Intestinal immune activation in juvenile idiopathic arthritis and connective tissue disease

Objectives: To examine the prevalence of immune activation in gastrointestinal (GI) mucosa in children with juvenile idiopathic arthritis (JIA) or connective tissue disease (CTD). Study design: We studied 27 children (15 girls, mean age 9.8±4.8 years) with JIA/CTD and GI symptoms, including nine with oligoarthritis, nine with polyarthritis, two with systemic arthritis, three with enthesitis‐related arthritis, and four with various CTDs. The control group consists of 54 children (31 girls, mean age 11.3±6.3 years) with GI symptoms but shown to have no significant GI or rheumatoid disorder. The subjects were examined by gastroduodenoscopy (22 patients, 50 controls) and colonoscopy (23 patients, 16 controls). Intraepithelial CD3⁺, α/β⁺, and γ/δ⁺ lymphocytes were counted from duodenal and ileal biopsies. Results: Five patients with JIA/CTD (19%) had ulcerative colitis. Lymphoid nodular hyperplasia (LNH) was more common in the patients [74% (20/27)] than in the controls [16% (8/50), p = 0.001], as well in the duodenal bulb [29% (7/24) vs. 10% (5/50)], terminal ileum [74% (14/19) vs. 38% (5/13)], and the colon [50% (11/22) vs. 14% (2/14)]. In the duodenum, CD3, α/β⁺, and γ/δ⁺ lymphocytes counts were higher in JIA/CTD (p<0.05). In the ileum, γ/δ⁺ cell numbers had increased in JIA/CTD (p<0.05). Either LNH, increased γ/δ⁺ count, or both were more common in JIA/CTD [89% (24/27)] than in the controls [13% (7/54), p<0.0001]. Conclusions: The majority of children suffering from JIA or CTD with GI symptoms show abnormalities consistent with activation of the intestinal immune system. The aetiology of this reaction remains unknown, but similar features are seen in delayed‐type food allergy.

The gastrointestinal (GI) mucosal immune system may be involved in the pathogenesis of rheumatoid arthritis in adults [1]. In children with juvenile idiopathic arthritis (JIA) or connective tissue disease (CTD), only fragmentary information on the abnormalities of GI mucosa has been available. Increased intestinal permeability in JIA has been reported [2] but the analyses of structural GI changes have focused on the adverse effects of treatment [3]. Children with spondylarthropathy show inflammation in the ileum and the colon [4].

The GI immune system may show structural changes when reacting to luminal stimulation such as microbes or nutritional factors. The excessive formation of lymphoid nodules in the mucosa (lymphonodular hyperplasia, LNH) and an increase in intestinal intraepithelial lymphocytes (IELs) are considered as markers of activation of the mucosal immune system [5], [6], [7]. LNH is defined as the appearance of lymphoid follicles in the duodenum or as an increase in the normal size and number of follicles in the ileal mucosa [6]. LNH is associated with delayed‐type food allergy in children, but is not specific [5], [6], [8]. An increase in IELs is seen in conditions such as coeliac disease and delayed‐type food allergy [8].

We have evaluated the occurrence of the signs of intestinal immune activation in children with JIA or CTD and GI symptoms. We determined the presence of LNH [6] and counted the subpopulations of IELs in duodenal and ileal biopsies [8].

In 1999–2004, 27 (15 girls, mean age 9.8±4.8 years) of the 187 children with JIA or CTD followed up at the Paediatric Rheumatology OPD of Oulu University Hospital, Finland, had GI complaints or symptoms, and were studied with endoscopy. The control group included 54 children (31 girls, mean age 11.3±6.3 years) with GI symptoms but assessed as having no significant GI disorders or rheumatic diseases. Indications for an endoscopy included abdominal pain (patients/controls: 14/30), diarrhoea (4/10), constipation (2/2), melena (5/3), melena and diarrhoea (2/0), vomiting (1/1), malaise (1/3), suspicion of coeliac disease (0/1), or flatulence (0/1). Of the 113 endoscopic examinations (48 patients, 65 controls), 74 (24 vs. 50) were gastroduodenoscopies, and 39 (23 vs. 16) colonoscopies.

The patients had been 7.1 (mean; range 0.4–15.8) years old at the onset of JIA/CTD. The diagnosis of JIA (n = 23) and CTDs (n = 4) was based on the International League of Associations for Rheumatology (ILAR) classification [11]. Seven children had oligoarthritis, two extended oligoarthritis, nine polyarthritis, two systemic arthritis, and three enthesitis‐related arthritis. The CTDs (n = 4) included one juvenile polyarteritis nodosa, one juvenile sarcoidosis, one uveitis/nephritis, and one uveitis.

