Kynurenic acid, an endogenous constituent of rheumatoid arthritis synovial fluid, inhibits proliferation of synoviocytes in vitro
Abstract
Kynurenic acid is an antagonist of ionotropic glutamate receptors. It has been found that glutamate antagonists inhibit proliferation of different human tu- mor cells. Since the hyperplasia of synovial fibroblasts is one of the most striking features of inflammatory arthritis, the main goals of this study were detection and quantification of kynurenic acid in synovial fluid obtained from patients with rheumatoid arthritis, and determina- tion of its effect on proliferation of synoviocytes in vitro. Presence of kynurenic acid was determined by HPLC in all 58 samples of synovial fluid. The mean concentration was 15.89 pmol/ml. Kynurenic acid inhibited synoviocyte proliferation with the IC50 value of 5.9 mM. In sub- threshold concentration of 0.3 mM it enhanced antipro- liferative action of celecoxib and nimesulide. In conclusion, the presence of kynurenic acid in synovial fluid was documented in patients with rheumatoid arthritis. Its potential role as an endogenous substance, controlling synoviocyte proliferation can be suggested.
Introduction
Rheumatoid arthritis (RA) is a chronic disorder pri- marily affecting the joints, and leading to their pro- gressive destruction. Its pathogenesis is largely unknown. RA is generally considered to be an autoim- mune disease. Until now, many investigators have focused attention on the pivotal role of T lymphocytes and monocytes in its pathogenesis. However, evidence is accumulating that the inflammatory process in the synovial tissue is dominated by activated monocytes and fibroblasts [1, 2]. There is an evidence that the synovial fibroblast is a direct effector of tissue injury and matrix remodeling in inflammatory synovitis [3]. Although, until recently, the non-immune constituents of the synovial lining were regarded as targets of the inflam- matory milieu and secondary players in the development of disease, more recent data provide serious grounds for considering these cells as dominant players in the aethiopathogenesis of RA [1, 2].
The hyperplasia of synovial fibroblasts is one of the most striking features of RA [4]. It is considered to be essential to the evolution of joint destruction in RA, but the mechanism underlying control of synovial fibroblast proliferation remains poorly understood. RA synovial fibroblasts are relatively resistant to apoptosis and ex- hibit dysregulated growth secondary to the action of growth factors [3, 5, 6].
Kynurenic acid is a broad-spectrum antagonist of all subtypes of ionotropic glutamate receptors [7], but is preferentially active at the strychnine-insensitive glycine allosteric site of the N-methyl-D-aspartate (NMDA) receptor [8], and is also a non-competitive antagonist at alpha7 nicotinic receptor [9]. Its presence has been documented in urine, serum, amniotic fluid [10], cere- brospinal fluid [11], brain tissue [11, 12] and retina [13]. Kynurenic acid possesses anticonvulsant and neuro- protective properties [14]. However, its potential role in the periphery remains unknown. Recently, it has been found that the blockade of NMDA receptors triggered widespread apoptotic neurodegeneration in the devel- oping rat brain [15] and that glutamate antagonists inhibit proliferation of different human tumor cells [16, 17]. These findings suggest anticancer potential of glutamate antagonists [16, 17].The main goals of this study were the detection and quantification of kynurenic acid in synovial fluid ob- tained from patients with RA and determination of its effect on proliferation of synoviocytes in vitro.
Material and methods
Kynurenic acid detection in synovial fluid.Samples of synovial fluid were collected from the knee joints of 58 patients of both sexes with RA diagnosed by American College of Rheumatism (ACR) criteria. All subjects were informed about the purpose of the study and their consent were obtained. Experimental protocol was approved by a local bioethical committee.
Synovial Drugs
Nimesulide (N-(4-nitro-2-phenoxyphenyl)-methanesulf- onamide) and kynurenic acid were purchased from Sigma (USA). Celecoxib (4-[5-(4-methylphenyl)-3-(tri- fluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide) was obtained from Searle (USA). All other chemicals were purchased from commercial suppliers and were of the highest available purity.
Data analysis
Statistical analysis of data was performed using Stu- dent’s t-test or ANOVA. The IC50 value (the concen- tration of drug necessary to induce 50% inhibition) together with confidence limits was calculated using computerised linear regression analysis of quantal log dose-probit functions according to the method of Litchfield and Wilcoxon [18].
fluid was frozen at –80°C for storage until use. Samples
were centrifuged at 10,000 rpm for 10 min and super- natants were collected. Proteins were precipitated and removed by centrifugation. Supernatants were applied to cation-exchange columns (Dowex 50 W+). Kynurenic acid content was measured using an HPLC system with fluorescence detection (excitation: 334 nm; emission: 398 nm).
