One-year follow-up of Coll2-1, Coll2-1NO2 and myeloperoxydase serum levels in osteoarthritis patients after hip or knee replacement.

OBJECTIVES: To determine Coll2-1, Coll2-1NO(2) and myeloperoxydase (MPO) levels in serum of patients with knee or hip osteoarthritis (OA) before the surgery, 3 months and 1 year after knee or hip replacement. METHODS: Coll2-1, Coll2-1NO(2) and MPO were measured in 103 patients with isolated symptomatic knee or hip OA candidates for joint replacement. Sera were taken the day before surgery, 3 months and 1 year after hip or knee replacement. Coll2-1 and Coll2-1NO(2) immunohistochemistry was performed on biopsies removed from cartilage lesions. RESULTS: Immunostainings revealed the extensive presence of Coll2-1 and Coll2-1NO(2) in the superficial layer of fibrillated cartilage and around some chondrocytes clusters. Three months after joint replacement, Coll2-1 and MPO serum levels were decreased and even reached the reference value for Coll2-1. By contrast, Coll2-1NO(2) levels remained elevated. At 1-year follow-up, Coll2-1 levels remained at the reference value, MPO levels were similar to those measured at 3 months, and Coll2-1NO(2) levels were unchanged and comparable to the pre-surgery values. However, in patients with pre-surgery values above the median (more than 0.42 nM), Coll2-1NO(2) levels significantly and progressively decreased post-operatively, but tended towards an increase in patients with pre-surgery Coll2-1NO(2) values below the median. CONCLUSIONS: The normalisation of Coll2-1 levels 3 months after surgery indicates that Coll2-1 is a disease-specific marker that is sensitive to the structural changes occurring in a single joint. Furthermore, the immunohistochemical findings are consistent with the concept that the major source of serum Coll2-1 is the damaged articular cartilage. Finally, serum MPO levels decreased after joint replacement indicating that neutrophil activation occurs in OA joints, even in the late stage of the disease.

The chemical biomarkers C2C, Coll2-1, and Coll2-1NO2 provide complementary information on type II collagen catabolism in healthy and osteoarthritic mice.

OBJECTIVE: Compared with wild-type (WT) mice, biglycan/fibromodulin double-deficient mice develop severe knee osteoarthritis. We undertook this study to compare type II collagen catabolism in the 2 genotypes and to compare the usefulness of 3 biomarkers of collagen degradation (C2C [also known as Col2-3/4C(long mono)] as well as the peptide Coll2-1 and its nitrated form, Coll2-1NO2) for evaluating collagen catabolism in vivo. METHODS: In 15 WT mice and 15 biglycan/fibromodulin double-deficient mice, we determined serum levels of C2C at ages 66 and 141 days, and we determined serum levels of Coll2-1 and Coll2-1NO2 at ages 49, 81, 95, and 141 days. Expression of the biomarkers in knee sections was examined using immunohistochemistry. RESULTS: The mean concentrations of C2C and Coll2-1 were higher in biglycan/fibromodulin double-deficient mice at all time points. For C2C and Coll2-1, the ratio of the serum concentration in biglycan/fibromodulin double-deficient mice to that in WT mice (the double-deficient:WT ratio) was constant over time and was approximately 1.63 and approximately 1.15, respectively. In contrast, the double-deficient:WT ratio for Coll2-1NO2 varied and, depending on age, was >1 or <1. No significant correlation was found between the expression of the different biomarkers, except for a weak, negative correlation between Coll2-1NO2 and C2C. In both genotypes, antibodies to each biomarker labeled some fibroblasts in the tendons and menisci as well as chondrocytes above the tidemark in articular cartilage. Growth plates were unstained. For each biomarker, extracellular staining was limited to fibrocartilage areas in the tendons and menisci in all mice and was limited to some focal lesions of the cartilage in biglycan/fibromodulin double-deficient mice. CONCLUSION: The different double-deficient:WT ratios observed with C2C, Coll2-1, and Coll2-1NO2 in the absence of any correlation between the expression of the 3 biomarkers indicate that these biomarkers give complementary, rather than redundant, information about in vivo type II collagen catabolism.

