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ISSN: 1426-3912
Central European Journal of Immunology
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4/2003
vol. 28
 
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The effect of taurine and its metabolites on the pathogenic functions of rheumatoid arthritis fibroblast-like synoviocytes

Ewa Kontny
,
Iwona Janicka
,
Janusz Marcinkiewicz
,
Magdalena Chorąży-Massalska
,
Weronika Rudnicka
,
Włodzimierz Maśliński

Centr Eur J Immunol 2003; 28 (4): 167-172
Online publish date: 2004/09/17
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Introduction
Rheumatoid arthritis (RA) is an autoimmune disease characterized by hyperplasia of synovial membrane, chronic synovitis and progressive destruction of joint catrilage and bone. Fibroblast-like synoviocytes (FLS), the cells of mesenchymal origin located in the joint intimal lining, are believed to contribute to all of these pathological processes resulting in the development of RA symptoms [1]. Therefore, the limitation of an excessive growth of these cells as well as the reduction of synthesis of pro-inflammatory mediators and tissue destructive factors originated from FLS are considered to be a promising therapeutic approach for the treatment of RA patients. We have recently reported that taurine chloramine (Tau-Cl) inhibits in vitro several pathogenic functions of RA FLS [for review see 2], and found this compound to give some protection against development of collagen-induced arthritis in mice, although the latter in vivo effect was short-term and reversible [3]. Taurine chloramine [for review see 4 and 5] is formed in the reaction between hypochlorous acid (HOCl), that represent one of the major product of the respiratory burst, and a dominant free amino acid taurine (Tau). Activated neutrophils are the major source of Tau-Cl. This physiologic compound has been reported to possess oxidative, bactericidal and immunomodulatory properties. Although Tau-Cl is rather stable oxidant, it is reduced to Tau and CL,SUP>– while oxidizing its targets [6] and it was also shown to be slowly decomposed to sulphoacetaldehyde (SA) [7]. Thus, Tau, Tau-Cl and SA may co-exist at a site of inflammation. It has been well documented that Tau, in contrast to Tau-Cl, despite its cytoprotective and antioxidant properties does not affect the functions of RA FLS tested in vitro [2, 9-11]. On the other hand, it is completely obscure whether SA influences any functions of cells implicated in the inflammatory response. Therefore, in the present study we have focused on the comparison of the effects of Tau, Tau-Cl and SA on RA FLS proliferation and synthesis of pro-inflammatory agents (IL-6, PGE2) by these cells.
Materials and methods
Synovial samples and synoviocyte cultures

