Services on Demand
- Cited by Google
- Similars in SciELO
- Similars in Google
Print version ISSN 1807-5932
Clinics vol.66 no.8 São Paulo 2011
Association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope and smoking status in Brazilian patients with rheumatoid arthritis
Michel Alexandre YazbekI; Silvia de Barros-MazonII; Cláudio Lúcio RossiII; Ana Carolina LondeI; Lilian Tereza Lavras CostallatI; Manoel Barros BertoloI
IDepartment of Rheumatology, University of Campinas (UNICAMP), Campinas, SP, Brazil
IIDepartment of Clinical Pathology, University of Campinas (UNICAMP), Campinas, SP, Brazil
INTRODUCTION: Epstein-Barr virus exposure appears to be an environmental trigger for rheumatoid arthritis that interacts with other risk factors. Relationships among anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status have been observed in patients with rheumatoid arthritis from different populations.
OBJECTIVE: To perform an association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status in Brazilian patients with rheumatoid arthritis.
METHODS: In a case-control study, 140 rheumatoid arthritis patients and 143 healthy volunteers who were matched for age, sex, and ethnicity were recruited. Anti-Epstein-Barr nuclear antigen-1 antibodies and anti-cyclic citrullinated peptide antibodies were examined using an enzyme-linked immunosorbent assay, and shared epitope alleles were identified by genotyping. Smoking information was collected from all subjects. A comparative analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status was performed in the patient group. Logistic regression analysis models were used to analyze the risk of rheumatoid arthritis.
RESULTS: Anti-Epstein-Barr nuclear antigen-1 antibodies were not associated with anti-cyclic citrullinated peptide antibodies, shared epitope alleles, or smoking status. Anti-cyclic citrullinated peptide antibody positivity was significantly higher in smoking patients with shared epitope alleles (OR = 3.82). In a multivariate logistic regression analysis using stepwise selection, only anti-cyclic citrullinated peptide antibodies were found to be independently associated with rheumatoid arthritis (OR = 247.9).
CONCLUSION: Anti-Epstein-Barr nuclear antigen-1 antibodies did not increase the risk of rheumatoid arthritis and were not associated with the rheumatoid arthritis risk factors studied. Smoking and shared epitope alleles were correlated with anti-cyclic citrullinated peptide-antibody-positive rheumatoid arthritis. Of the risk factors, only anticyclic citrullinated peptides antibodies were independently associated with rheumatoid arthritis susceptibility.
Keywords: Rheumatoid arthritis; Risk factors; Epstein-Barr virus; Epstein-Barr virus nuclear antigens; Brazilians.
Rheumatoid arthritis (RA) is an inflammatory autoimmune disease with a pathogenesis that is not fully understood. The pathophysiology of RA is complex and multifactorial and involves both genetic and environmental factors. Many studies have demonstrated the importance of certain human leukocyte antigens (HLA) molecules that contain the shared epitope (SE) as the major genetic risk factor for RA. Nevertheless, genetic factors fail to completely explain the occurrence of RA.1,2
In addition, antibodies to cyclic citrullinated peptides (anti-CCP) are highly specific predictors of RA. Anti-CCP have been observed many years prior to disease onset, indicating an important role for them in the development of RA. An association between the positivity of anti-CCP and the presence of the SE in RA has been reported by numerous researchers. The presence of SE alleles appears to be a risk factor related to anti-CCP production.1,3 Interactions between tobacco exposure and SE status in anti-CCP-positive patients have been demonstrated by several studies, whereas neither smoking nor SE status confers an increased risk of developing anti-CCP negative RA.4-10
Among other environmental factors, infectious agents, most notably viruses, have long been suspected of promoting the development of RA and other autoimmune diseases.11-13 Links between Epstein-Barr virus (EBV) and RA have been a focus of research for the last three decades. Studies have demonstrated that EBV is a potent B-cell stimulator, antibodies in RA cross-react with EBV nuclear antigens, and EBV glycoproteins share sequence similarities with the HLA-DRB1 SE. These viral properties support the theory that EBV is a strong risk factor for RA.12-15
It has been observed that the levels of some anti-EBV antibodies (anti-EBNA, anti-VCA, and anti-EA) are higher in RA patients than in controls.16,17 In particular, Epstein-Barr nuclear antigen-1 (EBNA-1) contains glycine/alanine sequences that constitute a dominant EBNA-1 epitope against which specific reactivity has been demonstrated.18,19 An antibody against this EBNA-1 sequence also reacted with a protein expressed by synovial lining cells in RA patients.20 Identical glycine/alanine repeat sequences of EBNA-1 are also present in collagen, cytokeratin, and actin.21
Many studies have suggested that some RA risk factors are highly associated with EBV in the complex etiopatho-geny of RA. Among the antibodies against EBV, anti-EBNA-1 appears to be the most prominent in the interactions with other risk factors. In the present study, we evaluated associations between anti-EBNA-1, anti-CCP, shared epitope alleles, and smoking status in Brazilian RA patients.
