IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2008;49:1979-1982.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-1393
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cordeiro, C. A.
Right arrow Articles by Teixeira, A. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cordeiro, C. A.
Right arrow Articles by Teixeira, A. L.

Interleukin-10 Gene Polymorphism (–1082G/A) is Associated with Toxoplasmic Retinochoroiditis

Cynthia A. Cordeiro,1 Paula R. Moreira,2 Mariana S. Andrade,1 Walderez O. Dutra,2 Wesley R. Campos,1 Fernando Oréfice,1 and Antônio L. Teixeira3

1From the Uveitis Section, Department of Ophthalmology, and the 2Departments of Morphology and 3Internal Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Experimental data have demonstrated a relevant role for IL-10, an anti-inflammatory cytokine, in the modulation of acute ocular toxoplasmosis. Therefore, this study was conducted to investigate the possible association between an IL10 gene polymorphism at position –1082 and toxoplasmic retinochoroiditis (TR) in humans.

METHODS. One hundred patients with diagnosed TR were recruited from the Uveitis Section, Federal University of Minas Gerais. For comparison, one hundred healthy blood donors with positive serology for toxoplasmosis and without retinal signs of previous TR were included in the study. Genomic DNA was obtained from oral swabs of individuals and amplified using polymerase chain reaction (PCR) with specific primers flanking the locus –1082 of IL10 (–1082G/A). PCR products were subjected to restriction endonuclease digestion and analyzed by polyacrylamide gel electrophoresis, to distinguish allele G and A of the IL-10 gene, allowing the detection of the polymorphism and determination of genotypes.

RESULTS. There was a significant difference in the genotype distribution between TR patients and control subjects ({chi}2 = 6.33, P = 0.04). Carriers of the IL10 –1082 A allele (AA+AG genotypes) were more often patients with TR than control subjects ({chi}2 = 5.97, P = 0.01, OR, 2.55; 95% CI, 1.11 < OR < 5.55). In a subgroup analysis, there was no significant difference in genotypes and allele carriage regarding visual acuity, involvement of both eyes and TR recurrence.

CONCLUSIONS. This study suggests that the genotypes related with a low production of IL-10 may be associated with the occurrence of TR.

Toxoplasmosis is a common infection worldwide, but the disease is often asymptomatic in immunocompetent individuals. Ocular lesions, or toxoplasmic retinochoroiditis (TR), develop in only a few infected subjects, but it is uncertain why they develop.1 TR is the most common identifiable cause of posterior uveitis in many parts of the world, including Brazil.2 3 4 The intensity of damage to the retina and choroid depends on the severity of the infection and the associated inflammatory reaction.2 4 5 The response to antibiotic therapy in combination with corticosteroids and the clinical presentation vary significantly, with some patients presenting with one episode of mild inflammation whereas others have multiple recurrences of severe uveitis, leading to loss of eyesight. The immune response is likely to play a role in determining the evolution of disease and possibly the response to conventional therapy.5 6

A group of inflammation-related molecules, the cytokines, is involved in the control of the immunopathology of uveitis.7 8 Cytokine production has been shown to be under genetic control. Polymorphisms in the promoter region of cytokine genes may determine lower or higher levels of their production in response to different stimuli. As a consequence, these polymorphisms may influence the susceptibility to inflammatory diseases or their severity.9

Interleukin (IL)-10 is a cytokine with a significant anti-inflammatory function.10 11 Approximately three quarters of interindividual variability in human IL-10 levels has been attributed to genetic variation, and there is a growing body of evidence suggesting a potential role for IL-10 in a range of human diseases.9 12 13 14 15 16 A polymorphism within the promoter region of the IL10 gene at position –1082 has been associated with low levels of IL-10 production.13 This polymorphism is characterized by the substitution of guanine (G) to adenine (A) nucleotides. The –1082 AA IL10 genotype, marked by lower IL-10 production, has been associated with the occurrence of Behçet’s disease,14 the development of steroid-dependent inflammatory bowel disease,15 and the presence of invasive pulmonary aspergillosis.16 In another study, it was found that the IL-10 haplotype (–1082/–819/–592) seems to have a protective role against the development of invasive pulmonary aspergillosis after allogeneic stem cell transplantation, but this protective role was not dependent on –1082A allele.17

Therefore, we sought to investigate the possible association between IL10 (–1082G/A) polymorphism and TR in humans.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Subjects
The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the local institutional review board. The patients were informed verbally and in writing of the potential benefits and risks of the study, and all patients signed a written form stating that they understood and consented to participate.