Synovial tissues were obtained from knee joints at the time of total joint surgery or synovectomy, performed as a normal part of clinical care, from 27 female patients who fulfilled the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) criteria for the diagnosis of RA [12]. All patients had stage III or IV disease [13]. The mean ± SEM age of the patients was 55.2 ± 2.8 years, and the mean ± SEM disease duration was 11.16 ± 2.7 years. FLS were isolated and cultured in vitro as described previously [9]. Cells were treated with medium alone (control) or were stimulated as described below. Either taurine (Tau) (Sigma, St. Louis, MO) or taurine chloramine (Tau-Cl) or sulphoacetaldehyde (SA) were added at physiologically relevant (50-500 mM) concentrations together with the simuli. Tau-Cl was prepared by chlorination of taurine [9]. Sulphoacetaldehyde was synthesized according to the method described previously [14]. To make the results more credible, the effect of all tested compounds on cell responses was estimated using FLS isolated from the same patients. The viability of cells was controlled by measurement of lactate dehydrogenase activity, using a lactate dehydrogenase assay kit (Takara Shuzo, Otsu, Japan).
Cell proliferation assay
Proliferation of the cells (5x103/0.2 ml/well) was determined by an assessment of DNA synthesis in FLS, based on the incorporation of tritriated thymidine, according to the method described previously [9]. Recombinant human basic fibroblast growth factor (bFGF) or recombinant human tumor necrosis factor-α (TNF-a) (both from R&D Systems, Abingdon, UK) were used as the stimuli at 1ng/ml or 10 ng/ml concentrations, respectively.
Measurement of interleukin-6 (IL-6) production
To trigger IL-6 production FLS (4x104/ml/well) were stimulated with 1 ng/ml of recombinant human interleukin-1b (R&D Systems). The concentration of IL-6 was determined in both culture supernatants (secreted form) and in cell lysates (cell-associated form) collected 24 hours after stimulation. The IL-6 specific enzyme-linked immunosorbent assay (ELISA) was applied as described detaily before [9, 15]. Briefly, goat polyclonal neutralizing antibody specific for human IL-6 (R&D Systems) was used to capture IL-6 from samples, while IL-6-specific rabbit polyclonal antibody (Sigma) was applied to detect the cytokine, followed by horseradish peroxidase-conjugated goat anti-rabbit immunoglobulins and 0-phenylenediamine dihydrochloride (OPD) (both from Sigma) as a substrate. Optical density was measured at 492 nm using an automatic ELISA reader (LP-400; Diagnostic Pasteur, Marnes-La-Coquette, France). The detection limit for IL-6 was 39 pg/ml.
Measurement of prostaglandin E2 (PGE2) synthesis
The concentration of PGE2 was determined in culture supernatants collected 24 hours after stimulation of the cells (4x104/ml/well) with 1 ng/ml rhIL-1b. The competitive acetylcholinesterase enzyme immunoassay (PGE2 EIA kit; Cayman Chemical, Ann. Arbor, MI) was applied according to the manufacturer`s protocol.
Statistical analysis
Repeated-measures analysis of variance, followed by Tukey`s test, was applied to evaluate the effects of the stimuli and tested compounds. Results are expressed as the mean ± SEM. P values less than 0.05 were considered significant.
Results
Spontaneous proliferation of the cells cultured in medium alone (4081 ± 758 cpm) was significantly raised in the presence of bFGF (6906 ± 1196 cpm; P=0.007) or TNF-a (6337 ± 1266; P=0.035). Tau-Cl inhibited proliferation of TNF-α stimulated cells in a dose dependent manner, while Tau did not affect the cell response (Fig. 1.). These results correspond to our previous observation showing inhibitory effect of Tau-Cl, but not Tau, on both spontaneous and bFGF-triggered RA FLS proliferation [9]. Similarly to Tau, SA had no significant effect on either spontaneous or bFGF- or TNF-a–triggered cell proliferation (Fig. 1.).Under these experimental conditions, neither Tau (Ł500 mM) nor SA (≤ 500 mM) nor Tau-Cl (≤400 mM) was cytotoxic. However, at a concentration of 500 mM Tau-Cl the cells were progressively damaged (mean ± SEM cytotoxicity was 8.7 ± 3.6 % and 41.8 ± 6.5% after 24 and 72 hours of treatment, respectively). Similar results were obtained in cell cultures treated with tested compounds alone or together with stimuli.
FLS produced IL-6 spontaneously (934.3 ± 213 pg/ml of secreted and 144.5 ± 31.2 pg/ml of cell-associated forms, respectively). In the presence of IL-1b production of IL-6 was significantly (P= 0.001 - 0.0001) elevated (8222.3 ± 992 pg/ml of secreted form and 1638 ± 351 pg/ml of cell-associated form; n=15). Consistently with previous results [9,10] Tau-Cl significantly and in a dose-dependent way inhibited IL-1b-triggered production of both secreted and cell-associated forms of IL-6. By contrast, neither Tau nor SA affected IL-6 production (Fig. 2.). The effect of all tested compounds on spontaneous IL-6 production was similar to that stated in IL-1b-stimulated cells (not shown).