METHODS AND MATERIALS
Patients and controls
A total of 140 RA Brazilian patients (including 122 females) aged 25-82 years (mean = 54.24 years), comprising 74 Caucasians, 20 Blacks, and 46 Mulattoes were evaluated at the University of Campinas Teaching Hospital in Brazil. All the patients fulfilled the 1987 RA revised criteria of the American College of Rheumatology. For the control group, we screened 143 healthy individuals who were matched for age, sex, and ethnicity and had no personal or family history of autoimmune disease. Smoking exposure was measured in pack-years, and all subjects were classified as never smoker, past smoker or current smoker. Clinical information was collected using a questionnaire at the time of enrollment. This study was approved by the ethics committee of the University of Campinas, Brazil, and informed consent was obtained from all patients and controls.
Serology and genotyping
Anti-EBNA1 antibodies were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit containing synthetic peptides (ETI-EBNA-G, DiaSorin, Saluggia, Italy). Anti-CCP antibodies were determined by ELISA (QUANTA Lite CCP3 IgG, Inova Diagnostics Inc., USA). Rheumatoid factor was evaluated by a nephelometric immunoassay (BN ProSPec System, Siemens, Marburg, Germany). All assays were performed according to the manufacturers' instructions.
HLA-DRB1 typing was undertaken by polymerase chain reaction (PCR), using specific primers and hybridization with sequence-specific oligonucleotides, as previously described.22 HLA-DRB1*01, HLA-DRB1*04, and HLA-DRB1*10 were the SE alleles studied.5 Patients carrying zero, one, or two alleles of SE were classified as SE -/-, SE+/-,orSE +/+, respectively.
The chi-squared and Fisher's exact tests were used to compare categorical variables between the groups. The differences between the mean or median values of two groups were analyzed using the Mann-Whitney test. The Kruskal-Wallis test was used when the subjects were separated into three or more groups. To evaluate RA risk, we calculated odds ratios (OR) with 95% confidence intervals (CI). A logistic regression analysis with univariate and multiple models was used with stepwise variable selection. The significance level for statistical tests was set at 5% (p<0.05). All statistical analyses were performed using SAS (Statistical Analysis System) software for Windows, version 9.1.3 (SAS Institute Inc., Cary, NC, USA).
Patient and control characteristics
The demographic, clinical, and laboratory characteristics of the RA cases and matched controls are listed in Table 1.
Anti-EBNA-1, anti-CCP, SE status, and smoking status in patients and controls
The rates of anti-EBNA-1 and anti-CCP positivity, as well as the mean antibody levels detected using these assays, along with the percentages related to SE status and smoking status in the two groups, are listed in Table 2. No significant differences were observed between the patient group and the control group in terms of the positivity and the mean levels of anti-EBNA-1 antibodies. The positivity and mean levels of anti-CCP antibodies were significantly higher in the patients than in the controls (p<0.001). The presence of the SE alleles (SE+/ - and SE+/+) and a smoking history of at least 15 pack-years were significantly higher in the patients than in the controls (p<0.001 and p = 0.003, respectively).
Anti-EBNA-1 profile according to anti-CCP, SE status, and smoking status in patients
Anti-EBNA-1 levels were standardized in quartiles (1 = ,63.71 U/ml; 2 = 63.71-125.26 U/ml; 3 = 125.27-333.44 U/ml; 4= >333.45 U/ml). No significant differences were found in the distribution of anti-EBNA-1 antibodies according to anti-CCP positivity, SE status, or smoking status (Figure 1).
