IL-10 Genotype Analysis in Patients with Behçet’s Disease

IL-10 Genotype Analysis in Patients with Behçet’s Disease Graham R. Wallace, Elly Kondeatis, Robert W. Vaughan, David H. Verity, Yuanneng Chen, Farida Fortune, Wafa Madanat, Charlie A. Kanawati, Elizabeth M. Graham, and Miles R. Stanford ABSTRACT: Behçet’s disease (BD) is a multisystem inflammatory disease characterized by recurrent orogenital ulceration, ocular inflammation, and skin lesions. The etiology of the disease is currently unknown but evidence suggests that there is a strong genetic component mediating the chronicity of the disorder. We have examined the association between polymorphisms at position ⫺1082, and ⫺819 in the promoter region of the gene encoding IL-10 in patients with Behçet’s disease from two distinct patient populations. The IL-10 ⫺1082AA genotype was weakly associated with BD when all patients were analyzed as a group (pc ⫽ 0.04, OR 1.4, 95% CI 1.1–1.9), but not in the UK or Middle Eastern (ME) cohorts of patients alone compared to local controls. An association with IL-10 ⫺819T was evident in all BD patients, (pc ⫽ 0.02, OR 1.5, 95% CI 1.1–2.0), and this ABBREVIATIONS BD Behcet’s disease

INTRODUCTION Behçet’s disease (BD) is a systemic vasculitis characterized by inflammatory lesions of the orogenital mucosa, eyes, skin, central nervous system, and joints. The triggering factor for BD is still unclear, although increasingly bacterial and viral infections of mucosal surfaces

From the Academic Unit of Ophthalmology, University of Birmingham (G.R.W.), Birmingham, United Kingdom, Departments of Clinical Transplantation Laboratory (E.K., R.W.V.) and Ophthalmology (D.H.V., Y.C., E.M.G., M.R.S.), Guy’s, King’s and St Thomas’ Hospital Medical Schools, London, United Kingdom, Department of Oral Medicine, Queen Mary’s (F.F.) , London, Jordan Hospital (W.M.), Amman, Jordan, and St. John Eye Hospital (C.A.K.), Jerusalem, Israel. Address reprint requests to: Dr. Graham Wallace, Academic Unit of Ophthalmology, Division of Immunity and Infection, University of Birmingham, Birmingham B15 2TT United Kingdom; Tel: ⫹01 21 414 6793; Fax: ⫹01 21 414 6794; E-mail: [email protected]. Received September 11, 2006; accepted November 9, 2006. Human Immunology 68, 122–127 (2007) © American Society for Histocompatibility and Immunogenetics, 2007 Published by Elsevier Inc.

was because of an association in the UK but not ME patients (pc ⫽ 0.0004, OR 2.1, 95% CI 1.4 –3.3). The ⫺1082A/⫺819T haplotype, which is linked to low production of this cytokine, was not significantly associated with Behçet’s disease. This link between BD, a chronic, relapsing, autoinflammatory condition, and a genotype associated with low IL-10 production provides evidence that abnormalities in the genetic control of cytokine levels may be relevant in influencing the immune response in Behçet’s disease in some patient groups. Human Immunology 68, 122–127 (2007). © American Society for Histocompatibility and Immunogenetics, 2007. Published by Elsevier Inc. KEYWORDS: IL-10; gene polymorphism; Behçet’s disease

SNP

single nucleotide polymorphism

have been implicated [1–3]. Evidence for chronic activation of the immune response has been demonstrated in patients with BD [4, 5]. Stimulation of peripheral blood mononuclear cells from patients with BD with either phytohemagglutinin or antiCD3/anti-CD40 all showed a strong polarization to Th1 cytokine production [6 – 8]. From these studies it is clear that the relative levels of cytokine production may be important in the determination of disease onset, progression, and outcome [9]. Cytokine production has been shown to be under genetic control. A haplotype formed by three single nucleotide polymorphisms (SNPs) at positions ⫺1082, ⫺819, and ⫺592, in the promoter region of the IL-10 gene have been reported [10]. In vitro studies showed that the presence of the ⫺1082/⫺819 AT haplotype resulted in a significantly lower concanavalin A-stimulated production 0198-8859/07/$–see front matter doi:10.1016/j.humimm.2006.11.010