One hundred patients (41 males, 59 females) with diagnosed TR were recruited from the Uveitis Section, Department of Ophthalmology, Federal University of Minas Gerais, Brazil, between August 2006 and November 2006. No subject enrolled in this study had autoimmune or systemic infectious disease. Active TR was defined by the presence of a gray-white focus of retinal necrosis next to a pigmented retinal scar or in the other eye in patients with positive serology for toxoplasmosis. A recurrent disease was defined as a new active focus of retinal necrosis after 3 months of an active episode.18 The following clinical data were also collected: age, sex, duration of follow-up, associated systemic disease, and number of recurrent episodes. All patients underwent a detailed ocular examination, including best-corrected visual acuity, applanation tonometry for intraocular pressure, slit lamp examination, and fundus examination with 78-D lens and indirect ophthalmoscope. The number and location of retinochoroidal lesions were documented in all patients by careful fundus drawings or photographs.

One hundred healthy blood donors from the Hemominas Foundation, matched by age and sex, were included as the control group. All of them had positive IgG antibody for toxoplasmosis without any history of uveitis. All control subjects also underwent an ocular examination to exclude the presence of retinal scars suggestive of previous TR.

Sample Collection and DNA Extraction
Epithelial cells from 200 individuals were obtained through an oral swab performed with a sterile plastic spatula and placed immediately in 1500 µL of Krebs buffer (NaCl 20%, KCl 2%, CaCl2 2%, H2O 2%, MgSO4, KH2PO4, and C6H12O6). DNA extraction was performed. A pellet of cells was obtained by centrifugation at 200g for 5 minutes. The supernatant was removed and 20 µL of silica (SiO2; Sigma-Aldrich, St. Louis, MO) and 450 µL of lyses buffer (6.0 M GuSCN, 65 mM Tris-HCl [pH 6.4], 25 mM EDTA, and 1.5% Triton X-100) were added to the microtubes. The samples were homogenized by vortexing and incubated for 30 minutes at 56°C. After incubation, the samples were subjected to another centrifugation, and the supernatant was discharged. The pellet obtained (with DNA adsorbed on the silica) was washed twice with 450 µL washing buffer (6.0 M GuSCN, 65 mM Tris-HCl [pH 6.4]), twice with 450 µL of 70% ethanol, and once with 450 µL acetone and dried at 56°C for 20 minutes. Finally, 100 µL of TE buffer (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA) was added and incubated at 56°C for 12 hours to release the DNA. After incubation, the solution was homogenized and centrifuged, and the supernatant containing DNA was transferred to a new tube.

Polymerase Chain Reaction and Restriction Endonuclease Digestion
IL10 (–1082) polymorphism was assessed by polymerase chain reaction (PCR) amplification followed by digestion with a specific restriction enzyme. The sequences of PCR primers were 5'-CCAAGACAACACTACTAAGGCTCCTTT-3'and 5'-GCTTCTTATATGCTAGTCAGGTA-3' with expected PCR product size of 377 bp. PCR was performed in a total volume of 50 µL, containing 10 µL of solution DNA, Pre-mix buffer (50 mM KCl, 10 mM Tris-HCl [pH 8.4], 0.1% Triton X-100, 1.5 mM MgCl2, deoxynucleoside triphosphates, Taq DNA polymerase), and primers (20 picomoles/reaction). The amplification conditions consisted of 94°C for 3 minutes followed by 35 cycles of 94°C for 30 seconds, 54°C for 35 seconds, and 72°C for 30 seconds. The run was terminated by final elongation at 72°C for 5 minutes. Amplification was performed in a thermocycler (PTC-100-60; MJ Research, Waltman, MA). The products were digested with 5 units of XagI enzyme (MBI Fermentas, Vilnius, Lithuania) at 37°C for 4 hours and digestion products of 280+97 and 253+27 bp were obtained for A and G alleles, respectively. The visualization was performed in a 10% polyacrylamide gel electrophoresis.