Anti-CCP profile according to SE status and smoking status in patients
Although it was not significant, there was a trend for patients with higher levels of anti-CCP to carry SE alleles (p = 0.059). Smokers had significantly higher levels of anti-CCP (p = 0.018). SE carriers and smoking patients had a significantly higher risk of being positive for anti-CCP (Figure 2, OR = 3.82; 95% CI = 1.28-11.40; p = 0.016).
Anti-EBNA1, anti-CCP, SE status, and smoking status in RA risk
The univariate logistic regression analysis showed that anti-CCP positivity carried a highly significant RA risk (p,0.001; OR = 247.49). The presence of one or two SE alleles increased the risk of RA 1.90-fold (p = 0.012) and 4.67-fold (p = 0.001), respectively. A smoking history of 15 or more pack-years led to a 3.17-fold increased risk of RA (p = 0.001). Anti-EBNA-1 levels were not found to be a risk factor for RA in this model.
In the multivariate logistic regression analysis with stepwise selection, anti-CCP was the only selected variable that was independently associated with RA.
There is increasing evidence of the roles of a number of environmental factors, including viruses exposure and smoking, in the pathogenesis of RA. EBV has been studied for many decades; however, it remains unclear how its interactions with other RA risk factors develop. In 1976, Alpsaugh and Tan observed a higher serologic reactivity to the nuclei of EBV-infected B cells in RA patients than in the controls.23 The target B cell antigen was named ''rheumatoid arthritis nuclear antigen'' (RANA). Reactivity to RANA was not observed in uninfected B cells and was not influenced by the HLA-DR phenotype or the presence of rheumatoid factor. Subsequently, it was established that RANA was, in fact, the same molecule as EBNA-1. The QKRAA sequence present in HLA-DRB1*0401 shares sequence homology with EBV gp110 protein, a target of the immune response to infection.24 Deblon et al. demonstrated that RA patients who carried the SE had a stronger humoral response to EBNA-1 than did SE-negative RA patients.25 These data are conflicting but suggest that EBV infection is related to the presence of HLA-DR molecules that contain the SE.
The anti-citrulline immune response is strongly involved in RA etiology, and anti-citrulline antibodies can precede RA development by several years. Anti-CCP antibodies are highly specific and are considered a valuable disease marker and a strong risk factor for RA. A gene-environment interaction among anti-CCP antibodies, SE status, and smoking has been observed in several studies. The presence of HLA-DR SE only confers a risk of RA to patients who are positive for anti-CCP.1,10,26,27 Smoking is associated with the increased presence of citrulline-modified proteins in genetically predisposed individuals who carry HLA-DR SE alleles.8-10 In this study, we did not perform the HLA-DRB1 subtyping to identify HLA-DR SE alleles, since Lundstrom et al. observed that interactions of SE status with anti-CCP antibodies and smoking were independent of HLA-DRB1 subtyping and occurred with *01, *04, and *10 groups, regardless of fine specificity.5
EBV antigens can also undergo citrullination, thus becoming targets of anti-CCP. Antibodies to citrullinated EBV peptides have been identified in sera from RA patients, and the viral-citrullinated-peptide antibody levels of these patients correlated with their anti-CCP levels.28 A relationship between EBV and citrullinated peptides was supported by the observation that anti-citrulline antibodies reacted with deiminated EBNA-1.29 Toussirot and Roudier suggested that EBV should be considered as an environmental factor associated with anti-CCP production, just as smoking is associated with anti-CCP development in RA patients.15
