Lower level of synovial fluid interferon-{gamma} in HLA-B27-positive than in HLA-B27-negative patients with Chlamydia trachomatis reactive arthritis

S. Bas, T. K. Kvien1, N. Buchs, T. Fulpius and C. Gabay

Division of Rheumatology, Department of Internal Medicine, University Hospital, Geneva, Switzerland and
1 Oslo City Department of Rheumatology, Diakonhjemmet Hospital, Oslo, Norway


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. To compare the synovial fluid (SF) concentrations of various cytokines in rheumatoid arthritis (RA) and in reactive arthritis, and to look for a correlation between cytokine levels and the presence of HLA-B27 antigen in reactive arthritis patients.

Methods. Concentrations of interleukin (IL) 10, IL-12, IL-18, interferon {gamma} (IFN-{gamma}) and tumour necrosis factor {alpha} (TNF-{alpha}) were determined by commercially available enzyme-linked immunosorbent assays (ELISA) in the SF from 48 patients with reactive arthritis, 33 with RA and 13 with osteoarthritis (non-inflammatory controls).

Results. The SF concentrations of IL-10 were significantly lower in patients with reactive arthritis (median 2.3 pg/ml) than in RA patients (median 14.6 pg/ml). The SF levels of IFN-{gamma} were not significantly different but the ratios of IFN-{gamma} to IL-10 were significantly higher in patients with reactive arthritis (median 9.2) than in RA patients (median 0.83). When the subset of patients with Chlamydia trachomatis reactive arthritis was considered, the SF concentration of IFN-{gamma} was significantly lower in HLA-B27-positive (median 2.9 pg/ml) than in HLA-B27-negative patients (median 42.4 pg/ml). After 2 yr of follow-up, two HLA-B27-positive patients, who had low SF levels of IFN-{gamma}, had a chronic course of arthritis, whereas after 1 yr all HLA-B27-negative patients had complete resolution of arthritis.

Conclusions. The lower IFN-{gamma} concentrations in HLA-B27-positive patients with C. trachomatis reactive arthritis could be related to the tendency of these patients to have more severe or chronic arthritis.

KEY WORDS: Cytokines, Synovial fluid, Reactive arthritis, Chlamydia trachomatis, Interferon {gamma}.


    Introduction
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
In reactive arthritis the mechanism of joint damage is unclear, but it is likely that cytokines play critical roles. A more prevalent Th1 profile [high levels of interferon {gamma} (IFN-{gamma}) and low levels of interleukin (IL) 4] has been reported [16], but several studies have shown relative predominance of IL-4 and IL-10 and relative lack of IFN-{gamma} and tumour necrosis factor {alpha} (TNF-{alpha}) in the synovial membrane [7, 8] and fluid (SF) [8, 9] of reactive arthritis patients compared with rheumatoid arthritis (RA) patients. A lower level of TNF-{alpha} secretion in reactive arthritis than in RA patients has also been reported after ex vivo stimulation of peripheral blood mononuclear cells (PBMC) [10].

It has been argued that clearance of reactive arthritis-associated bacteria requires the production of appropriate levels of IFN-{gamma} and TNF-{alpha}, while IL-10 acts by suppressing these responses [9, 1115]. Indeed, IL-10 is a regulatory cytokine that inhibits the synthesis of IL-12 and TNF-{alpha} by activated macrophages [1618] and of IFN-{gamma} by T cells [9, 19]. However, IFN-{gamma}-inducing cytokines, such as IL-12 and IL-18 [2026], may also play a role in reactive arthritis. Thus, Yin et al. [9] assigned a crucial role to the IL-10/IL-12 balance in the regulation of the cytokine pattern in the joints of patients with reactive arthritis.

In this study, we investigated the levels of IL-10, IL-12, IL-18, IFN-{gamma} and TNF-{alpha} in the SF of patients with well-characterized Chlamydia trachomatis reactive arthritis, various uro- or enteroarthritides, RA and osteoarthritis (non-inflammatory controls). The possible correlation between their levels and the presence of HLA-B27 antigen was examined in patients with reactive arthritis as this antigen seems to contribute to the severity of the disease [10, 2729].


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
Synovial fluid samples came from our collection and had been kept for various times at -70°C (after centrifugation 10 min at 1600 g). Diagnosis was taken from the chart at the time of collection of the SF. The patients were divided into the following three groups.

