Abstract
To investigate the possible role of tryptophan metabolism in immune regulation of primary Sjögren's syndrome (pSS) the serum concentrations of tryptophan and its metabolite kynurenine were measured by reverse-phase high-performance liquid chromatography (HPLC) in 103 patients with pSS, 56 patients with sicca symptoms and 309 healthy blood donors. The kynurenine per tryptophan ratio (kyn/trp), which reflects the activity of the indoleamine-pyrrole 2,3-dioxygenase (IDO) enzyme involved in tryptophan catabolism, was calculated. Both female and male patients with pSS had significantly higher serum kynurenine concentrations and kyn/trp than subjects with sicca symptoms or healthy blood donors. The median (quartile range) concentration of kynurenine in female patients with pSS was 2·41 µmol/l (1·86–3·26) compared with 1·85 µmol/l (1·58–2·38, P < 0·0001) in subjects with sicca symptoms and 1·96 µmol/l (1·65–2·27, P < 0·0001) in healthy blood donors. Their kyn/trp × 1000 was 34·0 (25·1–44·3) compared with 25·3 (21·1–31·5, P < 0·0001) in subjects with sicca symptoms and 24·3 (21·0–28·9, P < 0·0001) in healthy blood donors. Female pSS patients with high IDO activity (kyn/trp × 1000 ≥ 34·0) had significantly higher ESR, serum C-reactive protein, serum IgA and serum beta-2 microglobulin concentrations as well as higher serum creatinine levels, and they had positive antinuclear antibodies more frequently and presented with more American-European consensus group criteria than those with low IDO activity (kyn/trp × 1000 < 34·0). These data suggest that mechanisms dependent on tryptophan catabolism regulate immune responses in pSS. Tryptophan degradation is enhanced in patients with pSS, and high IDO activity is associated with severity of pSS.
Keywords: tryptophan, kynurenine, indoleamine 2, 3-dioxygenase, primary Sjögren's syndrome
Introduction
Tryptophan is an essential amino acid whose catabolism by the enzyme indoleamine-pyrrole 2,3-dioxygenase (EC 1·13·11·42, also known as indoleamine 2,3-dioxygenase, IDO) has recently been linked with immunoregulation [1–3]. The degradation of tryptophan takes place via two biochemical pathways: by tryptophan 5-hydroxylase enzyme to 5-hydroxytryptamine (serotonin) or by either the tryptophan 2,3-dioxygenase (EC 1·13·11·11, TDO) enzyme or by the IDO enzyme to N-formylkynurenine. This is then converted to kynurenine and further catabolized to antranilic acid with its derivatives. TDO regulates homeostatic serum tryptophan concentrations and IDO is up-regulated in response to infection and tissue inflammation. Dietary intake of tryptophan may affect its serum concentration and the kynurenine per tryptophan ratio (kyn/trp) is therefore considered a more reliable marker for IDO-induced tryptophan catabolism than serum tryptophan concentration. Mellor and Munn [1–3] have recently proposed that IDO-producing cells might play a role in preventing the initiation of autoimmune disorders, but once the autoimmune disorder is established the presence of chronic inflammation might provoke sustained IDO production.
Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder which primarily affects the salivary and lacrimal glands. The most characteristic manifestations of the disease are keratoconjunctivitis sicca and xerostomia. An interaction between genetic and environmental factors is considered to lead to autoimmunity in pSS. In an appropriate genetic background an unknown triggering factor, possibly viral, may lead to the immunologically mediated inflammatory mechanisms which result in the chronic exocrine gland lesions present in pSS [4]. T-cell-mediated autoimmune responses in the glandular tissue as well as apoptotic events are currently considered to be of major importance in the pathogenesis of SS [5].
IDO expression is associated with chronic inflammatory conditions [6–10]. Tryptophan degradation due to IDO activation has been suggested to represent a defence mechanism against viral or malignant challenge or against an attack by auto-reactive T-cells [1–3]. In pSS persistent cellular immune activation is present. We hypothesized that tryptophan degradation due to enhanced IDO activity might be altered in patients with pSS. In order to address this issue, we analysed markers of tryptophan degradation in 103 patients with pSS and, as control groups, in 56 subjects with sicca symptoms but no pSS, and in 309 healthy blood donors.
