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Rheumatology Advance Access originally published online on March 7, 2006
Rheumatology 2006 45(9):1129-1132; doi:10.1093/rheumatology/kel056
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Is the enthesitis-related arthritis subtype of juvenile idiopathic arthritis a form of chronic reactive arthritis?

N. Saxena, R. Misra and A. Aggarwal

Department of Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India.

Correspondence to: A. Aggarwal, Department of Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, India. E-mail: amita{at}sgpgi.ac.in


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objective. Enteric organisms are known to trigger reactive arthritis. The enthesitis-related arthritis (ERA) form of juvenile idiopathic arthritis (JIA) clinically resembles reactive arthritis. Therefore, we looked for a role of enteric bacteria in ERA.

Methods. Synovial fluid (SF) was obtained from 26 patients with ERA and 10 patients with rheumatoid arthritis (RA). Blood specimens were also obtained from patients with ERA and from 10 normal individuals. Lymphocyte proliferation assays were done on whole blood and SF mononuclear cells using as antigens crude lysates of the enteric bacteria Salmonella typhimurium, Yersinia enterocolitica, Shigella flexneri and Campylobacter jejuni. Crude lysate of Escherichia coli was used as a control antigen. HLA-B27 typing was done using the polymerase chain reaction. Homing of gut-specific T cells (CD103+) to the synovial compartment was studied using tri-colour flow cytometry. The antigen-specific cytokine profile was determined by flow cytometry.

Results. Antigen-specific lymphoproliferative responses were observed in 14 of 26 patients. Among these patients, 12 showed a response in SF (four each to S. typhimurium and C. jejuni, and in two each to S. flexneri and Y. enterocolitica), and two patients in blood (S. typhimurium in both). None of the healthy controls showed a response in the blood. Lymphoproliferative responses in SF were more frequent in patients with JIA (12/26) than in controls with RA (1/10, P<0.05). Patients with an antigen-specific response had a higher ratio of SF/blood CD103+ T cells compared with those with no antigen-specific response (P<0.01). Antigen-specific as well as mitogen-stimulated cytokine production showed a Th1 bias.

Conclusion. Enteric bacteria may have a role in exacerbation of disease in patients with ERA. The immune response in patients with ERA is Th1-dominant.

KEY WORDS: Infection, Autoimmunity, Cytokine, Juvenile rheumatoid arthritis.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The immunopathogenetic mechanisms in JIA [1] are ill understood. Possibly, genetic factors contribute to susceptibility, and environmental factors trigger and perpetuate the disease. Among the environmental factors, infectious agents are the most important [2]. The profile of JIA patients in India is different [3], the most common type being late-onset pauciarticular disease or the so-called enthesitis-related arthritis (ERA) [4]. Of children with ERA, 37–90% also fulfil European Spondyloarthropathy Study Group (ESSG) criteria [5, 6]. In view of the clinical similarities between ERA and reactive arthritis (ReA), we looked for a link between enteric bacteria implicated ReA and ERA.

Dominant Th1 immune responses are important in the clearance of enteric bacteria implicated in the causation of ReA. Also, most patients with ReA have a Th1-biased immune response [7]. Therefore, we also studied the Th1–Th2 balance in patients with ERA.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Twenty-six children who had ERA [1] with active synovitis were included. Patients with a history of diarrhoea in the preceding 4 weeks were excluded. Synovial fluid (SF) was collected during a therapeutic arthrocentesis after consent. Peripheral blood (PB) was also collected. HLA-B27 typing was done by the ARMS polymerase chain reaction [8].

SF obtained from 10 patients with rheumatoid arthritis (RA) and blood from 10 normal individual (all males) were used as controls for the lymphoproliferation assay. Sera from 50 age-matched healthy subjects and SF from 31 patients with RA were used as controls for antibody levels. Our institution's ethics committee approved the study.

Lymphocyte proliferation assay
Antigens were prepared as described previously [9]. In brief, Salmonella typhimurium, Shigella flexneri, Yersinia enterocolitica and Campylobacter jejuni were grown overnight at 37°C, pelleted, washed three times and lysed using a pulse sonicator. After centrifugation, the supernatant was stored at –80°C in aliquots. Escherichia coli crude lysate was used as a control antigen.

The lymphocyte proliferation assay with SF mononuclear cells (SFMCs) was done as described previously by us [10]. Whole blood was diluted (1:1) in RPMI medium and used for the assay [11]. The presence of an antigen-specific response was defined as a stimulation index (SI) ≥2.5 in response to a single antigen; or, if SI ≥2.5 was observed with more than one antigen, the highest SI had to be greater than 2.5 times the next highest SI value [12].

Analysis of gut-associated integrin {alpha}eß7 (CD103) of T cells
Cell surface expression of CD103 and CD45RO on T cells was evaluated using tri-colour flow cytometry with fluorescein isothiocyanate-labelled anti-CD4 or anti-CD8, phycoerythrin (PE)-labelled anti-CD103 and cychrome-labelled anti-CD45RO antibodies. Data were analysed using Cell-Quest software.