One-year increase of Coll 2-1, a new marker of type II collagen degradation, in urine is highly predictive of radiological OA progression.

OBJECTIVE: To analyse the relationship between the levels of urinary biochemical markers of type II collagen degradation and the clinical and radiological severity and progression of knee osteoarthritis (OA). METHOD: Seventy-five patients with primary knee OA were included in this 3-year follow-up study. Mean joint space width (JSW) of the medial compartment of the femorotibial joint was measured with a computer assisted method on standardized radiographs taken at baseline and after a 3-year follow-up. Pain, stiffness, and physical function subscales of the Western Ontario and McMaster Universities (WOMAC) were assessed at the same time points. Type II collagen peptides Coll 2-1 and Coll 2-1 NO(2), as well as pyridinoline (Pyr) and deoxypyridinoline (D-Pyr) were measured in urines at baseline, after 1 year and 3 years, with specific immunoassays. RESULTS: At baseline, significant correlations were found between the urinary Coll 2-1 and Coll 2-1 NO(2) levels and the global WOMAC score (Coll 2-1: r=0.28, P=0.01; Coll 2-1 NO(2): r=0.27, P=0.02) and its subscales for pain (Coll 2-1: r=0.27, P=0.01; Coll 2-1 NO(2): r=0.30, P=0.01) and function (Coll 2-1: r=0.29, P=0.01; Coll 2-1 NO(2): r=0.27, P=0.02). Pyr and D-Pyr levels were not significantly correlated with the WOMAC scores. One-year change in Coll 2-1 and Coll 2-1 NO(2) urinary levels were negatively correlated with a 3-year change in JSW (Coll 2-1: r=-0.31, P=0.03; Coll 2-1 NO(2): r=-0.31, P=0.03), indicating that an increase of Coll 2-1 or Coll 2-1 NO(2) over 1 year is predictive of subsequent joint space narrowing. Neither Pyr nor D-Pyr was correlated with radiological OA progression. CONCLUSIONS: At baseline, Coll 2-1 and Coll 2-1 NO(2) urinary levels were indicative of the clinical activity of knee OA and the increase of these peptides over 1 year was predictive of the radiological progression of knee OA.

Strontium ranelate increases cartilage matrix formation.

Based on previous studies showing that strontium ranelate (S12911) modulates bone loss in osteoporosis, it could be hypothesized that this drug also is effective on cartilage degradation in osteoarthritis (OA). This was investigated in vitro on normal and OA human chondrocytes treated or not treated with interleukin-1beta (IL-1beta). This model mimics, in vitro, the imbalance between chondroformation and chondroresorption processes observed in vivo in OA cartilage. Chondrocytes were isolated from cartilage by enzymatic digestion and cultured for 24-72 h with 10(-4)-10(-3) M strontium ranelate, 10(-3) M calcium ranelate, or 2 x 10(-3) M SrCl2 with or without IL-1beta or insulin-like growth factor I (IGF-I). Stromelysin activity and stromelysin quantitation were assayed by spectrofluorometry and enzyme amplified sensitivity immunoassay (EASIA), respectively. Proteoglycans (PG) were quantified using a radioimmunoassay. Newly synthesized glycosaminoglycans (GAGs) were quantified by labeled sulfate (Na2(35)SO4) incorporation. This method allowed the PG size after exclusion chromatography to be determined. Strontium ranelate, calcium ranelate, and SrCl2 did not modify stromelysin synthesis even in the presence of IL-1beta. Calcium ranelate induced stromelysin activation whereas strontium compounds were ineffective. Strontium ranelate and SrCl2 both strongly stimulated PG production suggesting an ionic effect of strontium independent of the organic moiety. Moreover, 10(-3) M strontium ranelate increased the stimulatory effect of IGF-I (10(-9) M) on PG synthesis but did not reverse the inhibitory effect of IL-1beta. Strontium ranelate strongly stimulates human cartilage matrix formation in vitro by a direct ionic effect without stimulating the chondroresorption processes. This finding provides a preclinical basis for in vivo testing of strontium ranelate in OA.

Modulation of human chondrocyte metabolism by recombinant human interferon.

