Pascale Gegout-Pottie

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 rats 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 Freunds 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-α 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.

Pefloxacin-Induced Achilles Tendon Toxicity in Rodents: Biochemical Changes in Proteoglycan Synthesis and Oxidative Damage to Collagen

ABSTRACT 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.

Obesity affects the chondrocyte responsiveness to leptin in patients with osteoarthritis.

IntroductionIncreasing evidence support the regulatory role of leptin in osteoarthritis (OA). As high circulating concentrations of leptin disrupt the physiological function of the adipokine in obese individuals, the current study has been undertaken to determine whether the elevated levels of leptin found in the joint from obese OA patients also induce changes in the chondrocyte response to leptin.MethodsChondrocytes isolated from OA patients with various body mass index (BMI) were treated with 20, 100 or 500 ng/ml of leptin. The expression of cartilage-specific components (aggrecan, type 2 collagen), as well as regulatory (IGF-1, TGFβ, MMP-13, TIMP 2) or inflammatory (COX-2, iNOS, IL-1) factors was investigated by real-time PCR to evaluate chondrocyte responsiveness to leptin. Furthermore, the effect of body mass index (BMI) on leptin signalling pathways was analyzed with an enzyme-linked immunosorbent assay for STATs activation.ResultsLeptin at 20 ng/ml was unable to modulate gene expression in chondrocytes, except for MMP-13 in obese OA patients. Higher leptin levels induced the expression of IGF-1, type 2 collagen, TIMP-2 and MMP-13. However, the activity of the adipokine was shown to be critically dependent on both the concentration and the BMI of the patients with a negative association between the activation of regulated genes and BMI for 100 ng/ml of adipokine, but a positive association between chondrocyte responsiveness and BMI for the highest leptin dose. In addition, the gene encoding MMP-13 was identified as a target of leptin for chondrocytes originated from obese patients while mRNA level of TIMP-2 was increased in leptin-treated chondrocytes collected from normal or overweight patients. The adipokine at 500 ng/ml triggered signal transduction through a STAT-dependent pathway while 100 ng/ml of leptin failed to activate STAT 3 but induced STAT 1α phosphorylation in chondrocytes obtained from obese patients.ConclusionsThe current study clearly showed that characteristics of OA patients and more expecially obesity may affect the responsiveness of cultured chondrocytes to leptin. In addition, the BMI-dependent effect of leptin for the expression of TIMP-2 and MMP-13 may explain why obesity is associated with an increased risk for OA.

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 [Nω‐monomethyl‐L‐arginine (L‐NMMA), Nω‐nitro‐L‐arginine methyl ester (L‐NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin‐1β (rhIL‐1β) 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‐1β 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 [Na235SO4] 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‐1β 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‐1β (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 μM) on NO production by cartilage cells stimulated with IL‐1β (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‐NMMASMT>AGL‐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‐1β, 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‐NAMEL‐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 μM) were unable to correct nitrite levels or proteoglycan synthesis in IL‐1β treated cells. L‐arginine (5000 μM) tended to reverse the correcting effect of L‐NMMA (1000 μM) 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 μM) (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‐1β 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‐1β 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.

Effects of Continuous Infusion of Interleukin 1β on Corticotropin-Releasing Hormone (CRH), CRH Receptors, Proopiomelanocortin Gene Expression and Secretion of Corticotropin, β-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.

Biotelemetry: an original approach to experimental models of inflammation

Abstract. 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 animals 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.

Synovial fluid levels of adipokines in osteoarthritis: Association with local factors of inflammation and cartilage maintenance

The role of body weight in the pathogenesis of osteoarthritis (OA) - previously considered the sole factor in the association between obesity and OA - is being re-evaluated as the contribution of adiposity to the cartilage degenerative process becomes clearer. The current study has been undertaken to better understand the role of adipose-derived proteins, namely adipokines, in OA. For this purpose, we investigated in patients with OA the relationships between the joint levels of leptin, adiponectin and resistin and those of factors involved in inflammation and cartilage maintenance. The sandwich enzyme-linked immunosorbent assays were used to determine in the synovial fluid (SF) from 35 OA patients, the concentrations of adipokines, interleukin-6 (IL-6) and transforming growth factor-β (TGF-β). The soluble form of leptin receptor (sOb-R) was also examined to evaluate the biological active free form of leptin. Correlation analysis indicate that IL-6 levels are positively related to the levels of resistin and adiponectin. Surprisingly, the free form of leptin, but not the total leptin, is negatively associated with IL-6. Beside, adiponectin is the single adipokine that is correlated with TGF-β. Interestingly, a sexual dimorphism is observed in the study as correlations between adipokines and IL-6 or TGF-β are found only with female OA patients. Taken together, these findings suggest that only adiponectin may contribute to the metabolic changes associated with OA. The three adipokines may also be involved in inflammation, but with opposite effects. Both resistin and adiponectin may exhibit pro-inflammatory activity while the free form of leptin may down-regulate the inflammation.