Th1/Th2 cytokine profile of PB and SF lymphocytes
Whole blood (100 µl/tube) or SFMCs (105 cells/tube) were stimulated with 50 ng/ml of phorbol myristate acetate (PMA) (Sigma, USA) and 1 µg/ml of ionomycin (Sigma) for 6 h; brefeldin A (Sigma) was added to prevent secretion of cytokines. Later, cells were stained with FITC-labelled anti-CD4 or anti-CD8 antibodies, fixed, permeabilized with Cytofix/Cytoperm kit (Becton Dickinson) and stained with intracellular cytokine PE-labelled anti-IFN-{gamma} or anti-IL-4 antibodies. Unstimulated specimens were used to measure spontaneous cytokine production.

Antigen-specific lymphocyte cytokine profile
The SFMCs (105 cells/tube) were stimulated with 10 µg/ml of the bacterial antigen to which they had shown a specific lymphoproliferative response in the lymphocyte proliferation assay, for 6 h in the presence of anti-CD28 antibody, instead of PMA and ionomycin. The rest of the procedure was as described above.

Measurement of antibody levels
Immunoglobulin (Ig) G and IgA antibodies to different enteric bacteria in serum and SF specimens were assayed using ELISA (enzyme-linked immunosorbent assay) as previously described [9].

Statistical analysis
Group data are summarized as median (range). The Mann–Whitney U-test was used for intergroup comparisons. For comparison of paired samples the Wilcoxon signed rank test was used.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
All 26 patients were male. The median age of onset was 12 (range 6–14) yr, age at the time of presentation was 16 (9–35) yr, duration of disease was 5.5 (0.5–26) yr and duration of relapse was 4 (1–36) weeks. Of these 26 patients, only two were receiving sulphasalazine and one each was getting methotrexate and low-dose prednisolone. Twenty-one patients were HLA-B27-positive.

SFMCs from 12 of 26 patients showed an antigen-specific response to enteric bacteria: four each to S. typhimurium and C. jejuni, and two each to S. flexneri and Y. enterocolitica. SFMCs from four patients showed a cross-reactive response to more than one bacterial antigen, and those from 10 patients showed no response. An antigen-specific response was more frequently observed in patients with ERA (12/26) than in patients with RA (1/10, P<0.05). In nine paired blood samples no antigen-specific response was seen (Fig. 1). All three patients on DMARDs showed an antigen-specific response.


Figure 1
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  		FIG. 1. Stimulation indices of synovial fluid cells (upper panel) and peripheral blood cells (lower panel) with different bacterial antigens in 14 patients showing antigen-specific responses. Stimulation indices ≥2.5 were considered significant.

 
Two patients showed an antigen-specific proliferative response to S. typhimurium in blood but not in SFMCs (Fig. 1); both these patients had disease exacerbation of short duration (2 and 6 weeks, respectively). None of the 10 healthy controls showed an antigen-specific response. Nine of 14 patients with an antigen-specific response and all the 12 without such responses were HLA-B27-positive (not significant).

CD103 ({alpha}eß7) expression was studied in 16 patients with ERA. CD103 ({alpha}eß7) expression was studied in 16 patients with ERA. In whole blood, the median frequency of CD8+CD103+ T cells was [4% (range 0.02–89)] higher than that of CD4+CD103+ T cells [1.3% (0.04–67); P<0.0001]. In synovial fluid, the median frequency of CD8+CD103+ T cells [17.4% (0.04–76.5)] was higher than that in CD4+CD103+ T cells [1.4 (0.02–32); P<0.0001]. Of the CD103+ cells in SF and peripheral blood, 93% (3–100%) and 82% (7–100%), respectively, co-expressed CD45RO, indicating that most of the CD103+ cells were of the memory phenotype. To study selective homing of CD103+ cells to the synovial compartment, the ratio of T cells staining positive for CD103 in SF and whole blood was calculated. The ratio exceeded 1 in eight of nine patients who showed antigen-specific lymphoproliferative responses, and in one of seven patients who did not show such responses (P<0.01).

Intracellular cytokine production was measured in five patients who had antigen-specific responses in SFMCs. The proportion of CD4+ and CD8+ T cells producing IFN-{gamma} after stimulation with PMA and ionomycin was higher than the proportion of those producing IL-4. Similarly, in the SF, the number of cells producing IFN-{gamma} was greater than that of cells producing IL-4. On antigen stimulation, the frequency of IFN-{gamma} secreting cells was higher than that of IL-4 secreting cells (Table 1). In spontaneous and E. coli-stimulated SF cells, cytokine production was negligible.


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  		TABLE 1. Percentage of cells showing intracellular staining for different cytokines

 
IgG and IgA antibody titres in serum as well as SF against crude lysate of all enteric bacteria were similar in patients and controls (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Our data show that a proportion of patients with ERA have lymphoproliferative responses against enteric bacteria, which have previously been implicated in the causation of ReA [13]. Further, we found that gut-educated (CD103+) T cells home to the synovial compartment in patients with antigen-specific responses. Also, the cytokine profile in patients with ERA showed Th1 dominance, akin to that observed in ReA. These observations suggest that enteric bacteria may play a role in exacerbation of ERA.