OBJECTIVES: Interferon gamma (IFN gamma) is found to be elevated in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis, suggesting its implication in joint disease pathogenesis. In this study, we investigated the effects of IFN gamma on the production of cytokines (IL-6, IL-8, IL-10), prostaglandin E(2)(PGE(2)), proteoglycans (PG), nitric oxide (NO), interleukin-1 receptor antagonist (IL-1ra) and stromelysin by non-stimulated and IL-1 beta-treated human chondrocytes. The role played by NO in the responses of chondrocytes to IFN gamma was also examined by incubation of chondrocytes with N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase. METHODS: Enzymatically isolated human chondrocytes were cultured for 48 h in the absence or presence of IL-1 beta, IFN gamma or N(G)-monomethyl-L-arginine (L-NMMA) added solely or in combination. The productions of IL-6, IL-8, IL-10, IL-1ra and stromelysin were measured by enzyme amplified sensitivity immunoassays (EASIA). PG and PGE(2)were quantified by specific radioimmunoassays (RIA). Nitrite concentrations in the culture supernatants were determined by a spectrophotometric method based upon the Griess reaction. RESULTS: As expected, IL-1 beta highly stimulated NO, IL-6, IL-8, IL-10, IL-1ra, PGE(2)and stromelysin synthesis, but dramatically decreased PG production. NO, IL-6, IL-1ra and PGE(2)production by non-stimulated chondrocytes was dose-dependently increased by IFN gamma while PG production was inhibited. In the absence of IL-1 beta, IL-10 was undetectable in the culture supernatants. At the doses of 10 and 100 U/ml, IFN gamma markedly inhibited the constitutive and IL-1 beta-stimulated IL-8, IL-10 and stromelysin productions. Interestingly, IFN gamma synergized with IL-1 beta to increase NO, IL-6, IL-1ra and to depress PG production. As previously reported, the inhibition of NO synthesis by the competitive inhibitor L-NMMA led to enhancement of IL-6, IL-8 and PGE(2)production by IL-1 beta treated chondrocytes, but did not significantly modify IL-10, PG and MMP-3 productions. Inhibition of NO synthase significantly inhibited the stimulating effect of IFN gamma on IL-6 and IL-1ra but did not affect the inhibitory effect of IFN gamma on IL-8, PG or stromelysin production. CONCLUSIONS: These findings suggest that IFN gamma and IL-1 synergistically stimulate the production of IL-6, IL-1ra, NO and PGE(2)and inhibit PG synthesis. By contrast, IL-1 beta and IFN gamma have opposite effects on IL-8, IL-10 and stromelysin productions. These effects are not reversed by L-NMMA, suggesting that NO is not the principal mediator involved in responses of chondrocytes to IFN gamma.

In vitro study of the antioxidant properties of nimesulide and 4-OH nimesulide: effects on HRP- and luminol-dependent chemiluminescence produced by human chondrocytes.

OBJECTIVES: Reactive oxygen species (ROS) are now recognized to play an important role in the pathogenesis of rheumatic diseases and constitute an interesting therapeutic target for drugs. This in vitro study was designed to evaluate the antioxidant properties of nimesulide (NIM), a nonsteroidal antiinflammatory drug of the sulfonanilide class, and its main metabolite 4-OH nimesulide (4-OHNIM). METHODS: The scavenging effects of NIM and 4-OH NIM on hydroxyl radical ((.)OH) and superoxide anions (O(minusd)(2)) were investigated by electron spin resonance (ESR), using 5, 5-dimethylpyrroline-N-oxide (DMPO) as the spin trap agent. The quenching properties of these drugs on hypochlorite anion was studied by luminol enhanced chemiluminescence. Finally, the effects of NIM and 4-OHNIM on the reactive oxygen species production by human articular chondrocytes were recorded by HRP and luminol-enhanced chemiluminescence. RESULTS: By this method it has been demonstrated that NIM and 4-OHNIM, at concentrations ranging from 10 to 100 microM, are potent scavengers of(.)OH whereas only 4-OHNIM was capable to scavenge O(minusd)(2). Chemiluminescence generated by HOCl was also significantly and dose-dependently inhibited by both NIM and 4-OHNIM. Nevertheless, at each concentration tested, the inhibitory effect of 4-OHNIM was significantly more marked, even at the highest concentration (100 microM). Furthermore, when chondrocytes were pre-incubated for 48-96 h with NIM or 4-OHNIM, the luminol- and HRP-dependent CL produced by the cells was significantly inhibited in a dose-dependent manner. CONCLUSIONS: NIM and 4-OHNIM may protect cartilage against oxidative stress, not only by scavenging ROS but also by inhibiting their production by chondrocytes.

