Association between adiponectin and cartilage degradation in human osteoarthritis.

OBJECTIVE: Conflicting findings raise questions about the role of adiponectin in osteoarthritis (OA). The current study aimed to investigate in OA patients the association between the production of adiponectin and the grade of cartilage destruction, and to provide functional evidence for a potential role of adiponectin in OA. DESIGN: The expression of adiponectin was examined by immunohistochemistry in cartilage obtained from healthy individuals (n = 2; ages 56 and 41 years; 1 male and 1 female) and OA patients (n = 11; ages 64-79 years; 2 male and 9 female). The association between its production in chondrocytes and the grade of cartilage destruction was established on full-depth cartilage biopsies. The functional activity of adiponectin in OA cartilage was determined from the relation between the expression of adiponectin, its receptor, cartilage-specific components and factors involved in matrix degradation, and from the chondrocyte response to the full-length or the globular form of adiponectin. RESULTS: Adiponectin was not detected in healthy cartilage. Conversely, the adipokine was up-regulated in damaged tissue, but no strong association with the grade of cartilage destruction was found. We showed a positive correlation between adiponectin and mPGES or MMP-13 while AdipoR1 was related to the expression of type 2 collagen, aggrecan and Sox9. The full-length form of adiponectin but not the globular isoform, stimulated the production of PGE2 and MMP-13 activity in cultured human chondrocytes. CONCLUSIONS: The elevated level of adiponectin found in chondrocytes from OA patients might contribute to matrix remodelling during OA, the full-length isoform being the single active form.

Modulation of IL-1-induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities.

Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [Nomega-monomethyl-L-arginine (L-NMMA), Nomega-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin-1beta (rhIL-1beta) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps. Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presence of rhIL-1beta with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na2(35)SO4] and NO production by cumulated nitrite release during the period of study. Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1beta challenge (0 to 250 U ml(-1) and 0 to 15 U ml(-1) respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL-1beta (2500 U ml(-1) and 30 U ml(-1) respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response curves. When studying the effect of NOS inhibitors (1 to 1000 microM) on NO production by cartilage cells stimulated with IL-1beta (25 U ml(-1) or 5 U ml(-1)), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU > L-NMMA > or = SMT > or = AG > or = L-NAME and (iii) AETU was cytotoxic when used in the millimolar range. When studying the effect of NOS inhibitors on proteoglycan synthesis by cartilage cells treated with IL-1beta, we observed that: (i) they had more marked effects on proteoglycan synthesis in chondrocytes than in cartilage samples; (ii) they could be roughly classified in the following rank order of potency: L-NAME > or = L-NMMA > > AG > SMT > > AETU and (iii) potentiation of the IL-1 effect by AETU was consistent with cytotoxicity in the millimolar range. D-isomers of L-arginine analog inhibitors (1000 microM) were unable to correct nitrite levels or proteoglycan synthesis in IL-1beta treated cells. L-arginine (5000 microM) tended to reverse the correcting effect of L-NMMA (1000 microM) on proteoglycan synthesis, thus suggesting a NO-related chondroprotective effect. However, data with L-NAME and SMT argued against a general inverse relationship between nitrite level and proteoglycan synthesis. Dexamethasone (0.1 to 100 microM) (i) failed to inhibit NO production in femoral head caps and chondrocytes beads whilst reducing it in patellae (50%) and (ii) did not affect or worsened the inhibitory effect of IL-1beta on proteoglycan synthesis. Such results suggested a corticosteroid-resistance of rat chondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production. In conclusion, our results suggest that (i) NO may account only partially for the suppressive effects of IL-1beta on proteoglycan synthesis, particularly in cartilage samples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint-derived cells and (iii) L-arginine analog inhibitors are more promising than S-substituted isothioureas for putative therapeutical uses.

Mono-iodoacetate-induced experimental osteoarthritis: a dose-response study of loss of mobility, morphology, and biochemistry.