  1. Reactive arthritis (n=48): asymmetrical mono/oligoarthritis. Eighteen patients had urethritis and evidence of C. trachomatis infection [in one patient urethral/endocervical C. trachomatis antigen was detected by direct immunofluorescence, 11 had a positive urethral/endocervical C. trachomatis culture, and six had positive urethral C. trachomatis DNA amplification (Amplicor test; Roche Diagnostic Systems, Branchburg, NJ, USA)]. Eighteen patients had urethritis but without proof or evidence of C. trachomatis infection; 12 of these patients had negative urethral/endocervical Chlamydia detection and in six patients no search was made for various reasons. Twelve patients had diarrhoea: four of these had a positive stool culture: two for Salmonella enteritidis, one for Salmonella typhimurium and one for Shigella; three had a negative culture and five had no culture for various reasons. Nineteen patients were HLA-B27-positive, 15 were HLA-B27-negative and 14 were not determined. The duration of arthritis, number of active joints and cell counts are indicated in Tables 3Go and 4Go according to HLA-B27 status. Twelve of the 18 patients with C. trachomatis reactive arthritis were followed up for 3, 6, 12, 24 or 48 months.
  2. Rheumatoid arthritis (RA) diagnosed according to the revised criteria formulated by the ACR [30] (n=33). Twenty-one had a positive latex test and 12 a negative one. Median disease duration was 12 months [interquartile range (IQR) 1–48] and the median erythrocyte sedimentation rate (ESR) was 37 mm/1st h (IQR 20–65).
  3. Osteoarthritis (n=13).


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TABLE 3. Levels of IL-10, IL-12, IL-18, IFN-{gamma} and TNF-{alpha} in the SF of patients with reactive arthritis according to the presence of HLA-B27 antigen

 

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TABLE 4. Clinical data and concentrations of IL-10 and IFN-{gamma} in the SF of patients with C. trachomatis reactive arthritis according to the presence of HLA-B27 antigen

 
The median age, age range and the percentage of female patients are shown for each group in Table 1Go.


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TABLE 1. Presence of IL-10, IL-12, IL-18, IFN-{gamma} and TNF-{alpha} in the SF of patients with various arthritides

 


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TABLE 2. Levels of IL-10, IL-12, IL-18, IFN-{gamma} and TNF-{alpha} in the SF of patients with various arthritides

 

Determination of IL-10, IL-12, IL-18, IFN-{gamma} and TNF-{alpha} concentrations in SF samples
All cytokine levels were determined by commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers' protocol: IL-10, IL-12, IFN-{gamma} and TNF-{alpha} Quantikine kits were from R & D Systems Research (Minneapolis, MN, USA) and the IL-18 kit was from MBL (Nagoya, Japan). When sufficient SF was available, assays were performed in duplicate, otherwise they were done singly with SF diluted 1:1.25 (IL-10, IL-12 and TNF-{alpha}), 1:2 (IFN-{gamma}) or 1:5 (IL-18).

Excepting IL-12, all cytokine determinations were performed at the same time. The intra-assay variation indicated by the manufacturer was 1–5% for IL-10, IL-12 and IFN-{gamma}, 4–7% for TNF-{alpha} and 5–11% for IL-18. The detection limits (pg/ml) were 3.9 for IL-10, 4.4 for TNF-{alpha}, 5 for IL-12, 8 for IFN-{gamma} and 12.5 for IL-18. To allow statistical analysis, cytokine concentrations were determined for all the optical densities obtained, by extrapolating linear standard curves, even when the cytokine level found was below the minimum detectable concentration indicated by the manufacturer of the kit.

Statistical analysis
Fisher's exact test was used to compare the proportions of patients with detectable levels of cytokine. Kruskal–Wallis and Mann–Whitney tests were used to compare the levels of cytokines in the three groups. A comparison between two groups was made only when the Kruskal–Wallis test yielded a statistically significant result. P values less than 0.05 were considered significant.


    Results
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 Patients and methods
 Results
 Discussion
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Number of patient samples with detectable SF levels of IL-10, IL-12, IL-18, IFN-{gamma} and TNF-{alpha}
Significantly more patients with RA (89%) than with reactive arthritis (41%) had detectable SF levels of IL-10 (P=0.0006). Except in four SF samples from RA patients, the levels of IL-12 were always lower than the detection limit indicated by the manufacturer. The number of patients with detectable SF levels of IFN-{gamma} or TNF-{alpha} was not significantly different in patients with reactive arthritis compared with RA patients (Table 1Go).

Synovial fluid levels of IL-10, IL-12, IL-18, IFN-{gamma} and TNF-{alpha}
To allow statistical analysis, cytokine concentrations were determined by extrapolating linear standard curves, even when cytokine levels were below the minimum detectable concentrations indicated by the manufacturers.

