Institute of Experimental and Clinical Medicine, Vilnius University, 1 Laboratory of Ecological Genetics, Vilnius University and 2 Laboratory of Immunology, Institute of Biotechnology, Lithuania.
Correspondence to: I. Butrimiene, Department of Rheumatology, Vilnius University Institute of Experimental and Clinical Medicine, Zygimantu 9, LT-2600, Vilnius, Lithuania. E-mail: irena.butrimiene{at}santa.lt
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Abstract |
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Methods. Cytokine production by peripheral blood and synovial fluid mononuclear cells (PBMCs/SFMCs) of 28 patients with AcReA, 27 patients with ChrReA, 26 patients with rheumatoid arthritis (RA) and 31 healthy controls was analysed by enzyme-linked immunosorbent assay (ELISA) and flow-cytometry. Production of tumour necrosis factor-alpha (TNF-), interferon-gamma (IFN-
) and interleukin (IL)-10 was measured by ELISA, while the percentages of TNF-
-, IFN-
- and IL-4-positive CD3+ cells were determined in the same groups of patients and healthy subjects using flow cytometry.
Results. Spontaneous TNF- production observed in PBMCs of ChrReA, but not of AcReA, patients was significantly higher (P<0.001) than in healthy controls. The percentages of TNF-
-positive CD3+ blood cells in ChrReA exceeded that of RA patients and healthy controls (P<0.05 and P<0.001, respectively). Also, the percentages of IFN-
-positive CD3+ cells were significantly higher in peripheral blood and synovial fluid of ChrReA patients (P<0.05 and P<0.05, respectively) as compared with AcReA. In ChrReA spontaneous IL-10 production in PBMCs was similar to that observed in healthy controls, while in RA and AcReA the production of IL-10 was significantly increased (P<0.05 and P<0.05, respectively). IL-4 production was low in all study groups with no significant differences detected.
Conclusions. High production of TNF- and IFN-
detected in ChrReA supports the possible use of anti-TNF-
treatment in ChrReA.
KEY WORDS: Reactive arthritis, Cytokines, TNF-, IFN-
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Introduction |
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Cytokines secreted by T helper (Th) cells play a key role in the pathogenesis of RA and ReA [2]. Pathogenesis of ReA is closely associated with an earlier bacterial infection mainly of the gastrointestinal or urogenital tract. It is known that cytokines of the Th1 type [interferon-gamma (IFN-), tumour necrosis factor-alpha (TNF-
) and others] are indispensable for effective defence against bacterial infection. In ReA, impaired Th1 cytokine production may cause a failure of effective bacterial elimination at the initiation of the disease and thereby play a part in the pathogenesis of ReA [3]. Indeed, a relative lack of Th1 cytokines was detected in peripheral blood and synovium of ReA patients in several studies [46]. Significant associations were also observed between low TNF-
secretion and a more chronic course of ReA in HLA-B27-positive patients [6]. However, in T-cell clones derived from the synovial fluid of patients with ReA, disease-related bacterial antigens predominantly induced Th1 cytokine secretion [79]. Conflicting data were also reported on Th2 cytokine [interleukin (IL)-4, IL-10] production in the inflamed joints of ReA patients [4, 5, 10]. This lack of general agreement on cytokines found in ReA patients warrants further cytokine investigation in ReA.
Recently developed anti-cytokine therapy allows direct control of cytokine production in autoimmune inflammatory diseases. Clinical studies revealed significant benefits from anti-TNF- therapy not only in RA but also in the spondyloarthropathies [11, 12]. In patients with ankylosing spondylitis anti-TNF-
therapy resulted in the rapid improvement of clinical symptoms and continued clinical benefit [13, 14]. In addition, anti-TNF-
therapy reversed abnormalities in Th cytokine pattern observed in the spondyloarthropathies [15, 16]. The first efforts to treat ReA and unclassified arthritis using anti-TNF-
therapy have recently appeared in the literature [17].
The main aim of our study was to analyse cytokine production in patients with ChrReA and compare it with acute ReA (AcReA), RA and healthy controls in order to better understand the pathophysiological mechanisms of ReA and search for new treatment possibilities. For that purpose the production of TNF-, IFN-
, IL-10 and IL-4 was measured using enzyme-linked immunosorbent assay (ELISA) and flow cytometry in peripheral blood and synovial cells of patients and in peripheral blood of healthy controls.
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Methods |
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All study subjects who participated in the investigations signed a written informed consent form. This informed consent was obtained according to the Declaration of Helsinki.
Cell collection
PBMCs were separated from heparinized blood (7 ml) by a standard procedure using Ficoll density gradient centrifugation. After three washes in serum-free RPMI-1640 medium (Biochrom, Germany), the mononuclear cells were resuspended in complete growth medium (RPMI supplemented with 2 mM L-glutamine, antibiotics and 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Germany) and processed for flow-cytometric analysis or cytokine assay as described below. SFMCs were isolated from the synovial fluid (7 ml) by centrifugation, washed three times with serum-free RPMI, resuspended in complete growth medium and processed for further analysis.