Cell culture
In vitro experiments were conducted on rabbit synovi- ocytes cell line HIG-82 obtained from ATCC (American Type Culture Collection, Menassas, VA, USA). Culture medium consisted of nutrient mixture F-12 Ham (Sigma Chemicals, St. Louis, MO, USA) supplemented with 10% of FBS (Life Technologies, Karlsruhe, Germany), 100 U/ml penicillin (Sigma) and 100 lg/ml streptomycin (Sigma). Cultures were kept at 37°C in humidified atmosphere of 95% air and 5% CO2.
Cell proliferation assessment
Cells were plated on 96 well microplates (NUNC) at a density of 1·104 cells/ml. The next day, culture medium was changed and cells exposed to serial dilutions of tested compounds. Cell proliferation was assessed after 96 h by means of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay (Cell proliferation kit I, Boehringer Mannheim). The yellow tetrazolium salt MTT is metabolized by viable cells to purple formazan crystals. HIG 82 cells were incubated for 3 h with MTT solution (5 mg/ml). Formazan crys- tals were solubilized overnight in SDS buffer, and the product quantified spectrophotometrically by measuring absorbance at 570 nm wavelength using E-max Micro- plate Reader (Molecular Devices).
Results
Identification of kynurenic acid
The samples of kynurenic acid isolated from synovial fluid were subjected for comparison with authentic kynurenic acid in three different mobile phase compo- sitions. In all cases, the shape and the latency to the peak of isolated and authentic kynurenic acid were identical. In synovial fluid samples to which authentic kynurenic acid was added, only one peak was recorded on the chromatograms (data not shown).
Kynurenic acid in synovial fluid
Presence of kynurenic acid was determined in all sam- ples of synovial fluid. The mean concentration was 15.89±8.45 pmol/ml (Fig 1).
Cell proliferation assessment
Kynurenic acid inhibited synoviocyte proliferation with the IC50 value (the concentration of drug necessary to induce 50% inhibition) of 5.9 mM (Fig. 2). Moreover, in a subthreshold concentration of 0.3 mM it enhanced antiproliferative action of antirheumatic drugs, celec- oxib and nimesulide (Fig. 3).
Discussion
The first report on the presence of kynurenic acid in synovial fluid obtained from five RA patients was published by Igari et al. in 1969 [19]. In this study kynurenine metabolites were determined by paper partition chromatography. The estimated concentration of kynurenic acid was 4.29 lg/ml [19]. Here, we isolated, identified and determined kynurenic acid by means of more precise analytical methods. In the present study, we reported its presence in synovial fluid obtained from RA patients of both sexes. Kynurenic acid was deter- mined in all 58 studied samples of synovial fluid, with the mean concentration of 15.89 pmol/ml. This is almost 1,400 times lower than that reported in the early study by Igari et al. [19] and corresponds well to the concen- trations found in human serum [10]. Moreover, its presence in nanomolar concentration range has been documented also in urine, amniotic fluid [10], cerebro- spinal fluid [11], brain tissue [11, 12] and retina [13]. The comparison of the concentration of kynurenic acid in RA synovial fluid to that in healthy controls was not possible for the ethical reasons.
Although kynurenic acid is present in the blood, its origin and role in the periphery is unknown. Kynurenic acid is a metabolite of tryptophan formed along the ky- nurenine pathway. The conversion from tryptophan to kynurenine is achieved by tryptophan-2,3-dioxygenase and indole-2,3-dioxygenase (IDO), and kynurenic acid is produced irreversibly from kynurenine by the action of kynurenine aminotransferases [20].
It is well established that IDO activity is elevated during infectious diseases. It was shown that IDO expression can be induced in many cell types by inter- feron-gamma and other bacterial and viral products [21]. Bertazzo and colleagues (1999) reported a high level of IL-1b, IL-6 and IL-8 and corresponding high activity of IDO in synovial fluid collected from patients with RA [22]. In the same patients, low levels of tryptophan in both synovial fluid and serum were found [22]. The lowered level of tryptophan [23, 24] and enhanced [23] or normal [24] concentration of kynurenine were recently reported in the serum of RA patients. There is no data available on the kynurenine aminotransferase(s) activity in RA. Only one report showed no change in plasma kynurenic acid concentration in patients with RA [23]. The role of kynurenic acid in tissues of the joint cavity remains completely unknown.