It remains unknown how EBV influences the risk of developing RA. Anti-EBNA-1 antibodies are increased in individuals who are exposed to EBV and remain increased for a lifetime. Multiple sclerosis (MS) is a good example of an autoimmune disease for which anti-EBNA-1 antibodies have been shown to be a strong risk factor. Interestingly, this risk is increased by smoking but is independent of the MS susceptibility HLA-DR15 allele.30,31 In the present study, we showed a high positivity of anti-EBNA-1 in both patients and controls, and there was no association between anti-EBNA-1 levels and anti-CCP levels, SE status, or smoking status in RA patients. The relative risk for RA was not increased in patients with higher levels of anti-EBNA-1. Thus, we have demonstrated that anti-EBNA-1 serology cannot evaluate the interactions between EBV and the most well-known RA risk factors. Further studies could assess the role of EBV in RA pathogenesis. The accurate quantification of EBV by real-time polymerase chain reaction and the evaluation of antibodies to viral citrullinated peptides are promising techniques that could aid in this process.28,32,33
Brazil has a heterogeneous population with a distinct genetic profile that has been influenced by various racial and ethnic groups, predominantly Africans and Europeans. As reported in more homogeneously Caucasian populations5-8, we showed that SE alleles and tobacco exposure are related to anti-CCP antibodies in RA patients. Several previous studies observed these same relationships in Brazilian RA patients.34,35
In this study, we demonstrated the contributions of anti-CCP antibodies, the presence of the SE, and smoking history to RA risk. However, through a multivariate logistic regression analysis, we showed of the risk factors, only anti-CCP antibodies were independently associated with RA. Anti-EBNA-1 antibodies were neither correlated with the other factors nor influenced disease risk, unlike in other autoimmune conditions, such as MS. Despite some supporting evidence, the role of EBV in the development of RA remains unclear and needs to be studied further. We emphasize the importance of establishing the role of EBV in conjunction withother factors involved in RA pathogenesis.
1. Klareskog L, Catrina AI, Paget S. Rheumatoid Arthritis. Lancet. 2009;373:659-72, doi: 10.1016/S0140-6736(09)60008-8. [ Links ]
2. Plenge RM. Recent progress in rheumatoid arthritis genetics. Curr Opin Rheumatol. 2009;21:262-71, doi: 10.1097/BOR.0b013e32832a2e2d. [ Links ]
3. Rantapaa-Dahlqvist S. What happens before onset of rheumatoid arthritis? Curr Opin Rheumatol. 2009;21:272-8, doi: 10.1097/BOR.0b013e32832a2e44. [ Links ]
4. Liao KP, Alferdsson L, Karlson EW. Environmental influences on risk for rheumatoidarthritis. CurrOpinRheumatol. 2009;21:279-83,doi: 10.1097/ BOR.0b013e32832a2e16. [ Links ]
5. Lündstrom E, Kallberg H, Alfredsson L, Klareskog L, Padyukov L. Gene-environment interaction between the DRB1 shared epitope and smoking in the risk of anti-citrullinated protein antibody-positive rheumatoid arthritis- All alleles are important. Arthritis Rheum. 2009;60:1597-603, doi: 10.1002/art.24572. [ Links ]
6. Karlson EW, Chang S-C, Cui J, Chibnik LB, Fraser PA, De Vivo I. Gene-environment interaction between HLA-DRB1 shared epitope and heavy cigarette smoking in predicting incident rheumatoid arthritis. Ann Rheum Dis. 2010;69:54-60, doi: 10.1136/ard.2008.102962. [ Links ]