Cytokine assay
For cytokine assay, mononuclear cells were seeded at a density of 1 x 106 cells/ml/well into a 24-well plate and cultured in a humidified CO2 incubator for 24 h. The supernatants were collected and stored at 20°C until assay. Cytokines IL-10, TNF- and IFN-
were analysed by a two-site ELISA. IL-10 was determined using an OptEIA hu IL-10 Set (BD PharMingen). For TNF-
detection, matched-paired monoclonal antibodies (Institute of Biotechnology, Vilnius) produced by clones NF-2 and NF-7 were used (sensitivity 100 pg/ml, standardization according to British Standard supplied by NIBSC, UK). For INF-
detection, matched-paired monoclonal antibodies (Institute of Biotechnology, Vilnius) produced by clones GIF-3 and GIF-10 were used (sensitivity 200 pg/ml, standardization according to British Standard supplied by NIBSC, UK). Analysis was performed on Nunc MaxiSorp microtitre plates. Enzymatic reaction was developed using TMB/plus substrate (Fermentas). The optical densities at 450 nm and at a correction wavelength 620 nm were measured on a microplate reader (DigiScan 400, Assys Hitech). Concentrations of cytokines were calculated using the KIM computer program. Results were expressed as median values and interquartile ranges (IQR).
Flow cytometry
For flow-cytometric analysis of intracellular cytokines, mononuclear cells were seeded at a density of 1 x 106 cells/well into a 96-multiwell plate pre-coated with anti-hu-CD3 (clone UCHT1, BD PharMingen) and incubated overnight in a humidified CO2 incubator. Cells were further incubated for 4 h with phorbol myristate acetate (PMA; Sigma, 10 ng/ml), ionomycin (Sigma, 1 mM) and the protein transport inhibitor Golgi-Plug (BD PharMingen, 3 mM). After stimulation, cells were harvested, washed once with Washing Buffer (BD PharMingen) and then permeabilized for 10 min in 4°C cold FACS permeabilizing solution (BD PharMingen). After washing, the cells were stained simultaneously with directly conjugated anti-cytokine and anti-CD3 antibodies (BD PharMingen). Cells were incubated for 20 min in the dark, washed and finally resuspended in Cell-Fix (BD PharMingen). IL-4, TNF- and IFN-
production was determined in CD3+ cells. The threshold for cytokine positivity was set using irrelevant isotype-specific antibodies (
1/
2a, BD PharMingen). Results for cytokine-positive cells [median (IQR)] were expressed as a percentage of the respective subpopulation.
Statistical analysis
The MannWhitney U-test was used to compare data on immunological analysis between different study groups. The value P<0.05 was accepted as statistically significant.
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Results |
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Spontaneous cytokine secretion by PBMCs
Spontaneous TNF- production by PBMCs measured using ELISA was higher in all patient groups than in the healthy control group. However, statistically significant differences in TNF-
production were observed in RA and ChrReA groups when compared with healthy controls (P<0.001 and P<0.01, respectively), while for the AcReA group this difference was not statistically significant (Fig. 1). The median concentrations of TNF-
in the different groups were 70 pg/ml (IQR 0150) in healthy controls, 142 pg/ml (IQR 30326) in AcReA, 256 pg/ml (IQR 50395) in ChrReA and 235 pg/ml (IQR 152501) in RA. TNF-
production in AcReA was statistically significantly (P<0.05) lower than in RA, while TNF-
concentrations observed in the ChrReA and RA groups were similar.
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The spontaneous production of IL-10 in all patient groups was higher than that found in the healthy controls. However, for the ChrReA group this difference was not statistically significant (Fig. 1). The median concentrations of IL-10 in the study groups were 65 pg/ml (IQR 0322) in healthy controls, 262.5 pg/ml (IQR 103.5412.5) in AcReA, 201 pg/ml (IQR 51372) in ChrReA and 265 pg/ml (IQR 133436) in RA. No statistically significant differences in cytokine production were observed between patient groups.
Cytokine-positive cells in peripheral blood
The highest percentages of TNF-- and IFN-
-positive CD3+ cells were detected in peripheral blood of patients with ChrReA (Fig. 2). The percentages of TNF-
-positive CD3+ cells were significantly higher in peripheral blood of ChrReA patients than in healthy controls [39.5 (IQR 21.748.8) vs 14.5 (IQR 8.438.2); P<0.001] and also than in patients with RA [39.5 (IQR 21.748.8) vs 15.4 (IQR 10.037.1); P<0.05]. Meanwhile, data comparison between AcReA and ChrReA groups revealed a statistically significant difference [8.4 (IQR 4.617.1) vs 14.2 (IQR 9.129.1); P<0.05] in percentages of IFN-
-producing CD3+ blood cells (Fig. 2).