IL-10 concentrations in SF were lower in patients with reactive arthritis (median 2.3 pg/ml, IQR 0.09–5.3) than in RA patients (median 14.6 pg/ml, IQR 7.3–22.4) (P<0.0001). The SF levels of IFN-{gamma} and TNF-{alpha} were not significantly different between patients with reactive arthritis and RA patients (median 14.9 pg/ml, IQR 0–60.4 vs median 7.9 pg/ml, IQR 0.95–20.9, P=0.32 for IFN-{gamma}; median 6.1 pg/ml, IQR 1.5–24 vs median 13.8 pg/ml, IQR 4.13–21.1, P=0.27 for TNF-{alpha}). To further investigate the possible imbalance between IFN-{gamma} and IL-10 levels in reactive arthritis compared with RA patients, the IFN-{gamma}/IL-10 ratio was calculated for each SF sample. The IFN-{gamma}/IL-10 ratios were significantly higher in reactive arthritis (median 9.2, IQR 0.4–29) than in RA patients (median 0.83, IQR 0.33–1.7) (Table 2Go and Fig. 1GoGo).



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FIG. 1. Levels of IL-10, IFN-{gamma} and TNF-{alpha} (pg/ml) in SF of patients with reactive arthritis (ReA), rheumatoid arthritis (RA) and osteoarthritis (OsteoA). The number of patients is shown for each graph. Levels were determined by ELISA. Horizontal bars within boxes show the median, boxes show the interquartile range and vertical bars show the 95% confidence interval (values above and below these levels were plotted separately).

 


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FIG. 2. Levels of IL-10 and IFN-{gamma} (pg/ml) in SF of patients with C. trachomatis reactive arthritis according to HLA-B27 status. The number of patients is shown for each graph. Levels were determined by ELISA. Horizontal bars within boxes show the median, boxes show the interquartile range and vertical bars show the 95% confidence interval (values above and below these levels were plotted separately).

 

Clinical data, cell counts and SF concentrations of IL-10, IL-12, IL-18, IFN-{gamma} and TNF-{alpha} according to the presence of HLA-B27 antigen in patients with reactive arthritis
The relationship between the SF levels of cytokines and the presence of HLA-B27 antigen was examined because this antigen has been associated with the severity of arthritis [10, 2729]. No significant difference according to HLA-B27 status was observed in the whole group of patients with reactive arthritis (Table 3Go).

Clinical data and synovial fluid concentrations of IL-10 and IFN-{gamma} according to the presence of HLA-B27 antigen in the subset of patients with C. trachomatis reactive arthritis
No significant differences were found between HLA-B27-negative and -positive patients for the duration of arthritis, the number of inflamed joints, the concentration of C-reactive protein, the ESR, the number of leucocytes/mm3 of SF, the number of patients with detectable SF levels of IL-10 or the concentration of IL-10. In contrast, detectable SF levels of IFN-{gamma} were observed in significantly more HLA-B27-negative patients (100%) than HLA-B27-positive patients (38%) (P=0.026). The SF level of IFN-{gamma} was also significantly higher in HLA-B27-negative patients (median 42.4 pg/ml, IQR 18.2–119) than in HLA-B27-positive patients (median 2.9 pg/ml, IQR 0.6–25.4, P=0.015). The time to complete resolution of arthritis was higher in HLA-B27-positive patients (median 7.5 months, IQR 3.0–30) than in HLA-B27-negative patients (median 3 months, IQR 2.8–5.3) but the difference was not significant (Table 4Go). However, only one out of six HLA-B27-negative but three out of six HLA-B27-positive patients had a time of remission longer than 3 months. In this last group, one patient had complete resolution of arthritis only after 4 yr and another still had one inflamed joint after 2 yr but was no longer followed up. The SF levels of IFN-{gamma} in these two HLA-B27-positive patients with chronic arthritis were low: 2.8 and 3 pg/ml.

HLA-B27 status did not correlate with the concentrations of other cytokines. However, it should be observed that no comparison was made regarding TNF-{alpha} levels in SF because the limited amount of SF available allowed its determination in only four samples.


    Discussion
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 Abstract
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 Patients and methods
 Results
 Discussion
 References
 
We studied T-cell responses in reactive arthritis compared with the RA joints. Significantly lower SF concentrations of IL-10, but not of IFN-{gamma} and TNF-{alpha}, were found in reactive arthritis than in RA patients. Higher SF levels of IL-10 in patients with RA were not associated with lower cytokine levels of IL-12, TNF-{alpha} or IFN-{gamma}. Thus, as already reported [2], no immunosuppressive effects of IL-10 appeared from these results.