7. Bang S-Y, Lee K-H, Cho S-K, Lee H-S, Lee KW, Bae S-C. Smoking increases rheumatoid arthritis susceptibility in individuals carrying the HLA-DRB1 shared epitope, regardless of rheumatoid factor or anti-cyclic citrullinated peptide antibody status. Arthritis Rheum. 2010;62:369-77. [ Links ]
8. Linn-Rasker SP, van der Helm-van Mil AHM, van Gaalen FA, Kloppenburg M, de Vries RRP, le Cessie S, et al. Smoking is a risk factor for anti-CCP antibodies only in rheumatoid arthritis patients who carry HLA-DRB1 shared epitope alleles. Ann rheum Dis. 2006; 65:366-71, doi: 10.1136/ard.2005.041079. [ Links ]
9. Baka Z, Buzas E, Nagy G. Rheumatoid arthritis and smoking: putting the pieces together. Arthritis Res Ter. 2009;11:238-51, doi: 10.1186/ar2751. [ Links ]
10. Klareskog L, Stolt P, Lundberg K, Kallberg H, Bengtsson C, Grunewald J, et al. A new model for an etiology of rheumatoid arthritis: smoking may trigger HLA-DR (shared epitope)- restricted immune reactions to autoantigens modified by citrullination. Arthritis Rheum. 2006;54:38-46, doi: 10.1002/art.21575. [ Links ]
11. Vaughan JH. Viruses and autoimmune disease. J Rheumatol. 1996;23:1831-3. [ Links ]
12. Vaughan JH. The Epstein-Barr virus in autoimmunity. Springer Semin Immunopathol. 1995;17:203-30, doi: 10.1007/BF00196166. [ Links ]
13. Toussirot E, Roudier J. Epstein-Barr virus in autoimmune diseases. Best Pract Res Clin Rheumatol. 2008;22:883-96, doi: 10.1016/j.berh.2008.09.007. [ Links ]
14. Costenbader KH, Karlson EW. Epstein-Barr virus and rheumatoid arthritis: is there a link? Arthritis Res Ther. 2006;8:204, doi: 10.1186/ar1893. [ Links ]
15. Toussirot E, Roudier J. Pathophysiological links between rheumatoid arthritis and the Epstein-Barr virus: an update. Joint Bone Spine. 2007;74:418-26, doi: 10.1016/j.jbspin.2007.05.001. [ Links ]
16. Inoue N, Harada S, Miyasaka N, Oya A, Yanagi K. Analysis of antibody titers to Epstein-Barr virus nuclear antigens in sera of patients with Sjogren's syndrome and with rheumatoid arthritis. J Infect Dis. 1991;164:22-8, doi: 10.1093/infdis/164.1.22. [ Links ]
17. Silverman SL, Schumacher HR. Antibodies to Epstein-Barr viral antigens in early rheumatoid arthritis. Arthritis Rheum. 1981; 24:1465-8, doi: 10.1002/art.1780241202. [ Links ]
18. Kouri T, Petersen J, Rhodes G, Aho K, Palusuo T, Heliövaara M, et al. Antibodies to synthetic peptides from Epstein-Barr nuclear antigen-1 in sera of patients with early rheumatoid arthritis and in preillness sera. J Rheumatol. 1990;17:1442-9. [ Links ]
19. Petersen J, Rhodes G, Roudier J, Vaughan JH. Altered immune response to glycine-rich sequences of Epstein-Barr nuclear antigen-1 in patients with rheumatoid arthritis and systemic lupus erythematosus. Arthritis Rheum. 1990;33:993-1000, doi: 10.1002/art.1780330711. [ Links ]
20. Fox R, Sportsman R, Rhodes G, Luka J, Pearson G, Vaughan J. Rheumatoid arthritis synovial membrane contains a 62,000-molecular-weight protein that shares an antigenic epitope with the Epstein-Barr virus encoded associated EBNA-1 antigen. J Clin Invest. 1986;77:1539-47, doi: 10.1172/JCI112469. [ Links ]
21. Baboonian C, Venables PJW, Williams DG, Williams RO, Maini RN. Cross-reaction of antibodies to a glycine/alanine repeat sequence of EpsteineBarr virus nuclear antigen-1 with collagen, cytokeratin, and actin. Ann Rheum Dis. 1991;50:772-5, doi: 10.1136/ard.50.11.772. [ Links ]
22. Kimura A, Sasazuki T. Eleventh International Histocompatibility Workshop reference protocol for the HLA DNA typing technique. In: Tsuji K, Aizawa M, Sasazuki T, eds. HLA 1991. Proceedings of the Eleventh International Histocompatibility Workshop and Conference,vol 1, Oxford: Oxford University Press. 1992:397-439. [ Links ]