Endoscopies were performed as described and LNH graded as mild (grade 1), or moderate/severe (grade 2) [6]. For immunohistochemistry, biopsies from the duodenal bulb (13 patients, 40 controls) and the terminal ileum (18 vs. 8) were frozen and stained for CD3+, α/β, and γ/δ TCR+ [8]. The patients were divided into groups with or without intestinal immune activation. The activation was present if either increased intraepithelial γ/δ+ T‐cell density in the duodenum (>2.5/mm) or in the ileum (>4.5/mm) [7] or grade 2 LNH in any part of the GI tract was detected.

At the time of endoscopy disease remission, the number of active joints by American College of Rheumatology (ACR) paediatric criteria and the activity of JIA/CTD with the visual analogue scale (VAS 0–100) were retrospectively assessed by one paediatric rheumatologist (PV) blinded for other information. The laboratory results included rheumatoid factor, anti‐nuclear antibodies, HLA‐B27 serology, erythrocyte sedimentation rate (ESR), and haemoglobin values. In children, the normal level of haemoglobin is age and sex dependent [12], hence deviation from the mean of the normal level was calculated individually.

The data were analysed with SPSS version 11.1. (SPPS Inc, Chicago, IL, USA). Fischer's exact test, the Mann–Whitney U‐test and Student's t‐test were used depending on the type of variable and the normality of distribution.

The protocol was approved by the Ethical Committee for Clinical Science of Oulu University Hospital.

Only 15% (4/27) of the patients with JIA/CTD showed normal endoscopic findings. Endoscopic oesophagitis [patients 19% (4/21) vs. controls (22%) 9/41, p = 0.79] and duodenitis [8% (2/24) vs. 2.5% (1/39), p = 0.55] were common in both patients and controls. Ulcerative colitis was diagnosed in five patients (19%), including one with oligoarthritis, three with polyarthritis, and one with CTD (chorioretinis). Two patients (7%) had delayed‐type milk allergy [8].

LNH (grade 1 or 2) in some part of the GI tract was diagnosed in 74% (20/27) of the patients and in only 16% (8/50) of the controls (p<0.001; Figure 1). In the duodenal bulb, LNH was present in 29% (7/24) of the patients and in 10% (5/50) of the controls (p<0.05), extending to the descending duodenum in three patients (13 %) and one control (3%, p = 0.13). In ileocolonoscopy, the patients had more LNH than the controls, including the ileum [74% (14/19) vs. 38% (5/13), p = 0.050], colon [50% (11/22) vs. 14% (2/14), p = 0.032], and rectum [20% (4/20) vs. 0% (0/22), p<0.001] (Figure 1). Grade 2 LNH was seen in 44% (12/27) of the patients, and in 8% (4/51) of the controls (p<0.001; Table 1).

Table 1. Intestinal immune activation in the different categories of JIA/CTD and in the controls: indications are the occurrence of lymphonodular hyperplasia (LNH) in any part of the gastrointestinal tract and an increase in intraepithelial γ/δ+ T‐cell densities in the duodenum (>2.5/mm) or the terminal ileum (>4.5/mm).

Graph: Figure 1 Prevalence of lymphoid nodular hyperplasia(LNH) in different parts of the gastrointestinal tract in the children with juvenile idiopathic arthritis or connective tissue disease (P) and in the controls (C). The significance of the differences was assessed with Fisher's exact test.

In the duodenum, patients showed higher densities of CD3+, γ/δ+, and α/β+ IELs than the controls but only γ/δ+ T‐cell densities were significantly higher in the ileum (Table 2). Increased intraepithelial γ/δ+ T cell densities (>2.5/mm) [7] were more common in the patients than in the controls in the duodenum (4/13 vs. 0/47, p = 0.004) and ileum (>4.5/mm) (12/19 vs. 2/8, p<0.05; Table 1). In the patients with the HLA‐B27 antigen, the density of α/β+ T cells in the ileum was lower (3.5±3.0; n = 6) than in the HLA‐B27 negative ones (6.7±3.1; n = 13; p = 0.048).

Table 2. Density of CD3+, α/β+, and γ/δ+ T‐cell receptor‐bearing intraepithelial lymphocytes (cells/mm; mean±SD), and γ/δ+/CD3+ ratios in biopsy samples from the duodenal bulb and terminal ileum in the children with juvenile idiopathic arthritis or connective tissue disease (JIA/CTD) and in the controls.

419 *p<0.05, **p<0.01: JIA/CTD vs. controls, Student's t‐test.

