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Cytokine Gene Polymorphism in Sympathetic Ophthalmia

¹From the Academic Unit of Ophthalmology, Clinical Sciences South Bristol, Bristol Eye Hospital, Bristol, United Kingdom; the ²Department of Ophthalmology, Royal College of Surgeons in Ireland, Royal Victoria Eye and Ear Hospital, Dublin, Ireland; the ³Department of Ophthalmology, University of Aberdeen, Scotland, United Kingdom; and the ⁴Department of Pathology and Microbiology, University of Bristol, United Kingdom.

	Abstract

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PURPOSE. Sympathetic ophthalmia (SO) is a prototypical autoimmune disease in which injury to one eye causes sight-threatening inflammation in the otherwise normal contralateral eye. Previous work found that human leukocyte antigen alleles HLA-DRB1*04 and DQA1*03 are markers of increased susceptibility and severity in British and Irish patients. Evidence is accumulating that single nucleotide polymorphisms (SNPs) in cytokine genes can also influence the development of autoimmune disease through their effect on levels of cytokine production. The purpose of this study was to determine whether polymorphisms in the cytokine genes are important markers for disease severity and outcome in patients with SO.

METHODS. Twenty-six British and Irish patients meeting well-defined criteria for the diagnosis of SO were compared with 48 matched controls. Genotyping of SNPs in the TNF, TNFß, and IL-10 genes was performed using the polymerase chain reaction and sequence-specific primers (SSP-PCRs) and of the CTLA-4 and TNF receptor 2 genes using restriction length polymorphism-PCR (RFLP-PCR).

RESULTS. Significant associations were found between the IL-10 –1082 SNP and disease recurrence from previously stable disease and the level of steroids required for maintenance therapy. In addition, the GCC IL-10 promoter haplotype (IL-10 –1082G, –819C, –592C) was found to be protective against disease recurrence.

CONCLUSIONS. These results show that cytokine gene polymorphisms are markers for the severity of disease in SO. They were found to be associated with recurrence of previously stable disease and with the level of maintenance steroid treatment required to control inflammatory activity.

Associations with specific human leukocyte antigen (HLA) alleles are well established for several autoimmune diseases, including SO, in which HLA DRB1*04 and DQA1*03 are significantly associated with disease susceptibility and severity in British and Irish patients.⁶ Polymorphisms in the HLA genes influence the initial presentation of disease-inducing peptides to T cells through their effect on HLA-peptide binding affinity and the development of the T cell repertoire during thymic maturation when autoreactive T cells are deleted.⁷ In addition, recent evidence has implicated single nucleotide polymorphisms (SNPs) in cytokine genes in modulating the susceptibility and severity of a number of autoimmune diseases.^{8 9 10 11} Studies seeking an etiological role for cytokine SNPs have found that many influence gene transcription rather than the molecular structure of the cytokines. Polymorphisms that result in the upregulation of proinflammatory cytokines, such as TNF (TNFB*2, TNF–308A),^{12 13} or the downregulation of anti-inflammatory cytokines, such as IL-10 (IL-10 –1082A),¹⁴ are predicted to create a more proinflammatory environment in the eye and consequently to worsen the severity of inflammation.

It is not possible to determine who will develop SO after ocular injury, nor is it possible to determine the outcome of disease based on clinical signs observed at presentation. This can result in inadequate treatment of the patient with sight-threatening disease or in overtreatment of the patient whose disease would otherwise have a benign course and in whom treatment through systemic immunosuppression may be complicated by potentially life-threatening adverse effects. However, patients with SO respond well to treatment, and, if adequate immunosuppression is initiated promptly in appropriate patients, the prognosis in SO can be very good.^{5 15 16}

The purpose of this study was to determine whether polymorphisms of the cytokine genes are associated with severity and outcome in patients with SO. We chose to genotype SNPs that functionally influence transcription levels and that are known to be associated with other autoimmune diseases: TNF–308G/A (dbSNP ID rs1800629), TNF–238G/A (rs361525), TNFß*1/2 (rs909253), IL-10 –1082G/A (rs1800896), IL-10 –819C/T (rs1800871), IL-10 –592C/A (rs1800872), TNF receptor 2 +196R/M (rs1061622, rs17883437), and CTLA-4 +49G/A (rs231775).

	Methods

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Patients and Controls
Twenty-seven patients (14 male, 13 female) with newly diagnosed and established SO and 48 controls were recruited from throughout the United Kingdom and Ireland, as previously described.^{5 6} Seventeen were patients with newly diagnosed SO who had been recruited through the British Ophthalmological Surveillance Unit (BOSU) of the Royal College of Ophthalmologists between July 1997 and September 1998 inclusive, and 10 were patients with established SO who had been recruited from uveitis clinics at Grampian University Hospitals, Aberdeen, and the Mater Hospital, Dublin.^{5 6} One patient (female) was excluded from the present study because her DNA had degraded and could not be used for reliable genotyping.

All patients had bilateral posterior uveitis or panuveitis with a definite history of ocular trauma followed by contralateral posterior uveitis with features clinically consistent with SO (anterior chamber activity with mutton-fat keratic precipitates, vitreitis, macular and disc edema, Dalen-Fuchs nodules, choroidal neovascularization, exudative retinal detachment or extraocular signs including CSF pleocytosis and hair and skin changes), and five patients demonstrated histologic features consistent with SO in enucleated exciting eyes.⁶

Clinical phenotype and markers of disease severity were assessed, including visual acuity, time interval between SO onset and last ocular injury, biomicroscopic features of intraocular inflammation, and immunosuppression therapy, as previously described.⁶ Disease recurrence was defined as an increase in disease activity with worsening of visual acuity, BIO score, or both requiring an increase in immunosuppression therapy from previously stable maintenance therapy for at least 2 months. Patients whose activity increased after a reduction of immunosuppression therapy were not defined as having disease recurrence. Disease recurrences were identified within the follow-up period of the original study, which had a median of 12 months (range, 6–354 months).⁶ Maintenance therapy was defined as the degree of immunosuppression required to control inflammation.⁶

Controls were matched by age, sex, and region. They were healthy, unrelated volunteers without any history of uveitis or other autoimmune disease who attended the same hospitals or primary care clinics as the patients.

Informed consent was obtained from patients and controls after explanation of the nature and possible consequences of the study. The local regional ethics committees granted approval, and the study was conducted in accordance with the tenets of the Declaration of Helsinki.

Genotyping
DNA was extracted using the salt extraction method, as previously described.^{6 17}

Sequence-Specific Primer PCR Genotyping of TNF, TNFß, and IL-10 Gene SNPs
The 25 µL PCR reaction mixtures contained 50 ng sample DNA, buffer (GeneAmp PCR Buffer II; Applied Biosystems, Warrington, Cheshire, UK), optimized MgCl₂ (Table 1) , 200 mM each dNTP (Applied Biosystems), 1 mM forward and reverse primers (Eurogentec; Romsey, Hampshire, UK) (Table 1) , 0.2 mM control primers (Eurogentec), and 0.5 U polymerase (AmpliTaq Gold DNA polymerase; Applied Biosystems).

The following cycling conditions were used on a thermal cycler (Peltier Thermal Cycler, model 220; MJ Research Systems, Watertown, MA): 94°C for 5 minutes, 30 cycles of 94°C for 1 minute, optimized Tm°C for 1 minute (Table 1) , 72°C for 1 minute, and 72°C for 5 minutes.

Agarose Gel Electrophoresis
PCR products were run on a standard 2% agarose gel in 0.5x TBE running buffer for 45 minutes at 100 V and 250 mA in an electrophoresis tank (Horizon 11.14 Horizontal Gel Electrophoresis Tank; Gibco/BRL Life Technologies Inc, Gaithersburg, MD) and were visualized after staining with ethidium bromide under UV light.

Method Reliability
For a subset of patients, the results of genotyping using the gold standard technique of DNA sequencing were compared with the results of SSP-PCR genotyping to ensure method reliability and accuracy.

Restriction Fragment Length Polymorphism Genotyping of the TNFR 2 and CTLA-4 Genes
TNFR 2 +196T/G.
The 25 µL PCR reaction mix contained 50 ng DNA, buffer (GeneAmp PCR Buffer II; Applied Biosystems), 1.0 mM MgCl₂, 200 mM each dNTP, 1 mM forward (5'-ACT CTC CTA TCC TGC CTG CT-3') and reverse (5'-TTC TGG AGT TGG CTG CGT GT-3') primers, and 0.5 U AmpliTaq Gold DNA polymerase, based on the method described by Al-Ansari et al.¹⁹

Cycling conditions were 94°C for 5 minutes, 35 cycles of 94°C for 1 minute, 57°C for 1 minute, 72°C for 1.5 minutes, and 72°C for 5 minutes. PCR products were run on a standard 2% agarose gel, as described, to check for amplification and contamination.

PCR product (7 µL) was mixed with 4 U NlaIII restriction enzyme, NEB4 buffer, and BSA (New England Biolabs, Hitchin, UK) in 10 µL and was incubated overnight at 37°C. The results of the digest were visualized on a standard 2% agarose gel, as described.

CTLA-4 +49 A/G.
The 25 µL PCR reaction mix contained 150 ng DNA, buffer (GeneAmp PCR Buffer II; Applied Biosystems), 2.0 mM MgCl₂, 200 mM each dNTP, 1 mM forward (5'-GTC AAG GGA CCA TTA GAA G-3') and reverse (5'-CTT TGC AGA AGA CAG GGA TGA A-3') primers, and 0.5 U AmpliTaq Gold DNA polymerase, based on the method described by Heward et al.²⁰

Cycling conditions were 94°C for 5 minutes, 30 cycles of 95°C for 1 minute, 55°C for 1 minute, 72°C for 1.5 minutes, and 72°C for 5 minutes. Each PCR product was run on a standard 2% agarose gel, as described, to check for amplification and contamination.

PCR product (5 µL) was mixed with 1 U BbvI restriction enzyme and NEB2 buffer (New England Biolabs) in 10 µL and was incubated for 2 hours at 37°C. Results of the digest were run on a standard 2% agarose gel, as described.

Statistical Analysis
The ² test or the Fisher exact test was used to compare patient and control groups and to determine associations between clinical phenotype and genotype within the patient group using SPSS version 11.5.0 (SPSS UK Ltd, Woking, UK) and Epi Info 6 version 6.04d software (Centers for Disease Control and Prevention, Atlanta, GA). Distributions of ordinal and continuous phenotypic characteristics were compared using the Mann-Whitney U or the Kruskal-Wallis nonparametric test and one-way ANOVA, respectively. P < 0.05 was significant.

	Results

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Comparison of SNP Genotype and Allele Frequency between SO Patients and Controls
There were no statistically significant differences in genotype or allele frequencies in any of the SNPs between patients and controls that were independent of the HLA class 2 alleles described previously.⁶ In addition, there was no association with IL-10 promoter haplotype.

Comparison of SNP Genotype and Allele Frequency with Clinical Features of SO and Markers of Disease Severity
Clinical Features of SO.
There were no statistically significant associations among SNP genotype, allele frequency, and IL-10 haplotype with any clinical features of uveitis.

Severity of SO.
IL-10.
There was a statistically significant association between IL-10 –1082 SNP and disease recurrence. Disease recurrence was associated with the AA and AG genotypes (P = 0.031) or with carrying an A allele at this locus (P = 0.020; Table 2 ). In addition, the GCC IL-10 promoter haplotype (IL-10 –1082G, –819C, –592C) was found to be protective against disease recurrence (P = 0.033; Table 3 ).

Overall, 11 of 12 patients taking more than 10 mg/d prednisolone were also taking more than one immunosuppression agent (seven patients taking prednisolone + cyclosporin A [one patient was later changed to tacrolimus], two patients taking prednisolone + azathioprine, two patients taking prednisolone + cyclosporin A + either azathioprine or mycophenolate mofetil), compared with 8 of 14 patients taking less than 10 mg/d prednisolone (five patients taking prednisolone + cyclosporin A, two patients taking prednisolone + azathioprine, and one patient taking prednisolone + cyclosporin A + alemtuzumab). Furthermore, patients with a G allele at the IL-10 –1082 locus required lower doses of maintenance steroid therapy (P = 0.033; Table 4 ). There was a nonsignificant trend for patients with the AG or GG genotype at the IL-10 –1082 locus to require lower doses of maintenance steroid therapy (P = 0.052), but there was no association with IL-10 promoter haplotype.

View this table: [in this window] [in a new window]   TABLE 5. TNF–308 SNP and Onset of SO

Nevertheless, of all the patients with early onset of disease, only 1 of 14 had poor final visual acuity at follow-up, and 13 of 14 remained at Snellen visual acuity of 6/12 or better, which was independent of the follow-up period. Fifty percent of patients with onset of disease more than 3 months from the last ocular injury had a final Snellen visual acuity worse than 6/12.

	Discussion

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Our results show that cytokine gene polymorphisms are markers for the severity of disease in sympathetic ophthalmia. We found that they are associated with disease recurrence and the level of maintenance steroid treatment required to control the inflammatory process.

These results are important because at this time it is not possible to determine who will develop SO after surgical or accidental trauma and how severe the disease is likely to be once established. The clinician is faced with the dilemma of how heavily to immunosuppress a patient with a potentially sight-threatening though treatable disease with drugs that can have life-threatening effects.

Objective assessment of the severity of uveitic disease is difficult, and there is no consensus on how assessments should be graded. No single clinical feature suffices, and a scoring system that grades disease activity at the time of examination—e.g., the Uveitis Scoring System,²² —is unsuitable because it is not designed to give a cumulative score for severity and because it gives an impression of the degree of activity at only a single time point, irrespective of time since onset, degree of immunosuppression, or visually significant complications. Hence, the severity of inflammation without validated composite scoring can be defined indirectly by markers such as amount of maintenance immunosuppression required to control inflammation, recurrence of inflammation despite previously stable maintenance treatment, and visual acuity. Given these limitations, our results, in conjunction with those from previous work on HLA risk factors in SO, allow a more complete genetic profile of a patient to be built. Polymorphisms of HLA class 1 and 2 genes affect MHC-peptide binding affinity and, therefore, predictably are linked to disease initiation and susceptibility in SO. During the chronic phase of disease, there is a gradual decline in antigen-induced proliferative responsiveness, when antigen specificity becomes less relevant and inflammation is mediated purely by cytokines and bystander recruitment.²³ The cytokine milieu becomes more important in sustaining the inflammatory response and maintaining pathogenic T cells,^{24 25} so polymorphisms in the cytokine genes are more likely to influence disease severity rather than susceptibility. Indeed, in the present study, we found no significant link between any of the cytokine polymorphisms and susceptibility to SO, but we did find that cytokine polymorphisms are linked to markers of severity.

IL-10 is a potent anti-inflammatory cytokine produced by T cells, macrophages, and retinal cells.²⁶ It strongly inhibits antigen-specific T cell proliferation, cytokine production, and MHC class 2 expression^{27 28} and is important in the induction of antigen-specific anergy or tolerance.^{29 30} Functional studies of IL-10 promoter polymorphisms have found that the IL-10 –1082A allele and the ACC/ATA promoter haplotypes are linked to the downregulation of IL-10 production, whereas the IL-10 –1082G allele and the GCC haplotype are linked to upregulation.^{14 31} We found that the IL-10 –1082 A allele and the AA/AG genotypes were associated with disease recurrence, even though the patients with recurrent disease were on more than one immunosuppressant drug. In addition, we found the GCC IL-10 promoter haplotype to be protective against disease recurrence. Furthermore, patients with the IL-10 –1082G allele required lower doses of steroids as maintenance treatment, though many of these patients (6/14) were on prednisolone alone. Thus, our results are in keeping with the experimental evidence for the importance of IL-10 in dampening the inflammatory process during the efferent phase of SO.

The role of TNF in the pathogenesis of uveitis is somewhat more complex. TNF is a multifunctional cytokine secreted by monocytes (TNF), lymphocytes (TNFß) and resident retinal cells.^{32 33} It has potent proinflammatory effects since blocking its action in experimental models of uveitis reduces structural damage to the retina³⁴ whereas the administration of TNF leads to worsening of disease.³⁵ In contrast, chronic TNF exposure suppresses the cytokine and proliferative responses of T cells and drives Fas-dependent apoptosis.^{23 36 37}

Because of the proximity of the TNF genes to class 1 and 2 genes within the MHC region of chromosome 6 and the linkage disequilibria that exist between them, independent associations with disease can be difficult to define. Certain polymorphisms may be considered as part of an extended haplotype that includes the HLA class 1 and 2 genes.¹³ However, independent associations have also been demonstrated.³⁸ We have previously found that SO patients who are HLA DRB1*04-DQA1*03 positive experienced earlier onset of disease,⁶ which is not surprising given the role of MHC-peptide binding in disease initiation. Yet these patients not only had better final visual acuity at follow-up, they were also all TNF–308G positive. Evidence suggests that TNF is less important in the early stages of disease, during antigen priming, because TNF-deficient mice do not show any difference in antigen-specific T cell responses compared with wild-type mice.³⁹ Hence, we could speculate that patients with the TNF–308G allele, linked to the downregulation of TNF production,¹³ tend to have better long-term visual outcomes because of low TNF production during the efferent phase of disease.

Although most of the activity of TNF is mediated through TNF-R1, many effects are also mediated through TNF-R2, including cellular proliferation and apoptosis. We did not find any association between patients with SO and TNF-R2 or CTLA-4 polymorphisms, though animal studies implicate these proteins in the pathogenesis of uveitis.^{40 41} It is possible that other polymorphisms within these genes have a stronger influence on disease pathogenesis and may be identified by further functional studies and investigation of cytokine polymorphisms in patients with uveitis or that this study was not statistically powerful enough to detect a difference. This is, however, the largest case series of patients with SO from the UK and Ireland, recruited after 15 months of nationwide surveillance; a larger study would require multinational collaboration.

Recent successes have been reported in the treatment of patients with uveitis through the novel use of anti–TNF monoclonal antibody (mAb). Not only have these been associated with an increase in serum CD4⁺ IL-10⁺ T cells exclusively in successfully treated patients,⁴² but also TNF genotype has been linked to response to anti-TNF treatment in patients with rheumatoid arthritis.⁴³ Anti-TNF therapies were introduced to the uveitis treatment repertoire after we recruited patients for the present study. The prospect of analyzing TNF genotype before starting treatment with anti–TNF mAb has important clinical and economic implications because patients who are genetically predisposed to be high TNF producers are likely to benefit most from this treatment.

In conclusion, identifying genetic markers associated with susceptibility to and severity of uveitis may provide insight into the pathogenesis of uveitis, the development of therapies that target specific elements of the ocular immune response (e.g., anti–TNF mAb), and an enhanced genetic profile of patients who have more aggressive disease to allow tailoring of treatment to the patient.

	Acknowledgements

 
The authors thank Tony Hughes, University of Bristol, for his advice regarding statistical analysis.

	Footnotes

 
Supported by The Wellcome Trust and National Eye Research Centre.

Submitted for publication January 31, 2005; revised May 24, 2005; accepted September 6, 2005.

Disclosure: D. Atan, None; S.J. Turner, None; D.J. Kilmartin, None; J.V. Forrester, None; J. Bidwell, None; A.D. Dick, None, A.J. Churchill, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.

Corresponding author: Denize Atan, Academic Unit of Ophthalmology, Bristol Eye Hospital, Lower Maudlin Street, Bristol BS1 2LX, UK; denize.atan{at}bristol.ac.uk.

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