OBJECTIVE: To characterize the dose-responsiveness of morphologic and biochemical chondral changes relative to mobility in mono-iodoacetate (MIA)-induced osteoarthritis (OA) in rats. METHODS: Rat mobility was assessed by biotelemetry. Articular lesions were characterized by macroscopic and histologic examinations. Cartilage proteoglycan metabolism was evaluated by the 1,9-dimethylmethylene blue dye binding assay and by radiosulfate incorporation in patellar cartilage. RESULTS: Spontaneous locomotor activity was rapidly, transiently, and dose-dependently decreased after MIA injection into rat knees (primary response). Thereafter, only high doses (0.3 mg and 3.0 mg) led to a secondary progressive long-term loss of spontaneous mobility on day 15, when subchondral bone was exposed. These 2 doses resulted in significant changes in cartilage proteoglycan concentration at day 15 and a strong inhibition of anabolism in the peripheral patellae by day 2, contrasting with the effects of lower doses (0.01, 0.03, and 0.1 mg). CONCLUSION: When a sufficient dose of MIA is used, this model can easily and quickly reproduce OA-like lesions and functional impairment in rats, similar to that observed in human disease. These parameters, as well as proteoglycan metabolism, could serve as indicators for studying chondroprotective drugs, or for evaluating the ability of imaging techniques to detect and evaluate chondral lesions.

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.

Effects of continuous infusion of interleukin 1 beta on corticotropin-releasing hormone (CRH), CRH receptors, proopiomelanocortin gene expression and secretion of corticotropin, beta-endorphin and corticosterone.

A number of recent studies suggest that interleukin 1 beta (IL-1 beta) is a major mediator of hypothalamo-pituitary-adrenal (HPA) responses following infectious aggression. We investigated whether IL-1 beta mediates long-term changes in HPA activity and studied the cellular regulation of the anterior pituitary. To mimic chronically elevated IL-1 beta production thought to occur during infectious diseases, osmotic pumps (Alzet type) were implanted in the peritoneal cavity of male rats and hIL-1 beta was infused continuously at rates of 1 or 3 micrograms/day. Effects of hIL-1 beta action on plasma ACTH, beta-endorphin (beta-EP) and corticosterone (CORT) secretion and on anterior pituitary (AP), ACTH and beta-EP content were followed. In addition, hypothalamic (HT) CRH mRNA and in AP, CRH receptor (CRH-Rc) mRNA, POMC nuclear primary transcript RNA, POMC nuclear intermediate processing RNA and POMC nuclear and cytoplasmic mRNA were quantified using a highly sensitive solution hybridization nuclease protection assay. Continuous infusion of hIL-1 beta stimulated the HPA axis at varying degrees. Increased HT CRH gene expression, AP POMC gene transcription, ACTH and beta-EP release occurred only during the first 3 days of the treatment. A long-lasting enhancement of ACTH and beta-EP synthesis and of POMC gene expression resulted from activated POMC gene transcription followed by an increased POMC mRNA stability and decreased POMC mRNA turnover. In the AP, stimulation of ACTH and beta-EP secretion and POMC gene transcription disappeared after continuous IL-1 beta treatment, possibly in part due to a refractory process mediated by decreased CRH-Rc gene expression in corticotropes.

Relations between functional, inflammatory, and degenerative parameters during adjuvant arthritis in rats.

We assessed the time-course of adjuvant arthritis (AA) in Lewis rats, using biotelemetry to monitor the rat's spontaneous locomotor activity and body temperature, and studied the evolution of the arthritic index, circulating concentrations of inflammation-promoting cytokines, cartilage proteoglycan synthesis, and the effect of indomethacin as a cyclooxygenase inhibitor to evaluate prostaglandin (PG) contribution in AA. The injection of complete Freund's adjuvant on day 0 (D0) induced a marked, transient loss of locomotor activity (D1-D4; initial phase) and then a second phase of hypomobility peaking on D15 and thereafter irreversible (D16-D20; arthritic phase). Fever peaked first on D1 and again between D13 and D17. The primary hyperthermia was associated with increases in plasma interleukin-6 and tumor necrosis factor-alpha concentrations and seemed to be partly PG dependent. Proteoglycan synthesis inhibition in the patellar cartilage increased gradually, spreading from the injected paw to the contralateral paw. It was corrected on D20 by preventive and curative indomethacin treatments. Indomethacin also greatly relieved hypomobility during the systemic phase of AA (D10-D15). The combination of information about cartilage metabolism, body temperature, locomotor activity, and cytokine in this study permits analysis of analgesic, antipyretic, anti-inflammatory, and chondroprotective properties of drugs in the various phases of AA. Thus, using a new methodology, we have discriminated the different phases of the disease and confirmed the symptomatic and systemic inhibitory effect of indomethacin on fever, activity, and cartilage metabolism.

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.