At the time of endoscopy, 44% (12/27) of the patients were in remission. Mean VAS was 18 and in JIA (n = 23) the mean number of active joints was 1.8. The use of anti‐rheumatic medication was as follows: 2/27 no medication, 7/27 only non‐steroidal anti‐inflammatory drugs (NSAIDs), 1/27 oxyclorin, 6/27 conventional (methotrexate or salazopyrin), 7/27 conventional + prednisolone, 4/27 conventional + tumour necrosis factor (TNF)‐α modulators.

The counts of IELs or features of LNH were not related to the duration or current activity of JIA/CTD, or to medical treatment. However, the presence of LNH in any part of the GI tract was associated with increased ESR [median (range): 10 (5–92) vs. 5 (2–52), p = 0.01, Mann–Whitney] and the trend for decreased haemoglobin concentration [median (range): −23.5 (−39.4 to 1) g/L vs. −6 (−32 to 4) g/L, p = 0.060, Mann–Whitney].

Intestinal immune activation, as indicated by the presence of grade 2 LNH or an increase in γ/δ+ T‐cell densities, was present in 63% (17/27) of the patients. It was not related to the duration of JIA/CTD or the number of active joints. The activation was associated with a higher disease activity score [median (range): 15 (3–85) vs. 3 (0–10), p = 0.008, Mann–Whitney] and with intensive medication (methotrexate or TNF modulator or both: 14/17 vs. 2/6, p = 0.045, Fisher's exact test). The activation was also associated with higher ESR [median (range): 13 (5–92) vs. 4 (2–7), p = 0.005, Mann–Whitney] and with a lower blood haemoglobin level [median (range): −24.0 (−39 to −1) g/L vs. −7.0 (−24 to 4) g/L, p = 0.012, Mann–Whitney].

Our observations indicate that almost 90% of the children with JIA/CTD suffering from GI symptoms show signs of activation of the intestinal immune system. LNH was present in 74% and an increased number of intraepithelial γ/δ+ T lymphocytes in 54% of the patients were found, differing significantly from those in the controls. We found five cases with inflammatory bowel disease (IBD) (19%; 3% of all children with JIA/CTD), indicating clearly increased prevalence [13]. The figures for mucosal immune activation would probably have been different if JIA/CTD patients without GI symptoms could have been included. However, because our patients represent almost 15% of all the children with any rheumatic disease in our region, our findings suggests that such microscopic or endoscopic abnormalities are common in rheumatic children.

The increment in mucosal intraepithelial T cell subsets in JIA/CTD was skewed towards the α/β+ T cells. The increase in α/β+ T cells was associated with untreated food allergy [7], [8] and coeliac disease [8]. Quantitative abnormalities of GI γ/δ+ T cells in rheumatic diseases have not been reported so far, but there is some evidence that γ/δ+ cells might be involved in the pathogenesis of synovial inflammation through the synovial homing of mucosal lymphocytes [14].

Children with an HLA‐B27 antigen showed significantly lower densities of γ/δ+ T cells than the HLA‐B27 negative ones. We speculate that in HLA‐B27‐positive subjects, γ/δ+ T lymphocytes have no significant influence on the entrance of arthritogenic bacterial components into synovial tissues [9]. By contrast, mucosal immune aberration involving γ/δ+ T cells might be important in the pathogenesis of arthritis in the HLA‐B27‐negative patients.

LNH in the GI mucosa was about three times more common in children with JIA/CTD than in the controls (Figure 1). LNH has a heterogeneous background, but in children it shows a close association with delayed‐type food hypersensitivity [6], [7], [8], [10].

Intestinal immune activation in JIA/CTD (massive LNH or an increase in γ/δ+ T cells) was associated with higher clinical activity and with intensive current medication, but not with the duration of JIA/CTD. LNH was associated with increased ESR. The inter‐relationship of these phenomena is not known. Mucosal immune activation, actual pathogenesis and activity of JIA/CTD may all share common background factors or they may have other pathogenetic links between them. Because the intestinal mucosa forms the largest immune organ of the human body, constantly exposed to environmental antigens, it is tempting to speculate that intestinal immune activation might have a predominant role in this complex network of immune activation and its manifestations.

In conclusion, the activation of the GI‐associated immunological system seems to be present in the majority of patients with JIA/CTD and GI symptoms. Whether this activation has any role in the pathogenesis of JIA/CTD or whether it is secondary to the rheumatic disease or the treatment is not known. As similar features in mucosal immune activation are seen in children with delayed‐type food hypersensitivity [6], [7], [15], the assessment of the significance of food‐borne antigens in the initiation of the pathological cascades would be interesting.

The immunohistochemical analyses were performed at the Coeliac Service Laboratory, University of Tampere, Tampere, Finland. This work was supported by Stiftelsen Alma and KA Snellmann.