Subjects and methods
Subjects
Serum samples were obtained after informed consent from 103 patients with pSS (96 female, 7 male) and 56 subjects presenting with sicca symptoms (46 female, 10 male) but not fulfilling the criteria for pSS. Two of the patients with sicca symptoms fulfilled the criteria for rheumatoid arthritis, one had an undifferentiated connective tissue disease (UCTD), three a seronegative spondylarthropathy and two a nonspecified oligoarthritis. The data collections have been described in detail elsewhere [11,12]. The pSS patients were originally selected by modified Californian criteria for probable or definite pSS [13] and the histological findings were graded on the Chisholm-Mason [14] scale, grades 3 and 4 being regarded as diagnostic. Ninety-three of the patients also fulfilled the revised American-European consensus group criteria for pSS [15]. The mean age of the patients with pSS was 60 ± 12 years, range 29–82 years, and of subjects with sicca symptoms 55 ± 13 years, range 28–80 years. The mean duration of sicca symptoms of the eyes in patients with pSS was 11 ± 8 years and of the mouth 13 ± 8 years.
Clinical methods
A careful clinical examination together with an in-depth interview with the patients covering previous and concurrent diseases and medications and duration of pSS had recently been conducted [11,12]. Special emphasis was laid on possible extraglandular manifestations of pSS (dermatological, endocrine, gastrointestinal, lymphoproliferative, musculoskeletal, neurological, renal, respiratory and vascular symptoms). Purpura was defined as a history of typical episodic palpable purpura lesions in the lower limbs or skin biopsy histology. Lymphadenopathy was defined as lymph node enlargement so persistent as to have indicated a nodal biopsy. Arthritis was defined as articular swelling observed by a clinician. Peripheral and central neurological symptoms were recorded from the history given by the patients as well as from data on possible neurological investigations from the case histories. The diagnosis of pulmonary fibrosis was based on findings in chest radiographs; that of alveolitis had been established by thorough investigations in a pulmonary unit.
Normal controls
Three hundred and nine healthy Finnish Red Cross Transfusion Service blood donors (139 females, 170 males) served as a control group. The mean age of these subjects was 45 ± 11 years, range 21–64 years.
Standard laboratory tests
The standard laboratory tests included erythrocyte sedimentation rate (ESR), serum C-reactive protein (CRP) and serum creatinine. Rheumatoid factor (RF) was determined by laser nephelometry (Behring Nephelometric Analyser, Marburg, Germany) and antinuclear antibodies (ANA) by indirect immunofluorescence using Hep-2 cells. Anti-SS-A and anti-SS-B antibodies were determined by enzyme immunoassay. Serum concentrations of immunoglobulin IgA, IgG and IgM were measured by laser nephelometry (Behring Nephelometric Analyser) and serum beta-2 microglobulin (β2m) by radioimmunoassay (Pharmacia beta-2-micro RIA kit, Pharmacia Diagnostics Uppsala, Sweden).
Tryptophan and kynurenine determinantions
Tryptophan and kynurenine concentrations in peripheral blood were measured by reverse-phase high-performance liquid chromatography as previously described [16]. Tryptophan was separated with Shimadzu liquid chromatograph LC-10AD VP (Shimadzu Co, Kyoto) using 50 mm BDS Hypersil C 18 5 µm column (Thermo Electron Co, Bellefonte, PA, USA). It was monitored by fluorescence with Shimadzu RF-10 A XL detector at 266 nm excitation and 366 nm emission wavelenghts. Kynurenine was separated with Hewlett Packard 1100 liquid chromatograph (Palo Alto, CA, USA) using Merck LiChroCart 55–4150 mm cartridge containing Purospher STAR RP-18 3 µm Column (Merck Co, Darmstadt, Germany). It was determined by ultraviolet absorption at 360 nm wavelength with Hewlett Packard G13144 detector. Kyn/trp was calculated by relating concentrations of kyn (µmol/l) to trp (µmol/l), this allowing an estimate of IDO activity.
Statistical analysis
Kruskall-Wallis anova and Mann–Whitney U-test were used in comparisons of continuous variables and χ2 test in comparisons of dichotomous variables. Logistic regression analysis by backward stepwise method was applied to analyse the independent effects of the intercorrelated variables on high IDO activity. Findings were considered statistically significant at P < 0·05. Statistical analyses were performed with SPSS 11·5 for Windows.