Kynurenic acid is a broad-spectrum antagonist of all subtypes of ionotropic glutamate receptors [7]. Gluta- mate and aspartate are the major neurotransmitters mediating synaptic excitation in the central nervous system. However, glutamate and its receptors were found also in peripheral nerves. In 2000, McNearney et al. found elevated levels of glutamate and aspartate in the synovial fluid of humans with active arthritis, sug- gesting that glutamate mediated events may contribute to the pathogenesis of this condition [25]. Recently, the possibility that glutamate may be released by neuronal endings in the inflamed knee joint was demonstrated in rats [26]. Moreover, its contribution to the hyperalgesic events initiated during the development of knee joint inflammation was strongly supported [26, 27]. It is known that kynurenic acid possesses antinociceptive properties in different animal pain models attributed to its antagonistic action on the glutamate receptors [28]. Based on these data and on our finding, it can be spec- ulated that endogenous kynurenic acid in synovial fluid may affect pain susceptibility by blockade of glutamate receptors on sensory terminals in the joint tissue.
In RA, evidence is accumulating that the inflammatory process in the synovial tissue is dominated by induce apoptosis and therefore to suppress the prolif- eration of RA synovial fibroblasts [29], colon adeno- carcinoma [30] and cholangiocarcinoma cells [31]. Similarly, nimesulide inhibited proliferation of colon adenocarcinoma [30] and pancreatic cancer cells [32].
The concentration of kynurenic acid effective in vitro ( 0.5 mM) was higher than that found in synovial fluid (16 nM) obtained from RA patients. These findings, however, do not necessarily exclude the possibility that kynurenic acid may contribute to the control of prolif- eration of synoviocytes in RA patients. First, the in vitro experiments were performed on rabbit synoviocytes cell line HIG-82, and therefore the antiproliferative activity of kynurenic acid should be determined on synoviocytes obtained from RA patients. Secondly, the concentration of kynurenic acid in the immediate vicinity of fibroblast in the synovial cavity in vivo cannot be estimated. In our study, the total level of kynurenic acid was calculated in the whole amount of synovial fluid evacuated during therapeutic arthrocentesis. Thirdly, we found that a subthreshold concentration of kynurenic acid enhanced the antiproliferative activity of celecoxib and nimesulide. Thus, it can be speculated that even in a low concen- tration, it may exert such activity in vivo acting in concert with other antiproliferative compounds.
In conclusion, we documented that kynurenic acid is an endogenous constituent of synovial fluid, and hypothesize that its action may be attributed to the control of synoviocyte proliferation in RA.
In this study, we found that kynurenic acid inhibited synoviocyte proliferation. Moreover, we evidenced that in a subthreshold concentration of 0.3 mM it enhanced antiproliferative action of antirheumatic drugs, celec- oxib and nimesulide. Recently, celecoxib was found to induce apoptosis and therefore to suppress the prolif- eration of RA synovial fibroblasts [29], colon adeno- carcinoma [30] and cholangiocarcinoma cells [31]. Similarly, nimesulide inhibited proliferation of colon adenocarcinoma [30] and pancreatic cancer cells [32].
The concentration of kynurenic acid effective in vitro ( 0.5 mM) was higher than that found in synovial fluid (16 nM) obtained from RA patients. These findings, however, do not necessarily exclude the possibility that kynurenic acid may contribute to the control of prolif- eration of synoviocytes in RA patients. First, the in vitro experiments were performed on rabbit synoviocytes cell line HIG-82, and therefore the antiproliferative activity of kynurenic acid should be determined on synoviocytes obtained from RA patients. Secondly, the concentration of kynurenic acid in the immediate vicinity of fibroblast in the synovial cavity in vivo cannot be estimated. In our study, the total level of kynurenic acid was calculated in the whole amount of synovial fluid evacuated during therapeutic arthrocentesis. Thirdly, we found that a subthreshold concentration of kynurenic acid enhanced the antiproliferative activity of celecoxib and nimesulide. Thus, it can be speculated that even in a low concen- tration, it may exert such activity in vivo acting in concert with other antiproliferative compounds.