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Cytokine production in synovial fluid
Cytokine production by SFMCs was also evaluated using ELISA and flow cytometry. No significant differences in spontaneous secretion of TNF- and INF-
by SFMCs were found between different study groups by using ELISA (data not shown). The percentage of IFN-
-expressing CD3+ cells was about four times higher in the synovial fluid of patients with ChrReA than in patients with AcReA. In the synovial fluid of ChrReA patients 23.5% (IQR 12.541.1) of CD3+ cells were IFN-
positive, while in AcReA only 4.8% (IQR 014.4) of cells displayed this positivity (P<0.05) (Fig. 3).
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In the AcReA group 12 patients were HLA-B27 positive and 16 patients were HLA-B27 negative. No significant differences in cytokine production measured by ELISA or flow cytometry were observed in AcReA according to HLA-B27 status.
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Discussion |
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Our study revealed significant differences in cytokine profiles of ChrReA and AcReA. This was indicated by a higher production of TNF- observed in PBMCs of patients with ChrReA than with AcReA. Also the percentage of IFN-
-expressing CD3+ cells in blood and synovial fluid was significantly higher in ChrReA than in AcReA. However, no difference in Th2 cytokine production (IL-4 and IL-10) was observed when ChrReA was compared with AcReA.
In several prospective studies patients with AcReA were evaluated for one or more years. Analysis of cytokine profiles in these studies revealed significant associations between prolonged disease duration and low TNF- or IFN-
secretion [6, 10]. Also, correlations were detected with a more chronic course of disease and HLA-B27 antigen expression. However, the conclusions of these studies were based on analysis of small numbers of cases. Our study design allowed comparison of cytokine profiles in two ReA groups of equal numbers with different stages of the disease.
The molecular mechanisms of pathogenesis of ReA were mostly analysed in comparison with RA. Significant differences in cytokine profiles were observed in such comparisons. A relative lack of Th1 cytokines and increased production of Th2 cytokines were observed in ReA patients as compared with RA [46, 19, 20]. It was concluded that the relative predominance of Th2 cytokines could be responsible for ineffective antibacterial defence in ReA and thereby associated with pathogenesis of disease. Similarly, in our study, the patients with AcReA had a statistically significantly (P<0.05) lower spontaneous TNF- level than patients with RA, while IL-10 production was similar in both groups. However, a different cytokine profile was observed in the ChrReA group. In this group the spontaneous production of IL-10 was slightly lower than in RA or AcReA, but the production of TNF-
was similar to that observed in RA. The percentages of TNF-
-positive CD3+ cells in the blood and IFN-
-positive CD3+ cells in synovial fluid of ChrReA patients were significantly higher than in RA. Our data support the idea that the relative lack of Th1 cytokines may play a significant role in pathogenesis of ReA at the acute phase of disease; however, in the chronic disease phase the repertoire of the cytokines differs and is similar to what is found in RA.
Expression of the antigen HLA-B27 is closely related to pathogenesis of ReA and other spondyloarthropathies [3, 21, 22]. A significant reduction in the expression of Th1 cytokines was detected in HLA-B27-positive patients with ankylosing spondylitis and also in healthy HLA-B27-positive controls as compared with HLA-B27-negative controls [23]. Our study revealed a similar trend in ChrReA, but not in the AcReA group. Decreased production of both TNF- and IL-10 was detected in PBMCs from HLA-B27-positive ChrReA patients as compared with HLA-B27-negative patients. However, such an association was not supported by intracellular cytokine measurement. While genetic factors play a part in the modulation of the immune response in ReA, their influence in cytokine regulation needs to be clarified. This is supported by other authors [24].
Treatment of spondyloarthropathies, particularly ankylosing spondylitis, with monoclonal antibody to TNF- (infliximab) is highly clinically effective [16, 25]. One study suggested short-term effectiveness and safety of anti-TNF-
therapy in ReA and unclassified arthritis [17]. Significant down-regulation of TNF-
and IFN-
production without any effect on Th2 cytokine production was observed in ankylosing spondylitis treated with infliximab [15, 16]. However, some other studies were unable to detect changes in serum pro-inflammatory cytokine level before and after anti-TNF-
treatment [24]. The data on cytokine changes during the anti-TNF-
treatment are contradictory and require further investigation in every single kind of spondyloarthropathy.
Our study revealed significant increase of TNF- and IFN-
production in ChrReA, as compared with AcReA. This supports the possibility that anti-TNF-
treatment in ReA during the chronic phase of the disease is beneficial. Without doubt, the search for latent infection in these patients should be fully carried out and if found must be eradicated before initiation of anti-TNF-
therapy.
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Acknowledgments |
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The authors have declared no conflicts of interest.
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References |
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