Ethical approval
The study protocol was approved by the Ethical Committee of Tampere University Hospital, Tampere, Finland and by the Ethical Committee of the Finnish Red Cross Transfusion Service, Helsinki, Finland.
Results
The demographic, clinical and immunoserological characteristics of the pSS patients are presented in Table 1.
Table 1. Demographic, clinical and immunological characteristics of 103 patients with primary Sjögren's syndrome.
| Characteristic | Value or frequency |
|---|---|
| Demographic | |
| Females: males | 96 : 7 |
| Age, mean ± SD (years) | 60 ± 12 |
| Duration of symptoms of the eyes, mean ± SD (years) | 11 ± 8 |
| Duration of xerostomia, mean ± SD (years) | 13 ± 8 |
| Clinical | |
| Labial salivary gland histological grade 3–4† | 72/97 (74) |
| Arthralgia | 75 (73) |
| Raynaud's symptom | 52 (51) |
| Myalgia | 52 (51) |
| Recurrent salivary gland swelling | 51 (50) |
| Arthritis | 21 (20) |
| Purpura | 19 (18) |
| Peripheral nervous system symptoms | 19 (18) |
| Pleuritis | 15 (15) |
| Lymphadenopathy | 10/77 (13) |
| Alveolitis or pulmonary fibrosis | 9/77 (12) |
| Central nervous system symptoms | 9 (9) |
| Myositis | 0 |
| Immunological | |
| ANA-positive | 82 (80) |
| RF-positive | 60 (58) |
| anti-SSA antibody-positive | 65/101 (63) |
| anti-SSB antibody-positive | 48/101 (47) |
Chisholm-Mason scale; ANA, antinuclear antibodies; RF, rheumatoid factor. The values indicate numbers of patients, unless otherwise indicated, and the figures in parenthesis are percentages of cases.
Tryptophan and kynurenine concentrations and kyn/trp
The analysis of the data was performed in women and men separately, as kynurenine concentrations and the kyn/trp differed significantly between the healthy women and men blood donors. The median serum tryptophan concentration in female patients with pSS was 75·0 µmol/l (quartile range 63·8–81·2) compared with 74·0 µmol/l (62·7–86·4) in patients with sicca symptoms and 80·5 µmol/l (69·8–91·4) in healthy blood donors (Fig. 1a). The median kynurenine concentrations were significantly higher (2·41 µmol/l, 1·86–3·26) in female pSS patients compared with patients with sicca symptoms (1·85 µmol/l, 1·58–2·38) or healthy blood donors (1·96 µmol/l, 1·65–2·27) (Fig. 1b). The kyn/trp × 1000 was higher (34·0, 25·1–44·3) in female patients with pSS than in patients with sicca symptoms (25·3, 21·1–31·5) or healthy blood donors (24·3, 21·0–28·9) (Fig. 1c).
Fig. 1.
Median (quartile range) serum tryptophan concentrations (µmol/l) (a), kynurenine concentrations (µmol/l) (b) and kynurenine/tryptophan (kyn/trp) × 1000 (c) in female patients with primary Sjögren's syndrome (n = 96), patients with sicca symptoms (n = 46) and healthy blood donors (n = 139). Statistical analysis: Kruskall-Wallis anova and Mann–Whitney U-test
In male pSS patients the median serum tryptophan concentration was 80·2 µmol/l (73·3–97·3) versus 76·3 (73·4–87·3) in patients with sicca symptoms and 78·8 µmol/l (70·4–86·3) in healthy blood donors (Fig. 2a). The median serum kynurenine concentration was significantly higher in male patients with pSS (2·84 µmol/l, 2·03–3·62) than in those with sicca symptoms (1·75 µmol/l, 1·56–2·15) or in healthy blood donors (2·14 µmol/l, 1·78–2·44) (Fig. 2b). The kyn/trp × 1000 in male patients with pSS (34·5, 29·3–38·7) was also higher than in patients with sicca symptoms (22·3, 20·7–27·9) or in healthy blood donors (27·0, 23·6–32·1) (Fig. 2c). The kyn/trp × 1000 was significantly lower in male sicca patients than in healthy controls (P= 0·024).
Fig. 2.