23. Alpsaugh MA, Tan EM. Serum antibody in rheumatoid arthritis reactive with a cell- associated antigen. Demonstration by precipitation and immunofluorescence. Arthritis Rheum. 1976;19: 711-9,doi: 10.1002/1529-0131(197607/08)19:4<711::AID-ART1780190409>3.0.CO;2-I. [ Links ]
24. Roudier J, Petersen J, Rhodes JH, Luka J, Carson DA. Susceptibility to rheumatoid arthritis maps to a T-cell epitope shared by the HLA-Dw4 DR beta-1 chain and the Epstein-Barr virus glycoprotein gp110. Proc Natl Acad Sci USA. 1989;86:5104-8, doi: 10.1073/pnas.86.13.5104. [ Links ]
25. Deblon N, Toussirot E, Auger I, Roudier J, Roudier C. Les proteins EBNA-1 et gp110 du virus d'EpsteinBarr sont les cibles d'une forte reponse anticorps chez les patients atteints de polyarthrite rhumatoi'de qui expriment l'epitope partage. Rev Rhum. 2005;72:978-9. [ Links ]
26. Szodoray P, Szabo Z, Kapitany A, Gyetvai A, Lakos G, Szanto S, et al. Anti-citrullinated protein/peptide autoantibodies in association with genetic and environmental factors as indicators of disease outcome in rheumatoid arthritis. Autoimmun Rev. 2010;9:140-3, doi: 10.1016/j. autrev.2009.04.006. [ Links ]
27. van der Helm-van Mil AH, Verpoort KN, Breedveld FC, Huizinga TW, Toes RE, de Vries RR. The HLA-DRB1 shared epitope alleles are primarily a risk factor for anti-cyclic citrullinated peptide antibodies and are not an independent risk factor for development of rheumatoid arthritis. Arthritis Rheum. 2006;54:1117-21, doi: 10.1002/art.21739. [ Links ]
28. Anzilotti C, Merlini G, Pratesi F, Tommasi C, Chimenti D, Migliorini P. Antibodies to viral citrullinated peptide in rheumatoid arthritis. J Rheumatol. 2006;33:647-51. [ Links ]
29. Pratesi F, Tommasi C, Anzilotti C, Chimenti D, Migliorini P. Deiminated Epstein-Barr virus nuclear antigen 1 is a target of anti-citrullinated protein antibodies in rheumatoid arthritis. Arthritis Rheum. 2006;54:733-41, doi: 10.1002/art.21629. [ Links ]
30. Simon KC, van der Mei IA, Munger KL, Ponsonby A, Dickinson J, Dwyer T, et al. Combined effects of smoking, anti-EBNA antibodies, and HLA-DRB1*1501 on multiple sclerosis risk. Neurology. 2010;74:1365-71, doi: 10.1212/WNL.0b013e3181dad57e. [ Links ]
31. De Jager PL, Simon KC, Munger KL, Rioux JD, Hafler DA, Ascherio A. Integrating risk factors: HLA-DRB1*1501 and Epstein-Barr virus in multiple sclerosis. Neurology. 2008;25;70:1113-8. [ Links ]
32. Balandraud N, Meynard JB, Auger I, Sovran H, Mugneir B, Reviron D, et al. Epstein-Barr virus load in the peripheral blood of patients with rheumatoid arthritis. Arhtritis Rheum. 2003;48:1223-8, doi: 10.1002/art.10933. [ Links ]
33. Anzilotti C, Riente L, Pratesi F, Chimenti D, Delle Sedie A, Bombardieri S, et al. IgG, IgA, IgM antibodies to a viral citrullinated peptide in patients affected by rheumatoid arthritis, chronic arthritides and connective tissue disorders. Rheumatology (Oxford). 2007;46:1579-82, doi: 10.1093/rheumatology/kem193. [ Links ]
34. Louzada-Junior P, Freitas MV, Oliveira RD, Deghaide NH, Conde RA, Bertolo MB, et al. A majority of Brazilian patients with rheumatoid arthritis HLA-DRB1 alleles carry both the HLA-DRB1 shared epitope and anti-citrunillated peptide antibodies. Braz J Med Biol Res. 2008;41:493-9, doi: 10.1590/S0100-879X2008005000021. [ Links ]
35. Oliveira RD, Junta CM, Oliveira FR, Silva LM, Donadi EA, Louzada-Junior P. Share epitope, citrullinated cyclic peptide antibodies and smoking in Brazilian rheumatoid arthritis patients. Clin Rev Allergy Immunol. 2008;34:32-5, doi: 10.1007/s12016-007-8017-2. [ Links ]
Received for publication on May 11, 2011
First review completed on May 19, 2011
Accepted for publication on May 30, 2011
Tel.: 55 19 3521-7106