Untreated control cells secreted little amount of PGE2 (95 ± 40 pg/ml), while in IL-1b stimulated cell cultures PGE2 concentration (2081 ± 827 pg/ml) was significantly (P=0.036) higher. Similarly to previously published results (5) Tau had no effect, while TauCl inhibited IL-1b-triggered PGE2 synthesis (Fig. 3.) in a dose-dependent manner. Interestingly, SA failed to influence this cell response (Fig. 3.).
Discussion
We have confirmed our previous observations [2, 9-11] that Tau-Cl inhibits in vitro several pathogenic functions of RA FLS: (i) cell proliferation, (ii) synthesis of IL-6, and (iii) generation of PGE2. These inhibitory effects of Tau-Cl were statistically significant at non-cytotoxic (300-400 mM) concentrations of this compound. Present results clearly show that neither Tau nor SA possess such inhibitory properties. The inability of SA to influence RA FLS functions is our new original finding.
Consistently with the opinion of others [16] we propose that during inflammatory response generation of Tau-Cl may represent negative regulatory loop to damp inflammation. Consequently, low Tau-Cl concentration at the inflammatory site may favour chronicity of the response. Interestingly, our recent data suggest impaired ability of RA synovial fluid neutrophils to generate Tau-Cl [17]. Thus, it is likely that in RA Tau-CL,SUP>-mediated downregulation of inflammatory response is not effective enough to prevent transition from the acute to the chronic phase. Compensation of this defect seems to be another therapeutic approach to treat RA patients. In this connexion our finding that SA neither damage nor affect activity of RA FLS is informative.
The mechanism of anti-inflammatory action of Tau-Cl is not fully understood. This compound was reported to affect either transcription or translation of genes encoding crucial mediators of the inflammatory response [for review see 2 and 5]. Importantly, Tau-Cl diminishes DNA-binding activity of AP-1 and NFkB transcription factors [10, 16, 18], known to be the key regulators of a broad range of genes implicated in the inflammatory response. It seems that oxidative properties of Tau-Cl may account for this, because it has recently been revealed that oxidation but not chlorination of IkBa inhibitor by Tau-Cl stabilizes the inhibitor and results in NFκB inactivation [19].
Because of the myriad of Tau actions, one can not exclude possibility that in vivo also this amino acid may importantly regulate the inflammatory response. Taurine was shown to protect cells against oxidative injury not only by scavenging highly toxic HOCl and formation of anti-inflammatory Tau-Cl, but also by e.g. limiting the availability of biological membrane lipids for lipid peroxidation or by acting as an important organic osmolyte and regulator of cellular Ca+2 homeostasis, and thus preventing cells from necrotic or apoptotic death [for review see 8]. Despite this, we have failed to observe Tau protection against RA FLS apoptosis triggered in vitro by anti-Fas antibody (data not shown). It is well documented that in vivo Tau treatment protects against tissue injury in a variety of animal models that share inflammation as a common pathogenic feature. However, this beneficial effect of Tau seems to be mediated mostly via generation of anti-inflammatory Tau-Cl [20]. Recent data have shown that also in animal model of allergic asthma Tau treatment produces anti-hyperreactivity and anti-inflammatory effects [21]. Although the precise mechanism of this protection needs further explanation, authors propose that it seems to be mediated via Tau-Cl, at least partially. Interesting new findings reveal Tau to be an important regulator of bone metabolism, both in vivo and in vitro [for review see 22]. Due to its ability to stimulate bone formation (e.g. via activation of osteoblasts) and inhibition of bone loss (e.g. via inhibition of osteoclasts formation) Tau is proposed to be introduced as an alternative to bisphosphonate therapy in osteoporosis and to prevent inflammatory bone resorption in periodontal disease.
Based on these data it is rational to consider in the future treatment of inflammatory diseases either by application of Tau (if the ability of neutrophils to generate Tau-Cl is retained) or Tau-Cl (in patients with a disturbed neutrophil myeloperoxidase-H2O2-halide system). Present results give further support to this proposal.

Acknowledgements
This work was supported by grants from the State Committee for Scientific Research of Poland (No. P05A 104 19) and the Institute of Rheumatology. The Institute of Rheumatology is supported by a core grant from the State Committee for Scientific Research of Poland.
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Copyright: © 2004 Polish Society of Experimental and Clinical Immunology This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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