Median (quartile range) serum tryptophan concentrations (µmol/l) (a), kynurenine concentrations (µmol/l) (b) and kynurenine/tryptophan (kyn/trp) × 1000 (c) in male patients with primary Sjögren's syndrome (n = 7), patients with sicca symptoms (n = 10) and healthy blood donors (n = 170). Statistical analysis: Kruskall-Wallis anova and Mann–Whitney U-test
Comparison of female pSS patients with high or low IDO activity
The detailed clinical data on the pSS patients are reported only for women in view of the small number of men. We divided the female pSS patients into two subgroups: those with high IDO activity, i.e. kyn/trp × 1000 ≥ 34·0and those with low IDO activity, i.e. kyn/trp × 1000 < 34·0. The mean ages of the pSS patients or duration of sicca symptoms of the eyes or xerostomia did not differ significantly between the groups (Table 2).
Table 2. The clinical features of female patients with primary Sjögren's syndrome (pSS) grouped by high (≥ median, 34·0) or low (< median) ratio of kynurenine and tryptophan (kyn/trp) × 1000.
| Female pSS patients with kyn/trp | |||
|---|---|---|---|
| High n = 48 | Low n = 48 | P-value | |
| Age, mean ± SD (years) | 61 ± 14 | 58 ± 10 | 0·051 |
| Duration of sicca symptoms of the eyes, mean ± SD (years) | 12 ± 7 | 11 ± 9 | 0·646 |
| Duration of xerostomia, mean ± SD (years) | 13 ± 9 | 12 ± 7 | 0·834 |
| LSG histological grade 3–4† | 37/46 (80) | 29/45 (64) | 0·088 |
| ≥ 4/4 of Californian criteria | 25 (52) | 16 (33) | 0·063 |
| ≥ 5/6 American-European criteria | 31 (65) | 16 (33) | 0·002 |
| Arthralgia | 31 (65) | 38 (79) | 0·112 |
| Arthritis | 11 (23) | 9 (19) | 0·615 |
| Raynaud's symptom | 22 (46) | 28 (58) | 0·220 |
| Purpura | 11 (23) | 8 (17) | 0·442 |
| Recurrent salivary gland swelling | 25 (52) | 24 (50) | 0·838 |
| Lymphadenopathy | 3/39 (8) | 7/35 (20) | 0·122 |
| Peripheral nervous system symptoms | 12 (25) | 6 (13) | 0·117 |
| Central nervous system symptoms | 3 (6) | 6 (13) | 0·294 |
| Alveolitis or pulmonary fibrosis | 5/39 (13) | 3/35 (9) | 0·557 |
| Pleuritis | 6 (13) | 9 (19) | 0·399 |
| Current corticosteroid usage | 3 (6) | 14 (29) | 0·003 |
| Current DMARD usage | 3 (6) | 5 (10) | 0·460 |
Chisholm-Mason scale. LSG, labial salivary gland; DMARD, disease-modifying antirheumatic drug. The values indicate numbers of patients, unless otherwise indicated, and the figures in parenthesis are percentages of cases. Statistical analysis: Mann—Whitney U-test or χ2 test.
There were no differences between the female pSS patients with high or low IDO activity in the frequency of such clinical manifestations of pSS as a history of arthralgia, arthritis, myalgia, salivary gland swelling, Raynaud's symptom, purpura, lymphadenopathy, pleuritis, alveolitis or pulmonary fibrosis, peripheral or central nervous system symptoms. The histological grade in labial salivary gland biopsies (Chisholm and Mason scale) or the number of Californian criteria for pSS [13] fulfilled did not differ significantly between female pSS patients with high or low IDO activity, although there was a trend towards more severe presentation in patients with high IDO activity (Table 2). However, when compared in terms of the number of the American-European consensus criteria [15] fulfilled, the patients with high IDO activity had significantly more often ≥5 criteria than those with low IDO activity (Table 2).
There were several differences in laboratory parameters between female pSS patients with high or low IDO activity: significantly higher ESR, serum CRP, serum IgA and serum beta-2 microglobulin levels were observed in those with high IDO activity compared with those with low. Moreover, they also had positive ANA and positive anti-SSB antibodies more frequently than those with low IDO activity, although the difference was significant only regarding ANA. The pSS patients with high IDO activity had higher serum creatinine levels than those with low activity. The female pSS patients with high IDO activity were currently using corticosteroid more frequently than the others (P= 0·003) (Table 3), although the total proportion of patients using corticosteroids was low (18%). When the patients receiving corticosteroids were excluded from the analysis, ESR, CRP, serum IgA and serum beta-2 microglobulin remained significantly higher in those with high IDO activity compared to those with low, but serum creatinine did not (P= 0·061) (data not shown).
Table 3. Serological findings in female patients with primary Sjögren's syndrome (pSS) grouped by high (≥ median, 34·0) or low (< median) ratio of kynurenine and tryptophan (kyn/trp) × 1000.
| Female pSS patients with kyn/trp | |||
|---|---|---|---|
| High n = 48 | Low n = 48 | P-value | |
| ESR (mm/h, mean ± SD) | 35·0 ± 24 | 21·0 ± 12 | 0·007 |
| CRP (µg/ml, mean ± SD) | 7·2 ± 5·0 | 6·3 ± 3·9 | 0·023 |
| S-creatinine (µmol/l, mean ± SD) | 90·0 ± 35 | 76·0 ± 12 | 0·038 |
| S-IgA (g/l, mean ± SD) | 3·34 ± 1·98 | 2·51 ± 1·02 | 0·028 |
| S-IgG (g/l, mean ± SD) | 18·9 ± 7·8 | 16·7 ± 5·6 | 0·214 |
| S-IgM (g/l, mean ± SD) | 1·49 ± 0·72 | 1·50 ± 0·96 | 0·531 |
| S-β2m (mg/l, mean ± SD) | 3·59 ± 1·65 | 2·12 ± 0·45 | < 0·0001 |
| ANA-positive | 42 (88) | 33 (69) | 0·026 |
| RF-positive | 36 (75) | 29/47 (62) | 0·163 |
| anti-SSA antibody-positive | 33 (69) | 30/46 (65) | 0·716 |
| anti-SSB antibody-positive | 28 (58) | 18/46 (39) | 0·063 |
ESR, erythrocyte sedimentation rate; CRP, c-reactive protein; ANA, antinuclear antibodies; RF, rheumatoid factor; s-β 2m, serum beta-2 microglobulin. The values indicate numbers of patients, unless otherwise indicated, and the figures in parenthesis are percentages of cases. Statistical analysis: Mann—Whitney U-test or χ2 test.
As several of the parameters associated with high IDO activity were likely to be intercorrelated we performed a logistic regression analysis with high IDO activity as a dependent factor and age, serum beta-2 microglobulin, serum creatinine, serum IgA, ESR and current corticosteroid usage as independent factors. The only factors that remained associated with high IDO activity in this model were serum beta-2 microglobulin (P < 0·0001, OR 13·9, CI 3·96–49·1) and current corticosteroid treatment (P= 0·038, OR 0·17, CI 0·03–0·91).
Discussion
Our results show that tryptophan degradation is increased in patients with pSS compared with a control group of healthy blood donors. The enhanced tryptophan degradation could result from the activation of either IDO or hepatic TDO. IDO seems a more likely activator of the enhanced tryptophan degradation in pSS, as TDO is known to regulate homeostatic serum tryptophan concentrations and IDO is up-regulated in response to inflammatory conditions. The tryptophan concentration in serum may be influenced, e.g. by the dietary intake of the essential amino acid, but the concentration of kynurenine as well as the kyn/trp, which were significantly increased in patients with pSS compared with healthy blood donors, indicate activity of the IDO enzyme, which is responsible for tryptophan degradation.
The serum concentration of kynurenine and the kyn/trp were increased in patients with pSS also when compared with a control group of patients with sicca symptoms but no pSS. It is therefore evident that the increased IDO activity is not associated with sicca symptoms per se but with the immunological phenomena involved in the various manifestations of pSS. Enhanced IDO activity has been related to many diseases in which chronic immune activation persists, i.e. to infectious and malignant diseases [2] but also to SLE [9]. The possibility cannot be excluded that the enhanced IDO activity in pSS patients is purely a marker of inflammation. However, the difference in IDO activity observed between pSS patients and the sicca control group, which also included patients with other inflammatory rheumatic conditions, contradicts this possibility.