IL-10 gene polymorphisms in BD

of IL-10 from mononuclear cells than mononuclear cells taken from patients with the GC haplotype. In patients infected with Helicobacter pylori, carriers of the GC haplotype had higher levels of mucosal IL-10 mRNA and more virulent bacterial strains than AT haplotype carriers [11]. Moreover, atheroscelerotic renal vascular disease was associated with the IL-10 AT haplotype suggesting that IL-10 may be protective in this condition [12]. Given the chronic overstimulation of the immune system in BD [1, 2], we sought to define the associations between IL-10 haplotypes and disease in two different patient groups We have investigated polymorphisms at positions ⫺1082 and ⫺819, but did not investigate the ⫺592 position, because this is reported to be in linkage disequilibrium with the ⫺ 819 position [13, 14]. Our results show that the IL-10 ⫺819T genotype was associated with BD in patients from the UK and all patients taken together compared to matched controls. The AT haplotype, which has been linked to low production of IL-10, was significantly associated with all BD patients but this significance was lost after correction. These results suggest that genotypes associated with low production of the anti-inflammatory cytokine IL-10 are more prevalent in patients with BD but this may differ in different patient cohorts. MATERIALS AND METHODS Patients After informed consent, blood samples were collected by venipuncture from two geographically separated patient groups of Caucasoid patients with Behçet’s disease, (n ⫽ 178), 63 attending the Medical Eye Unit at St. Thomas’ Hospital, London, and 115 of Arab descent, including 62 Jordanian patients attending the Jordan Hospital, Amman, and 53 Palestinian patients attending St John’s Hospital, Jerusalem. All patients fulfilled the International Study Group criteria for diagnosis [15] and underwent a prospective clinical and ophthalmologic examination. Any patients of ME or Afro-Caribbean origin in our UK cohort were not analyzed in this study. Normal controls without Behçet’s disease were drawn from respective populations (UK n ⫽ 182, ME n ⫽ 113). This study received the approval of the Ethics Committee of St Thomas’ Hospital. Cytokine Gene Polymorphisms DNA was prepared by proteinase K digestion, and salt extraction [16] and stored at ⫺70°C until use. Polymorphisms in the promoter region of the IL-10 gene at positions ⫺1082G/A and ⫺817C/T were detected by a polymerase chain reaction–single strand polymorphism (PCR-SSP) assay using four primer mixes [17]. ⫺1082G 5= CAgTGCAACTgAgATTTgg 3= antisense 5= CTACTAAggCTTCTTTgggAg 3= sense

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⫺1082A 5= CAgTGCAACTgAgAATTTg 3= antisense 5= ACTACTAAggCTTCTTTgggAA 3= sense ⫺819C 5= AggATgTgTTCCAggCTCCT 3= antisense 5= CCCTTgTACAggTgATgTAAC 3= sense ⫺819T 5= AggATgTgTTCCAggCTCCT 3= antisense 5= ACCCTTgTACAggTgATgTAAT 3= sense The final volume of all PCR reactions was 10 ␮l; the PCR reaction mixtures consisted of 67 mM Tris base pH 8.8, 16.6 mM ammonium sulphate, 2 mM magnesium chloride, 0.01%(v/v) Tween20, 200 ␮M of each dNTP, 1– 4 ␮M of each allele-specific primer, 0.1 ␮M of control primer, 0.1 ␮g of DNA, and 0.1875 units of Taq Polymerase (Abgene, Epsom,UK). All reactions were carried out in a Geneamp PCR System 9700 thermal cycler (PE Biosystems, Foster City, CA, USA). PCR cycling conditions consisted of 5 cycles of 96°C for 25 seconds, 70°C for 45 seconds, and 72°C for 20 seconds; followed by 21 cycles of 96°C for 25 seconds, 65°C for 50 seconds, and 72°C for 45 seconds; and finally 4 cycles with 96°C for 25 seconds, 55°C for 60 seconds, and 72°C for 120 seconds. The PCR products were visualized by electrophoresis through a 1% agarose gel in 0.5⫻ TBE buffer containing 1␮g/l ethidium bromide. The relative size of the PCR products was determined by comparison of the migration of a 100 –1,000 bp DNA molecular weight ladder (Abgene). A permanent visual image was obtained using a UV illuminator and a UVP Imagestore 7500 (UVP Products Ltd., Cambridge, UK). The ⫺1082 primers resulted in an amplicon of 258 bp and the ⫺819 primers an amplicon of 233 bp. The results were scored if clear bands were present for both the IL-10 and the control primers. Analysis of Data Associations with disease were sought between genotype and haplotype frequencies. Chi-squared analysis and calculation of odds ratios (OR) with 95% confidence limits (95% CI) were carried out using EpiStat. Where numbers in the 2 ⫻ 2 table were small, Fisher’s exact test was used to calculate probability. Subanalysis was performed relating haplotype to the presence of ocular disease. Analyses were performed for all patients versus all controls, and for UK patients and ME patients separately against their respective controls. A Bonferroni correction TABLE 1 Patients details

Male/Female Age Range % eye involvement No. Blind (%)

UK patients (n ⫽ 63)

Middle Eastern patients (n ⫽ 115)

32/31 21–76 (mean 42) 67 4 (7%)

95/20 15–65 (mean 32) 75 9 (8%)

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TABLE 2a IL-10 ⫺1082 gene polymorphism/genotype frequencya IL-10 –1082