Synovial fluid concentrations of cytokines do not confirm previous results obtained upon stimulation of SF mononuclear cells with specific bacteria [9] or at the single-cell level after intracellular staining of cytokines [8], and do not confirm the similar secretion of IL-10 but lower TNF-{alpha} secretion in reactive arthritis than in RA patients, after ex vivo stimulation of PBMC [10]. However, the different methods used can explain these disagreements, such as variations among immunoassays used to detect cytokines [3133], which may be affected by the presence of various cytokine binding proteins [3438]. The cytokine-secreting cell population is also different in SF and peripheral blood, as the antigen-specific frequency has been estimated to be about 10 times higher in SF than in peripheral blood [39]. The polyclonal activation of PBMC with phytohaemagglutinin can also generate vigorous cytokine secretion, which does not necessarily reflect physiological responses. Phytohaemagglutinin skews the pattern of cytokine secretion towards Th1, as it cross-links the T-cell receptor and CD2 [40], which induces IFN-{gamma} secretion by T cells [41]. Furthermore, disagreement between the in vivo production of cytokines by cells present in the joint and the number of single cells with positive intracellular staining can be explained by the wide variation among T-cell clones in the ability to secrete cytokines [42].

However, in spite of these methodological differences, the presence of the lower SF concentrations of IFN-{gamma} in HLA-B27-positive than in HLA-B27-negative patients with C. trachomatis reactive arthritis and the association of low levels of SF IFN-{gamma} with a chronic course of arthritis in two HLA-B27-positive patients is consistent with a previous report [10]. Indeed, Braun et al. [10] have reported a low level of IFN-{gamma} secretion by PBMC from patients with a long disease duration and a high frequency of HLA-B27 positivity. They also reported a lower level of TNF-{alpha} secretion by HLA-B27-positive than by HLA-B27-negative patients and a significant correlation between low TNF-{alpha} secretion and a more protracted course of arthritis [10]. In the present study, the concentrations of TNF-{alpha} in SF were also lower in HLA-B27-positive than in HLA-B27-negative patients, but the difference was not statistically significant, probably because of the small number of patients.

Therefore, our results and those of Braun et al. [10] lead to the conclusion that cytokines such as IFN-{gamma} and TNF-{alpha}, which play an important role in host defence against a wide range of infections, are produced in lower quantities in HLA-B27-positive patients. In addition, the relatively low levels of these cytokines correlate with a more protracted course of arthritis. This conclusion is supported by the lower level of production of IFN-{gamma} by HLA-B27-positive ankylosing spondylitis patients and healthy HLA-B27-positive controls than by healthy HLA-B27-negative controls [43]. Taken together, these results suggest that the inability to produce an appropriate IFN-{gamma} response in HLA-B27-positive individuals may contribute to impaired clearance of infectious agents and increased susceptibility to the development of more severe or chronic arthritis.

In conclusion, we report for the first time that IFN-{gamma} levels in vivo were significantly lower in HLA-B27-positive patients with C. trachomatis reactive arthritis, and that these low levels of IFN-{gamma} were associated with a more chronic course of arthritis in two HLA-B27-positive patients. This finding can be related to the postulated protective and pathological functions of IFN-{gamma} in C. trachomatis infection. Indeed, as for other intracellular bacteria, this cytokine can contribute to the resolution of infection by activating macrophages to destroy phagocytosed C. trachomatis elementary bodies [44]. In addition, studies with an in vitro cell culture system have shown a dose-dependent effect of IFN-{gamma} on C. trachomatis growth and differentiation with presence of persistently infected cells at low concentrations [45, 46]. The low levels of IFN-{gamma} in the SF of HLA-B27-positive patients with C. trachomatis reactive arthritis can contribute to the persistence of infected cells, which can serve as depots of antigen capable of stimulating a sustained inflammatory response.

Our result was only observed in the subgroup of patients with well-defined C. trachomatis reactive arthritis but not in the whole group of patients with reactive arthritis, probably because this group was too heterogeneous (arthritides due to various infectious agents, unidentified in the majority of cases except C. trachomatis.


    Acknowledgments
 
The following investigators of the EULAR-sponsored trial of long-term azithromycin treatment in reactive arthritis are warmly thanked: Drs Hill Gaston, Thomas Vischer, Martin Altwegg, Thomas Bardin, Ben Dijkmans and Pierre-Alain Plan, who provided some SF samples from patients with reactive arthritis. The technical assistance of Ursula Spenato is gratefully acknowledged. This study was supported by grants 32-47299.96 and 3200-061500.00 from the Fonds National Suisse de la Recherche Scientifique.


    Notes
 
Correspondence to: S. Bas, Research Laboratory, Division of Rheumatology, University Hospital, 1211 Geneva 14, Switzerland. E-mail: sylvette.bas{at}hcuge.ch. Back


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 11 July 2002; Accepted 9 October 2002