Moreover, high IDO activity in pSS patients was associated not only with markers of inflammation such as ESR and CRP, but also with immune responses like positive ANA, high serum IgA and serum beta-2 microglobulin concentrations. Increased production of serum beta-2 microglobulin is observed in conditions involving neoplastic proliferation of lymphoid B-cells or in inflammatory disorders with an activation of the lymphopoetic system, and has previously been associated with lymphoproliferative complications of pSS [12,17].
Our data suggest that the mechanisms activated by enhanced IDO activity play a role in immune regulation in pSS. The increased IDO activity might reflect an attempt to control the chronic antigen stimulation, which in pSS sometimes also triggers malignant transformation. Enhanced IDO activity is clearly not specific for pSS and it remains to be seen whether IDO activity is a true marker for tryptophan catabolism dependent T-cell immunoregulatory mechanisms in autoimmune diseases. It is difficult to evaluate how the enhanced IDO activity observed in pSS patients is connected with the already established pathogenetic events in pSS. It might, however, be surmised that the increased IDO activity in pSS is a mechanism the immune system is using in order to down-regulate the ongoing T cell-mediated autoimmune reaction. If this is the case, the cytokine interleukin-10 (IL-10) could play a decisive role. We have shown that the genetic polymorphism of IL-10 affects susceptibility to pSS, the genotype with high IL-10-producing capability (containing allele G at position–1082, C at –819 and C at–592) being associated with an increased risk of the disease [18]. This finding has also been confirmed in other cohorts of patients with pSS [19,20]. It has been suggested [3] that IDO-positive dendritic cells (DC) exert their regulatory function also by inducing the production of immunosuppressive cytokines such as IL-10 and transforming growth factor-β (TGFβ). It could thus be hypothesized that the IL-10-dependent regulatory function of IDO-positive DCs is more potent in persons with a capability of high IL-10 production. These elevated IL-10 levels would not be sufficient to totally inhibit the autoimmune reaction but would, as a side-effect, cause activation of B lymphocytes, which could be manifested by positive ANA and by elevated serum beta-2 microglobulin and IgA concentrations as reported here.
No differences in the various clinical manifestations of pSS were observed between patients with high or low IDO activity. However, IDO activity clearly seemed to be related to the severity and activity of pSS reflected in a higher number of American-European consensus criteria fulfilled in the high IDO activity group and in more severe labial salivary gland histological findings, although the latter finding did not reach statistical significance. In contrast, in a study of SLE patients only a weak association between IDO activity and the activity index SLEDAI was observed [9].
High IDO activity was associated with elevated serum creatinine concentrations. Hyperkynurinemia has indeed been found in patients with chronic glomerulonephritis with renal failure [21]. However, when the patients receiving corticosteroids were excluded from the data, serum creatinine was not significantly higher in pSS patients with high IDO activity compared with those with low activity. Moreover, when logistic regression analysis was performed, IDO activity was not independently associated with creatinine.
It has been suggested that clarification of the mechanisms of tryptophan catabolism in immune regulation might also permit novel immunotherapeutic interventions to alleviate autoimmune diseases [2]. Here, it could be established that the IDO activity was significantly lower in patients currently treated with low-dose corticosteroids than in those without corticosteroid treatment. Six-months’ treatment with methotrexate or corticosteroids did not alter the profiles of tryptophan metabolites in patients with rheumatoid arthritis in spite of significant therapeutic responses [22]. However, it seems probable that corticosteroid treatment is able to reduce IDO activity in pSS patients. Whether this finding would also have clinical implications and relevance should naturally be evaluated in a proper prospective controlled study.
In conclusion, our data show that tryptophan degradation in patients with pSS is increased due to enhanced IDO activity compared both with healthy blood donors and with patients with sicca symptoms, and further, that IDO activity is associated with severity of pSS. These data suggest that mechanisms dependent on tryptophan catabolism regulate immune responses in pSS as indicated by high levels of markers of inflammation, high serum beta-2 microglobulin and serum IgA and positive ANA in pSS patients with high IDO-activity.
Acknowledgments
This study was supported by the Medical Research Fund of Tampere University Hospital, Tampere, Finland, The Academy of Finland and the Tampere Tuberculosis Foundation. We thank Ms. Raija Repo and Ms. Sinikka Repo-Koskinen for skilful technical assistance.
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