UK BD patients UK controls ME patients ME controls All BD Patients All controls

AA

GA

GG

Ab

Gb

27 (43%) 59 (32%) 50 (43%) 46 (41%) 77 (43%) 105 (36%)

23 (37%) 75 (41%) 50 (43%) 40 (35%) 73 (41%) 115 (39%)

13 (20%) 48 (27%) 15 (14%) 27 (24%) 28 (16%) 75 (25%)

77 (61%) 193 (53%) 150 (65%) 132 (58%) 227 (64%) 325 (55%)

49 (39%) 171 (47%) 80 (35%) 94 (42%) 129 (36%) 265 (45%)

p value NS NS Pc ⫽ 0.04

IL-10 ⫺1082A is associated with BD in the combined patient groups. ⫺1082A versus ⫺1082G UK ⌾2. 2.16, p ⫽ 0.14, OR 1.4 (0.9 –2.1) NS; ME ⌾2 1.96, p ⫽ 0.16, OR 1.34 (0.9 –2) NS; All ⌾2. 6.5, p ⫽ 0.01, OR 1.4 (1.1–1.9) pc ⫽ 0.04.

a

b

factor has been applied to give a p value corrected for the number of analyses performed. RESULTS This study enrolled 178 patients with BD whose characteristics are shown in Table 1. In the UK and ME populations the prevalence of ocular involvement (approx 70%) was similar to other series [1]. In all cases ⫺1082G was linked to ⫺819C, although ⫺1082A was associated with both ⫺819C and ⫺819T. The ⫺1082 genotype was in Hardy-Weinberg equilibrium between both patient/control groups, and the haplotype frequency was similar to previous reported studies [13, 14]. Comparisons of the genotype frequencies of IL-10 gene polymorphisms showed a weak linkage (pc ⫽ 0.04, OR 1.4, 95% CI 1.1–1.9) when all patients were analyzed together. This was mainly because of UK patients who showed a greater prevalence of ⫺1082A than ME patients, but this did not reach significance (Table 2a). Similarly, there was an increased prevalence of ⫺819T in all patients when analyzed compared to controls (pc ⫽ 0.02, OR1.5, 95% CI 1.1–2), and this reached significance in the UK (pc ⫽ 0.0004, OR 2.1, 95% CI 1.4 –3.3) but not in ME patients alone (Table 2b).

When the patients were separated into different patient populations and compared to local controls the AT haplotype was not significantly associated with BD, in either UK or ME (33% vs 25% p ⫽ 0.2; 32% vs 28% p ⫽ 0.4 respectively) (Table 3). In all patients with Behçet’s disease there was a significantly higher prevalence of the AT (33% vs 26%) and lower GC (36% vs 45%) (p ⫽ 0.05) haplotypes compared to all controls However, on correction this significance was lost. Haplotype analysis showed that homozygosity for the AT haplotype was not associated with disease in any patient group, indicating that any differences between UK and ME patients were because of heterozygotes (data not shown). Similarly, there was no association with ocular disease or gender in these patient groups (data not shown). DISCUSSION Cytokines act in a complex interplay to mediate immune responses and the role of IL-10 is an immunomodualtory one. Up to 75% of interindividual variability in human IL-10 production has been attributed to genetic variation[18]. Previous work has shown that the IL-10 AT haplotype results in lower production of protein IL-10,

TABLE 2b IL-10 ⫺819 gene polymorphism/genotype frequencya IL-10–819

UK BD patients UK controls ME patients ME controls All BD patients All controls

CC

CT

TT

Cb

Tb

28 (44%) 102 (56%) 43 (37%) 60 (53%) 71 (40%) 162 (55%)

28 (44%) 67 (37%) 61 (53%) 43 (38%) 89 (50%) 110 (37%)

7 (12%) 13 (7%) 11 (10%) 10 (9%) 18 (10%) 23 (8%)

84 (57%) 271 (74%) 147 (64%) 163 (72%) 231 (65%) 434 (74%)

63 (43%) 93 (26%) 83 (36%) 63 (28%) 125 (35%) 156 (26%)

p value pc ⫽ 0.0004 NS pc ⫽ 0.02

IL-10 ⫺819T is associated with BD in UK and combined patient groups. ⫺819T vs ⫺819C UK: ⌾2. 14.8 p ⫽ 0.0001, OR 2.1 (1.4 –3.3), pc ⫽ 0.0004; ME: ⌾2. 3.2, p ⫽ 0.07, OR 1.46 (1–2.2), pc ⫽ 0.3; All: ⌾2. 7.6 p ⫽ 0.006, OR 1.5 (1.1–2), pc ⫽ 0.02. a

b

IL-10 gene polymorphisms in BD

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TABLE 3 The AT haplotype does not associate BD with any of the patient groupsa IL-10 Haplotype AT UK BD patients UK controls ME BD patients ME controls All BD patients All controls

