L-arg) analog nitric oxide synthase (NOS) inhibitors and isothioureas to restore the interleukin-1 (IL-1) induced inhibition of proteoglycan (PG) synthesis in rat.Methods Chondrocytes beads and patellae were challenged with IL-1betain vitro and monitored for NO production and proteoglycan synthesis. Rats injected with IL-1beta in knee joints were monitored for NO(2)( - )+NO(3)( - )levels in joint tissues and ex-vivo(35)S sulfate incorporation in patellae. NOS inhibitors were either added to culture medium or injected concomittantly to IL-1beta. Results Ability of NOS inhibitors to reduce NO(2)( - )levels decreased from chondrocytes beads to patellae. Partial restoration of PG synthesis was restricted to L-arg analogs in patellae. After IL-1 injection, NO was produced from patella and synovium. L-arg analogs restored partly PG synthesis when decreasing significantly NO(2)( - )+NO(3)( - )levels in synovial fluid. Isothioureas were ineffective. Conclusions NO accounts importantly for IL-1 induced inhibition of cartilage anabolism in rat. L-arg analog NOS inhibitors are more effective than isothioureas in restoring PG synthesis and have chondroprotective potency when administered locally in diseased joint.