Effects of nimesulide and indometacin on COX-1 and COX-2: a comparative study.

Evidence of the existence of two forms of cyclooxygenases and the clinical relevance of COX-2 inhibition led to the development of COX-2 selective NSAIDs. In order to evaluate this selectivity, we have developed and validated an enzymatic method. The precision and reproducibility of the assay were determined and COX-2 selectivity examined using nimesulide and indometacin.

Effects of nimesulide and sodium diclofenac on interleukin-6, interleukin-8, proteoglycans and prostaglandin E2 production by human articular chondrocytes in vitro.

OBJECTIVES: The aim of this study was to investigate the effects of two nonsteroidal anti-inflammatory drugs (NSAIDs), nimesulide and sodium diclofenac, on the production of proteoglycans (PG), prostaglandin E2 (PGE2) and cytokines (IL-6 and IL-8) by human articular chondrocytes in vitro. METHODS: Enzymatically isolated chondrocytes were cultured under constant agitation in a well defined culture medium. Specific radioimmunoassays were used to quantify PG and PGE2 production. Cytokine production (IL-6 and IL-8) was assayed by enzyme amplified sensitivity immunoassays (EASIAs). RESULTS: At a concentration of 3 micrograms/ml, nimesulide did not affect the PG production by chondrocytes. This concentration was superior to the highest level of nimesulide found in the synovial fluid of patients with rheumatoid arthritis 3 hours after the last oral administration of nimesulide (100 mg twice daily for 7 days). At 6 micrograms/ml a significant reduction in the PG content was obtained in the cellular phase in 5 out of the 8 cultures investigated. No similar effect was observed in the culture supernatants. Above this concentration nimesulide inhibited PG production in a dose-dependent manner. At concentrations ranging from 0.005 to 1 microgram/ml diclofenac did not significantly alter PG production. At therapeutic concentrations PGE2 production was totally inhibited by nimesulide, thus suggesting that PG inhibition is not linked to PGE2 production. Nimesulide inhibited PGE2 production by unstimulated (IC50 = 6 ng/ml) and IL-1 beta-stimulated (IC50 = 6.9 ng/ml) chondrocytes. At these concentrations, PGE2 production was fully inhibited by diclofenac. Furthermore, both nimesulide and diclofenac at therapeutic concentrations significantly decreased spontaneous and IL-1 beta-stimulated IL-6 production by human chondrocytes, but did not modify IL-8 production. CONCLUSION: From the results of this study we conclude that nimesulide and diclofenac at therapeutic concentrations are potent inhibitors of PGE2 and IL-6 production while they do not modify proteoglycan or IL-8 production.

Nitric oxide downregulates interleukin 1beta (IL-1beta) stimulated IL-6, IL-8, and prostaglandin E2 production by human chondrocytes.

OBJECTIVE: To investigate the effects of endogenously produced nitric oxide (NO) on interleukin 6 (IL-6), IL-8, prostaglandin E2 (PGE2), and proteoglycan production by human chondrocytes. METHODS: Human articular chondrocytes were isolated from their extracellular matrix by triple successive enzymatic digestion of the cartilage and cultured 48 h in a well defined culture medium. IL-6 and IL-8 were directly assayed into culture media by specific enzyme amplified sensitivity immunoassays. Proteoglycans and PGE2 were quantified by specific radioimmunoassays. Cell culture media were assayed for NO2 using a spectrophotometric assay based upon the Griess reaction. RESULTS: Unstimulated chondrocytes produced low levels of NO, IL-6, IL-8, and PGE2. Production was significantly stimulated by IL-1beta and lipopolysaccharide (LPS). As well, proteoglycan synthesis was profoundly inhibited by IL-1beta and LPS. Inhibition of NO synthesis with the competitive inhibitor NG-monomethyl-L-arginine (L-NMMA) led to enhancement of IL-6, IL-8, and PGE2 production stimulated by either IL-1beta alone or in combination with LPS, whereas the inhibition of proteoglycan production by IL-1beta was not modified by L-NMMA. CONCLUSION: LPS and IL-1beta stimulated IL-6, IL-8, and PGE2 production are downregulated by endogenously produced NO, which could limit the inflammatory reaction occurring in arthritis.