In India, the profile of JIA is quite different from that seen in Western countries [3]. Late-onset pauciarticular disease or ERA is the commonest subtype seen. Oligoarticular disease, which occurs in young girls, is distinctly rare in hospital-based studies [4]. Since we did not have this subgroup we have used adult RA synovial fluid as an inflammatory disease control.

An increased lymphoproliferative response of SF to bacteria, especially when the response is directed specifically against a single bacterial agent, has been used as a marker of microbial triggering in ReA [11, 14, 15]. In our population, antibody-based assays have a limited role in the diagnosis of ReA due to high background antibody levels among controls [10]. In contrast to antibody responses, cellular immune responses to enteric bacteria are shorter-lived in blood [15]. Thus, we used SF lymphoproliferative responses to study the role of enteric bacteria in ERA.

The presence of lymphoproliferative responses to enteric bacteria in 14 of our 26 patients with ERA suggests that enteric bacteria may be responsible for disease exacerbation in this disease. Of these 14 patients, 12 showed an antigen-specific response in SF whereas only two showed such a response in PB. The higher response in SF could be due to long disease duration [15], more efficient antigen presentation by SF antigen-presenting cells [16] and a higher frequency of antigen-specific T cells in synovial compartment [7]. Use of disease-modifying drugs did not inhibit the lymphoproliferative response.

S. typhimurium was the commonest bacterial trigger in our study. This is similar to our data in patients with undifferentiated spondyloarthropathy and ReA [8, 9]. Salmonella DNA has been detected in SF of a few patients with juvenile spondyloarthropathy [17]. In a European study [12], patients with late-onset pauciarticular juvenile chronic arthritis showed increased proliferative responses to Chlamydia and Yersinia, the organisms implicated in ReA in Europe. Concerns have been expressed regarding the cross-reactivity of lymphoproliferative responses among members of family Enterobacteriaceae [11]. In our study, cross-reactivity was found in only a few (4 of 26) specimens.

Another link between gut and joint could be that the intestinal T cells activated by antigens migrate to the joint and induce synovial inflammation. CD103 is expressed on more than 90% of CD 8+ T cells and around 40% of CD4+ lamina propria T cells and intraepithelial lymphocytes [18, 19]. CD103 expression was present in a significant proportion of T cells in the SF of JIA patients. The expression of CD103 was much higher on the CD8+ T cells compared with CD4+ T cells. Increased homing of CD103+ T lymphocytes to synovium could occur as a result of non-specific recruitment due to expression of its ligand E-cadherin following inflammation [20]. However, the selective homing in patients with an antigen-specific response compared with those without such response suggests that it is not linked to inflammation. The other reason could be selective trafficking of these antigen-primed T cells (most of them expressing CD45RO) due to the presence of bacterial antigens in the synovium. The possibility of local expansion of these antigen-primed T cells cannot be totally ruled out, especially if the relevant antigen is present in the local milieu. Another recent study also found an increased number of CD103+ T cells in the synovial compartment compared with peripheral blood in JIA [21]. Recently, CD103 has also been shown to be a marker for regulatory T cells [22]. It would be interesting to study the co-expression of CD103 with CD25 or Foxp3, markers of regulatory T cells, on SF T cells.

Since Salmonella, Yersinia and Campylobacter are facultative intracellular organisms, they require a dominant Th1 response for clearance. Our data, based on in vitro intracellular cytokine synthesis following respective bacterial antigen stimulation, show the presence of a strong Th1 response in patients with ERA. This is similar to the data available on antigen-specific cytokine responses in ReA [7]. In addition, most T-cell clones generated from Yersinia-induced ReA were also of the Th1 type [23]. We have previously shown that the cytokine profile in the synovial compartment in patients with ERA is Th1-biased, with high levels of IFN-{gamma} and undetectable IL-4 [24].

Our data suggest that microbial triggers may have a role in the exacerbation of ERA. Recognition of microbial products such as peptidoglycan, lipopolysaccharide and flagellin by macrophages using Toll-like receptors leads to the production of pro-inflammatory cytokines and IL-12 p40. IL-12p40 is the key cytokine responsible for Th1 polarization of T-helper cells [25].

The results of this study may have implications for the classification of patients with JIA, because the ILAR classification of JIA is based on the inclusion of arthritides without any known cause. Our findings of specific cellular immune responses against enteric bacteria and the high prevalence of HLA-B27 in patients with ERA suggest that, despite the absence of a history of a symptomatic enteric infection, these patients may have a disease resembling chronic ReA. Thus, our study supports the suggestion made by Burgos-Vargas et al. that some patients with ERA may have a forme fruste of chronic ReA [26].

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
The work was funded by a project to A.A. from the Indian Council of Medical Research, New Delhi, India. N.S. was supported by a fellowship from the Council of Scientific and Industrial Research, New Delhi, India.

The authors have declared that there are no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 9 December 2005; revised version accepted 24 January 2006.
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