Statistical Analysis
The study groups were tested for Hardy-Weinberg equilibrium comparing the expected with the observed genotype frequencies. Statistical analyses were performed with commercial software (SPSS for Windows, ver. 11.0.1; SPSS, Inc., Chicago, IL). Associations with TR were investigated between genotype and allelic frequencies. {chi}2 analysis and calculation of odds ratio (OR) with 95% confidence interval (CI) were performed. The level of statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
The clinical characteristics of subjects enrolled in this study are shown in Table 1 . Most patients had two or more episodes of the disease, and 37.1% had visual acuity equal or inferior to 20/200. Forty-four patients had lesions in both eyes. Only two patients had an associated systemic disease, including hypertension and diabetes mellitus. The ocular complications reported in this sample included retinal detachment (2%), total cataract (2%), glaucoma (1%), and epiretinal membrane (1%).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Clinical Characteristics of Patients with TR and Control Subjects

 
Table 2 presents genotype and allelic distributions of the IL10 –1082 G/A polymorphism in TR patients and control subjects. No difference was found between observed and expected distributions of genotypes for the control group, and therefore it was considered to be in Hardy-Weinberg equilibrium. There was a significant difference in the genotype distribution between groups ({chi}2 = 6.33, P = 0.04), indicating that the frequency of AG genotype may be related to TR occurrence. Furthermore, TR patients were more frequently carriers of the IL10 –1082 A allele (AA+AG genotypes) than control subjects ({chi}2 = 5.97, P = 0.01, OR, 2.55; 95% CI, 1.11 < OR < 5.55). The data demonstrate an association between the presence of the A allele and the occurrence of TR.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Genotype and Allele Carriage Frequencies of IL10 –1082

 
In intragroup analysis, there was no association between genotype ({chi}2 = 11.50, P = 0.31) or allele carriage distribution ({chi}2 = 5.72, P = 0.33), and the levels of visual acuity in the more compromised of the two eyes in the individual. No significant difference was found in either genotype ({chi}2 = 1.45, P = 0.48) or allele carriage ({chi}2 = 0.39, P = 0.52) frequency when patients with lesions in only one eye were compared with patients with lesions in both eyes.

We also assessed whether this polymorphism was related with TR recurrence. No association was observed among the frequencies of genotypes (P = 0.19) or A allele carriage (P = 0.11), and an incidence higher than 0.5 TR episode per person-year of follow-up (1 episode/2 years of follow-up).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In our study, the presence of IL10 –1082 A allele was associated with the occurrence of the TR. This link between TR and a genotype associated with an intermediate or lower IL-10 production provides evidence that abnormalities in the genetic control of cytokine levels may be relevant in influencing the immune response in TR. It is unlikely that confounding factors account for differences between the groups in the present study; nevertheless, one should bear in mind that additional factors that are difficult to control in this setting, such as duration of infection, may also have an effect on ocular disease.2

The pathogenesis of TR remains largely unknown. There is great controversy regarding which factors are responsible for the occurrence or recurrence of TR. Among the proposed factors are immune response, strains of parasites, and hormonal changes.1

IL-10 is a cytokine with important anti-inflammatory and immunosuppressive properties, among which are pleiotropic effects on cell-mediated immune system, commonly associated with inhibition of cell-mediated immune responses.10 11 It has been proposed that IL-10 may downregulate protective immune response to several intracellular pathogens, including Toxoplasma gondii,19 through the TNF-{alpha} and IL-12 production control.11 Experimental models of T. gondii infection have show that IL-10 is essential in damping an ongoing immune response and thereby controlling the extent of tissue damage.20

The role of the IL-10 polymorphism in the regulation of inflammation in TR has already been demonstrated in an animal model of acute ocular toxoplasmosis. In this study, we observed that lower levels of endogenous IL-10 determined increased cellular infiltration and necrosis in the eyes of the infected mice.21 In humans, one study showed that IL-10 levels were higher in ocular fluid from patients with TR when compared with control subjects with no inflammatory ocular disease.22 Thus, the analysis of IL-10 gene polymorphism in TR may represent an advance in the study of TR pathogenesis.