Effects of three avocado/soybean unsaponifiable mixtures on metalloproteinases, cytokines and prostaglandin E2 production by human articular chondrocytes.

The in-vitro effects of avocado and soybean unsaponifiable residues on neutral metalloproteinase activity, cytokines and prostaglandin E2 (PGE2) production by human articular chondrocytes were investigated. Avocado and soybean unsaponifiable residues were mixed in three ratios: 1:2 (A1S2), 2:1 (A2S1) or 1:1 (A2S2). Freshly isolated human chondrocytes were cultured for 72 h in the absence or presence of interleukin-1beta, (IL-1beta) (17 ng/ml), with or without unsaponifiable residue mixtures at a concentration of 10 microg/ml. A/S unsaponifiable residues were also tested separately at concentrations of 3.3, 6.6 and 10 microg/ml. All A/S unsaponifiable mixtures reduced the spontaneous production of stromelysin, interleukin-6 (IL-6), interleukin-8 (IL-8) and prostaglandin E2 (PGE2) by chrondrocytes. At concentrations of 3.3 and 6.6 microg/ml, A/S residues, tested separately, were potent inhibitors of the production of IL-8 and PGE2. Nevertheless, only avocado residue inhibited IL-6 production at these concentrations. A/S unsaponifiable mixtures had a more pronounced inhibitory effect on cytokine production than avocado or soybean residues added alone. As anticipated, IL-1beta induced a marked release of collagenase, stromelysin, IL-6, IL-8 and PGE2. A/S unsaponifiable mixtures partially reversed the IL-1 effects on chrondrocytes. These findings suggest a potential role for A/S unsaponifiable extracts in mitigating the deleterious effects of IL-1beta: on cartilage.

Effects of exogenous IL-1 beta, TNF alpha, IL-6, IL-8 and LIF on cytokine production by human articular chondrocytes.

Cytokines are potent regulators of the chondrocyte functions. Some of them are produced by chondrocytes and interact to regulate cartilage metabolism. In this study, we investigated the production of interleukin-1 beta (IL-1 beta), IL-6, IL-8 and leukemia inhibitory factor (LIF) by human chondrocytes and examined the modulation of their secretion by exogenous cytokines. Human articular chondrocytes were isolated from their extracellular matrix by a triple successive enzymatic digestion of the cartilage. Subsequently, chondrocytes were stimulated by increased amounts of human recombinant cytokines [IL-1 beta, tumour necrosis factor alpha (TNF alpha), IL-8, LIF, IL-6]. IL-1 beta, IL-6, IL-8 and LIF were assayed into culture media and inside cell extracts by specific enzyme amplified sensitivity immunoassays (EASIAs). Under these experimental conditions, we have identified various interactions between cytokines. IL-beta and TNF alpha highly stimulated IL-6, LIF and IL-8 productions. IL-6 decreased IL-8 synthesis and increased LIF production. IL-8 slightly enhanced IL-6 production. Finally, LIF stimulated IL-1 beta, IL-6 and IL-8 productions. Using neutralizing antibodies against IL-1, we demonstrated that the effects of LIF were secondary to the stimulation by LIF of IL-1 beta production by the chondrocytes. In conclusion, chondrocytes secrete a variety of immunocompetent cytokines including IL-1 beta, IL-6, IL-8 and LIF that can interact to regulate chondrocytes metabolism. These results also define new biological activities of LIF and IL-6, and raise questions concerning their role in the pathogenesis of joint diseases.