[n [n [n [n [n [n

⫽ ⫽ ⫽ ⫽ ⫽ ⫽

AC

GC

63] 42 (33%) 35 (28%) 49 (39%) 182] 93 (25%) 100 (28%) 171 (47%) 115] 74 (32%) 76 (33%) 80 (55%) 113] 63 (28%) 69 (30%) 94 (42%) 178] 116 (33%) 111 (31%) 129 (36%) 295] 156 (26%) 169 (29%) 265 (45%)

pc ⫽ NS. a IL-10 ⫺1082A/⫺819T versus other haplotypes, UK: ⌾2. 2.46, p ⫽ 0.116, OR 1.46 (0.9 –2.3); ME: ⌾2. 0.81, p ⫽ 0.4, OR 1.23 (0.8 –1.9); All: ⌾2. 3.8, p ⫽ 0.05, OR 1.34 (1–1.8).

whereas the GC haplotype results in greater production [10]. In the current study only the IL-10 ⫺819T genotype was associated with disease in UK patients and, to a lesser extent, when all patients were analyzed as a group. The ⫺1082A/⫺819T haplotype was raised but not significantly in patients with BD. It is more commonly reported that the IL-10 ⫺1082A genotype is associated with disease; however, ⫺819T (indicative of low IL-10 production) has also been independently reported as a risk factor in human disease [19, 20]. The difference between patient groups could be explained by the fact that all patients of ME, or AfroCaribbean origin in our UK cohort were removed prior to analysis leaving only white patients in this group. The finding that one or other SNP of various genes segregate in different patient populations is consistent with previous work For instance, TNF gene polymorphisms that have been linked to a higher or lower production of the protein, have shown associations with BD in both Arab and UK patients, although the particular SNP were different in the two groups [21, 22]. Similarly, analysis of the prevalence of the Factor V Leiden mutation in patients with BD found that it was strongly associated with retinal occlusive disease in Arab, but not in UK patients [23, 24]. A second possibility is that there may be selection bias in our study as the majority of patients tested had ocular disease, which is considered to be a severe manifestation of BD and such patients are likely to be kept under review. It will be important to extend the study to analyze patients with milder forms of the disease. However, there was no association between IL-10 haplotypes and ocular disease in our patient groups. A third possibility is that the gender differences between the UK and the ME patients could influence our results. The ME group had a strong bias towards males (83%), whereas the UK group was evenly balanced. We have previously described chemokine receptor polymorphisms that segregate with gender in patients with BD [25].

However when analyzed based on gender there was no link between the AT haplotype or individual genotypes between males and females in the UK cohort suggesting that the increased prevalence of the ⫺819T genotype was not being skewed by the larger number of females in this group. IL-10 has been demonstrated pathologically in the active lesions of BD and from stimulated T cells and serum from patients with the disease and may have a role in one of three ways [26]. Firstly, IL-10 may act in the initial stage of bacterial or viral infection or at sites of tissue damage, as a potent stimulator of NK cells and of macrophage recruitment [27]. Failure of an adequate innate response at this stage might lead to a failure to clear an infective focus resulting in prolonged immunologic stimulation. Secondly, as the adaptive immune response develops, IL-10 suppresses the pro-inflammatory function of macrophages by antagonizing expression of costimulatory molecules and the release of proinflammatory cytokines thus limiting the stimulation of Th1 cells [28]. A failure to down-regulate this part of adaptive immunity properly would result in a strong Th 1 profile, shown to be present in the disease [6 – 8]. Finally, IL-10 has been shown to be a pivotal mediator of regulatory T cells [29], and the AT haplotype may be associated with reduced numbers or effectiveness of such cells. In support of this scenario, alterations in the CD4-CD8 T-cell ratio and a defect in T-cell mediated suppression are both present in BD, and serum levels of IL-17, IL-18, and IFN␥ showed a striking increase in patients with active disease [30 –32]. Therefore the IL-10 ⫺819T genotype may act as a severity factor in BD because of reduced effectiveness of regulatory T cells, but would not appear to have a significant role in the onset of disease. The results of this study support the likelihood that levels of different cytokines either locally in the eye or systemically in leukocyte activation may have a role to play in BD. The above results suggest that a complex interplay of immune mediators governed at the genetic level may be responsible for the diverse spectrum of disease seen in patients with BD. However, the finding that the IL-10 ⫺819T haplotype is associated with disease in different patient groups indicates a possible role for this cytokine in Behçet’s disease. Confirmation of a role for IL-10 in BD will require larger cohorts of patients derived from population based studies and the analysis of other SNP that may be important in production of this immunosuppressive cytokine. ACKNOWLEDGMENTS This work was supported by grants from the Guide Dogs for the Blind Association and The Iris Fund for the Prevention of Blindness.

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