In line with our findings, besides the association between IL-10 polymorphisms and several inflammatory systemic diseases,9 12 13 14 15 16 there are some studies suggesting their role in human uveitis. A recent study observed the association of the IL10 gene polymorphism –1082G/A with occurrence and severity of disease in patients with sympathetic ophthalmia. It was also found to be associated with recurrence of previously stable disease and with the level of steroid treatment necessary to control inflammatory activity.23 In another study, that this gene polymorphism was associated with idiopathic intermediate uveitis.24

This study is the first to demonstrate the association between the genetic polymorphism and occurrence of TR in humans. The lack of association with visual acuity, bilaterality, or short-term incidence of recurrences does not rule out the possibility of a role for IL-10 in the pathogenesis of TR. Visual acuity is not necessarily a good measure of disease severity, because it depends on location of lesions, and bilateral disease is uncommon. Recurrence rates vary greatly. Thus, sample sizes may be too small and follow-up too short to identify the impact of IL-10 on these parameters. Confirmation of a role for IL-10 in TR will require larger cohorts of patients and the analysis of other polymorphisms or haplotypes.


   Footnotes
 
Submitted for publication October 28, 2007; revised December 14, 2007; accepted March 5, 2008.

Disclosure: C.A. Cordeiro, None; P.R. Moreira, None; M.S. Andrade, None; W.O. Dutra, None; W.R. Campos, None; F. Oréfice, None; A.L. Teixeira, 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: Cynthia A. Cordeiro, Rua Gilberto Siqueira 87 apto 502, Campos, RJ 28010-400 Brazil; cordeiro.cy{at}gmail.com.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Holland GN. Ocular toxoplasmosis: a global reassessment. Part I: epidemiology and course of disease. Am J Ophthalmol. 2003;136:973–988.[Web of Science][Medline][Order article via Infotrieve]
  2. Oréfice F. Uveíte: Clínica e Cirúrgica: Texto e Atlas. 2005; 2nd ed. 1484. Cultura Médica Rio de Janeiro, Brazil.
  3. McCannel CA, Holland GN, Helm CJ, Cornell PJ, Winston JV, Rimmer TG. Causes of uveitis in the general practice of ophthalmology. UCLA Community-Based Uveitis Study Group. Am J Ophthalmol. 1996;121:35–46.[Web of Science][Medline][Order article via Infotrieve]
  4. Henderly DE, Genstler AJ, Smith RE, Rao NA. Changing patterns of uveitis. Am J Ophthalmol. 1987;103:131–136.[Web of Science][Medline][Order article via Infotrieve]
  5. McMenamin PG, Dutton GN, Hay J, Cameron S. The ultrastructural pathology of congenital murine toxoplasmic retinochoroiditis. Part I: The localization and morphology of Toxoplasma cysts in the retina. Exp Eye Res. 1986;43:529–543.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  6. Vallochi AL, Nakamura MV, Schlesinger D, et al. Ocular toxoplasmosis: more than just what meets the eye. Scand J Immunol. 2002;55:324–328.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  7. Takase H, Futagami Y, Yoshida T, et al. Cytokine profile in aqueous humor and sera of patients with infectious or noninfectious uveitis. Invest Ophthalmol Vis Sci. 2006;47:1557–1561.[Abstract/Free Full Text]
  8. Goncalves RM, Rodrigues DH, Camargos da Costa AM, et al. Increased serum levels of CXCL8 chemokine in acute toxoplasmic retinochoroiditis. Acta Ophthalmol Scand. 2007;85:871–876.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  9. Lazarus M, Hajeer AH, Turner D, et al. Genetic variation in the interleukin 10 gene promoter and systemic lupus erythematosus. J Rheumatol. 1997;24:2314–2317.[Web of Science][Medline][Order article via Infotrieve]
  10. Groux H, Bigler M, de Vries JE, Roncarolo MG. Inhibitory and stimulatory effects of IL-10 on human CD8+ T cells. J Immunol. 1998;160:3188–3193.[Abstract/Free Full Text]
  11. Neyer LE, Grunig G, Fort M, Remington JS, Rennick D, Hunter CA. Role of interleukin-10 in regulation of T-cell-dependent and T-cell-independent mechanisms of resistance to Toxoplasma gondii. Infect Immun. 1997;65:1675–1682.[Abstract]
  12. Lazarus R, Klimecki WT, Palmer LJ, et al. Single-nucleotide polymorphisms in the interleukin-10 gene: differences in frequencies, linkage disequilibrium patterns, and haplotypes in three United States ethnic groups. Genomics. 2002;80:223–228.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  13. Turner DM, Williams DM, Sankaran D, Lazarus M, Sinnott PJ, Hutchinson IV. An investigation of polymorphism in the interleukin-10 gene promoter. Eur J Immunogenet. 1997;24:1–8.[Web of Science][Medline][Order article via Infotrieve]
  14. Wallace GR, Kondeatis E, Vaughan RW, et al. IL-10 genotype analysis in patients with Behcet’s disease. Hum Immunol. 2007;68:122–127.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  15. Castro-Santos P, Suarez A, López-Rivas L, Mozo L, Gutierrez C. TNFalpha and IL-10 gene polymorphisms in inflammatory bowel disease: association of –1082 AA low producer IL-10 genotype with steroid dependency. Am J Gastroenterol. 2006;101:1039–1047.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  16. Sainz J, Hassan L, Perez E, Romero A, et al. Interleukin-10 promoter polymorphism as risk factor to develop invasive pulmonary aspergillosis. Immunol Lett. 2007;109:76–82.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  17. Seo KW, Kim DH, Sohn SK, et al. Protective role of interleukin-10 promoter gene polymorphism in the pathogenesis of invasive pulmonary aspergillosis after allogeneic stem cell transplantation. Bone Marrow Transplant. 2005;36:1089–1095.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  18. Jabs DA, Nussenblatt RB, Rosenbaum JT. Standardization of Uveitis Nomenclature (SUN) Working Group; standardization of uveitis nomenclature for reporting clinical data—results of the First International Workshop. Am J Ophthalmol. 2005;140:509–516.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  19. Hunter CA, Abrams JS, Beaman MH, Remington JS. Cytokine mRNA in the central nervous system of SCID mice infected with Toxoplasma gondii: importance of T-cell-independent regulation of resistance to T. gondii. Infect Immun. 1993;61:4038–4044.[Abstract/Free Full Text]
  20. Gazzinelli RT, Wysocka M, Hieny S, et al. In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-gamma and TNF-alpha. J Immunol. 1996;157:798–805.[Abstract]
  21. Lu F, Huang S, Kasper LH. Interleukin-10 and pathogenesis of murine ocular toxoplasmosis. Infect Immun. 2003;71:7159–7163.[Abstract/Free Full Text]
  22. Ongkosuwito JV, Feron EJ, van Doornik CE, et al. Analysis of immunoregulatory cytokines in ocular fluid samples from patients with uveitis. Invest Ophthalmol Vis Sci. 1998;39:2659–2665.[Abstract/Free Full Text]
  23. Atan D, Turner SJ, Kilmartin DJ, et al. Cytokine gene polymorphism in sympathetic ophthalmia. Invest Ophthalmol Vis Sci. 2005;46:4245–4250.[Abstract/Free Full Text]
  24. Stanford MR, Vaughan RW, Kondeatis E, et al. Are cytokine gene polymorphisms associated with outcome in patients with idiopathic intermediate uveitis in the United Kingdom?. Br J Ophthalmol. 2005;89:1013–1016.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cordeiro, C. A.
Right arrow Articles by Teixeira, A. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cordeiro, C. A.
Right arrow Articles by Teixeira, A. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS