Secondary Logo

Journal Logo

Association between serum nitric oxide and Epstein–Barr virus in patients with systemic lupus erythematosus: a case–control study

Elsaied, Moustafa A.; Abdel Aziz, Abeer M.; Mesbah, Abeer; Aldiasty, Amani; Hegazy, Asmaa; Fathy, Amal

Journal of the Egyptian Women's Dermatologic Society: May 2016 - Volume 13 - Issue 2 - p 106–110
doi: 10.1097/01.EWX.0000481051.27875.10
Original articles
Free

Background Epstein–Barr virus (EBV) infection may act as an environmental trigger for induction of systemic lupus erythematosus (SLE), and nitric oxide (NO) mediates many different cell functions at sites of inflammation.

Objective To explore the relative frequencies of EBV and the associations between serum NO, a proinflammatory cytokine causing vasodilatation, oedema, cytotoxicity and tissue destruction, and EBV in SLE patients.

Patients and methods Thirty-eight SLE adult patients and 32 healthy controls were included in this case–control study. SLE activity was assessed by systemic lupus erythematosus disease activity index (SLEDAI). EBV immunoglobulin (Ig) G and IgM antibodies, real-time PCR for EBV (EBV-DNA) and NO levels were evaluated for all participants.

Results Twenty-seven (71.1%) SLE patients were positive for EBV-DNA, with a significantly higher median level (41.75 IU/ml) compared with controls (2.5 IU/ml). Frequency of EBV IgG antibody was significantly higher in SLE patients than in controls [37/38 (97.4%) vs. 26/32 (81.25%), P=0.001]. All SLE patients and controls were negative for EBV IgM antibody. EBV IgG (median) was significantly higher in SLE patients than in controls (26.25 vs. 11.35 IU/ml). EBV-DNA-positive patients showed significantly higher SLEDAI than EBV-DNA-negative patients (31.59±25.4 vs. 15.73±24.85, respectively). Serum NO was significantly higher in SLE patients than in controls (median 65.5 vs. 17 μmol/l). NO level was significantly higher in positive EBV-DNA patients than in negative ones (81 vs. 58 μmol/l).

Conclusion Adult SLE patients had higher frequencies of EBV-DNA, EBV IgG and NO levels compared with healthy controls. Exposure to EBV could be associated with the increased activity of SLE (higher SLEDAI) rather than the development of the disease, and the increased NO could be a mediator of this.

Departments of aDermatology

bClinical Pathology

cInternal Medicine (Rheumatology & Immunology)

dChest Medicine, Faculty of Medicine, Mansoura University, Mansoura, Egypt

Correspondence to Moustafa A. Elsaied, MD, Department of Dermatology, Faculty of Medicine, Mansoura University, Mansoura, Egypt Tel: +20 101 950 9962; fax: +20 502 231 191; e-mail: maelsaied@mans.edu.eg

Received May 11, 2015

Accepted November 24, 2015

Back to Top | Article Outline

Introduction

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by widespread immune dysregulation with hyperproduction of numerous autoantibodies and immune complexes, resulting in chronic systemic inflammation and potential damage to multiple organs 1. SLE occurs when an environmental trigger induces an immunological dysfunction in a genetically predisposed individual, leading to the loss of tolerance towards native proteins 2. Among the environmental triggers, viruses including Epstein–Barr virus (EBV), cytomegalovirus, parvovirus B19 and human endogenous retroviruses were postulated 3–6. Multiple investigators have suggested a strong association between previous EBV infection and SLE among children and adults in different populations 4,7–10.

Immune activation by a putative viral superantigen, epitope spreading, antigenic mimicry, polyclonal B-cell activation and viral reactivation with immune suppression are hypothesized to be the possible factors contributing to the role of EBV in SLE pathogenesis 11.

The production of nitric oxide (NO) plays a vital role in the regulation of physiological processes, host defence, inflammation and immunity. The proinflammatory effects of NO include vasodilation, oedema, cytotoxicity and cytokine-dependent tissue destruction. NO-dependent tissue injury has been implicated in a variety of rheumatic diseases, including SLE, rheumatoid arthritis and osteoarthritis 12. In addition, NO inhibits reactivation of EBV in the infected epithelium, leading to EBV latency 13.

The increased NO production in SLE patients could be attributed to activated endothelial cells and keratinocytes through the upregulation of inducible NO synthase 14. The aim of this study was to explore the relative frequencies of EBV (antigen and antibodies) and NO level in adult SLE patients and their correlation with systemic lupus erythematosus disease activity index (SLEDAI).

Back to Top | Article Outline

Patients and methods

This case–control study included 38 consecutive patients with SLE from Mansoura University Hospitals. All patients fulfilled the 1997 American College of Rheumatology revised criteria for the classification of SLE 15. Patients with autoimmune disease such as rheumatoid arthritis, dermatomyositis and thyroiditis as well as pregnant women were excluded.

The study was conducted from January 2014 to December 2014.

Thirty-two healthy age-matched and sex-matched persons were included as a control group. Every patient and control provided written informed consent, and the Research Ethics Committee for experimental and clinical studies at Faculty of Medicine, Mansoura University, approved the study.

Disease activity was assessed by SLEDAI, a validated clinical activity test that scores a weighted index of nine organ systems, including central nervous, vascular, renal, musculoskeletal, serosal, dermal, immunologic, constitutional and haematological systems. The range of possible SLEDAI scores is from 0 to 105 16.

Data on patients’ treatments, including use of immunosuppressive drugs (azathioprine, methotrexate and cyclophosphamide), hydroxychloroquine and the dose of steroids (prednisolone mg/day), were reported.

All patients provided 6 ml venous blood, which was measured out into two tubes: one EDTA tube for plasma EBV-DNA and another plain tube for serum: EBV immunoglobulin (Ig) G, EBV IgM antibodies and NO level. Samples were transferred as soon as possible for separation. Plasma and serum were frozen at −20°C until analysis.

Back to Top | Article Outline

EBV-DNA

EDTA plasma was used for purification of DNA from samples using the INSTANT Virus DNA Kit (cat no.: 845-KS-4150050; AJ Innuescreen, Jena, Germany). Steps of purification included lysis, binding, washing and finally elution of DNA into elution tubes.

Reverse transcription-PCR was used for detection of EBV-DNA by means of the RoboGene-EBV Quantification Kit intended for use with ABI PRISM 7300SDS (cat no.: 027300304; AJ Roboscreen GmbH, Leipzig, Germany) 17. The quantification standard consists of eight tubes coated with the given amount of synthetic EBV-DNA, which were amplified, in parallel with samples.

The amplification was associated with generation of a fluorescence signal measurable in FAM channel resulting in a sigmoid growth curve. EBV-DNA was determined on the basis of the threshold cycle (Ct) values for the samples resulting from analysis of quantitation standards. Forty cycles were obtained, and included Taq activation at 45°C for 10 min, melting at 45°C for 30 min and stem formation, annealing, synthesis and fluorescence detection (FAM) at 59°C for 1:30 min.

The EBV-DNA quantification kit is designed for in-vitro quantification of EBV genomes by means of the gene coding for the nuclear antigen [Epstein–Barr virus nuclear antigen-1 (EBNA-1)]. The kit components included EBV-D4, which is a reagent mix lyophilized with EBV/internal control, specific primers, probes and dNTPs. Positive and negative controls were included in each run for EBV-DNA detection. The measuring range is from 10 to 1 000 000 IU/ml. Results below 10 IU/ml were considered negative.

EBV IgG was detected with SERION ELISA classic EBV EBNA-1 IgG (cat no.: ESR 1362G; Institut Virion\Serion GmbH, Würzburg, Germany) 18.

Back to Top | Article Outline

Detection of EBV IgM

EBV IgM was detected using the SERION ELISA classic EBV VCA IgM Kit (cat no.: ESR1361M; Institut Virion\Serion GmbH, Würzburg, Germany) 19.

NO assay was carried out for all patients using Griess reaction with the Thermo Scientific NO Kit (cat no.: EMSNO, Thermo Scientific, Waltham, Massachusetts, USA). This NO assay kit is for quantitative determination of nitrite (NO2) and nitrate (NO3). The kit uses the enzyme nitrate reductase to convert nitrate to nitrite. Nitrite is then detected as a coloured azo dye product of the Griess reaction that absorbs visible light at 540 nm. The sensitivity of the kit is up to 0.222 μmol/l for nitrite and 0.625 μmol/l for nitrate 20.

Back to Top | Article Outline

Statistical analysis

Statistical analyses were carried out using SPSS for Windows, release 20 (SPSS Inc., Chicago, Illinois, USA). Quantitative data were presented as mean±SD or as median and range, and qualitative data were presented as frequency and percentage. The χ2 and Fisher’s exact tests were used to determine the relationship between qualitative data. Quantitative data were compared with the Mann–Whitney U-test. P values less than 0.05 were considered statistically significant.

Back to Top | Article Outline

Results

Thirty-eight SLE patients (36 female and two male) with a mean age of 28.5±9.4 years and disease duration of 5.49±5.1 years were included in this study.

The control group included 32 healthy participants (30 female and two male) with a mean age of 29.4±10.5 years. Table 1 shows the clinical data of the patients.

Table 1

Table 1

There were no significant differences between the two groups regarding sex and age (P=0.86 and 0.06, respectively).

EBV-DNA was positive in 27/38 (71.1%) SLE patients but in none of the controls.

EBV-DNA was significantly higher in SLE patients than in controls (P<0.001, Table 2).

Table 2

Table 2

All patients with SLE and controls were negative for EBV IgM antibody. The frequency of EBV IgG antibody and its serum levels were significantly higher in SLE patients than in controls.

NO level was significantly higher in SLE patients compared with controls (P<0.001, Table 2).

No significant differences of age and sex were found between positive and negative EBV-DNA patients. Disease duration was significantly longer in EBV-DNA-negative patients compared with positive ones (P=0.03, Table 3). EBV-DNA-positive patients showed higher SLEDAI and serositis compared with EBV-DNA-negative patients (P=0.05, Table 3).

Table 3

Table 3

No significant differences were found between the EBV-DNA-positive and EBV-DNA-negative groups regarding steroid dose and the frequency of use of other immunosuppressive drugs (P>0.05, Table 3). EBV IgG antibody was higher but not statistically significant in EBV-DNA-positive SLE patients compared with negative ones. NO level was significantly high in EBV-DNA-positive SLE patients compared with the negative group (P=0.004, Table 3). SLEDAI showed significant positive correlation with EBV-DNA, EBV IgG antibody and NO levels (Table 4).

Table 4

Table 4

Back to Top | Article Outline

Discussion

SLE patients with positive EBV-DNA (71.1%) showed significantly high SLEDAI compared with negative patients. This indicated significant organ involvement with increased disease severity and organ damage in EBV-DNA-positive SLE patients. The high prevalence of positive IgG antibodies against EBV in our patients (97.4%) is comparable to that of other published studies from different populations 8,21–25. James et al.22 reported a prevalence of 99.6% in their patients associated with higher SLEDAI. The prevalence of IgG antibody to EBV in healthy controls in our study was lower than that of other studies in different countries (82.25 vs. 94.7% 22; 95% 21). Mohamed et al.10 in his Egyptian study in a different governorate showed a slightly higher prevalence than that in our study of EBV IgG antibody in both SLE patients and controls (100 vs. 97.4% and 83.3 vs. 81.25%, respectively). The variability of EBV seroprevalence in different countries and in different governorates in Egypt may explain the differences in these results. In contrast to previous studies, Barzilai et al.26 found no statistically significant increase in EBNA-1 IgG titres in their SLE patients. Also, recently Hanlon et al.3 in their meta-analysis of controlled studies, found no statistically significant association of SLE with anti-EBNA-1 in spite of a higher proportion of anti-EBNA-1-positive lupus cases than controls (92.5 and 84.9%, respectively).

The negative EBV IgM antibody in both SLE patients and controls in our study is in agreement with the findings of Mohamed et al.10. The discrepancy between negative results of IgM and PCR results could be explained by the altered T-cell responses with defective control of latent EBV infection in SLE patients 27. Furthermore, Maurmann et al.28 reported that the EBV viral load together with viremia occurred more frequently as opposed to serological reactivation in healthy carriers, suggesting a different kinetics of serology and virologic markers to EBV. In addition, an earlier report by Kimura et al.29 indicated that the presence of EBV genomes does not always indicate an active EBV infection in healthy individuals with latent infection. Finally, the replication of EBV in the absence of an effective immune response is central to the pathogenesis of the disease 30. Therefore, the increased IgG reactivity to EBV might reflect a chronic state of EBV infection, whereas the low IgM reactivity may be due to a defective immune response 31.

Our SLE patients showed higher positive EBV-DNA than that reported by Mohamed et al.10 and Lu et al.32 (71.1 vs. 51.5 and 42%, respectively). This result supported Kang et al.27 and Moon et al.33, who found a 40-fold and more than 15-fold increase in EBV-DNA load in the peripheral blood mononuclear cells of SLE patients compared with healthy controls in American and Korean patients, respectively.

The higher SLEDAI score in the EBV-DNA-positive group compared with the EBV-DNA-negative group was in contrast with the findings of Mohamed et al.10, who found significantly low SLEDAI score in EBV-DNA-positive patients. Also, Zandman-Goddard et al.34 found that infection with EBV might be associated with a milder disease phenotype. The discrepancy between studies could be explained by altered immune response to EBV infection in different patients according to different EBV gene expressions 35,36. Peripheral blood mononuclear cells from SLE patients had greater expression of latent genes as well as increased expression of both latent and lytic genes after infection 37. However, a large cohort study of SLE patients is needed to validate this assumption in clinical practice. In spite of the nonsignificant difference of steroid dose between EBV-DNA-positive and EBV-DNA-negative groups, we could not exclude the effect of the immunosuppressive therapy. However, Babcock et al.37 and Gross et al.31 found the same frequency of EBV-infected cells in SLE patients irrespective of the treatment with immunosuppressive agents.

In our study, the higher NO level in SLE patients compared with controls and in positive EBV-DNA compared with negative EBV-DNA was associated with more viremia and more disease activity (SLEDAI). These results were in agreement with many studies measuring either NO 14,38,39 or its marker (serum nitrate plus nitrite) 40. Also, Nagy et al.41 found that T cells and monocytes of SLE patients produce more NO compared with controls. These results supported the role of elevated levels of reactive nitrogen species such as NO and peroxynitrite in the pathogenesis of SLE by alteration of proteins leading to the development of autoantibodies 42.

Back to Top | Article Outline

Conclusion

In a subset of adult SLE patients, the exposure to EBV infection (high frequencies of EBV-DNA and high EBV IgG) could be associated with the increased activity of SLE (higher SLEDAI) rather than the development of the disease, and the increased NO could be a mediator for this.

Prospective studies with larger sample sizes and a long follow-up period that would allow analysis of the relative timing of infection and the development of SLE are still needed to elucidate the role of EBV and NO in SLE pathogenesis.

Back to Top | Article Outline

Acknowledgements

Conflicts of interest

There are no conflicts of interest.

Back to Top | Article Outline

References

1. Habibi S, Saleem MA, Ramanan AV. Juvenile systemic lupus erythematosus: review of clinical features and management. Indian Pediatr 2011; 48:879–887.
2. Rigante D, Mazzoni MB, Esposito S. The cryptic interplay between systemic lupus erythematosus and infections. Autoimmun Rev 2014; 13:96–102.
3. Hanlon P, Avenell A, Aucott L, Vickers MA. Systematic review and meta-analysis of the sero-epidemiological association between Epstein–Barr virus and systemic lupus erythematosus. Arthritis Res Ther 2014; 16:R3.
4. Berkun Y, Zandman-Goddard G, Barzilai O, Boaz M, Sherer Y, Larida B, et al.. Infectious antibodies in systemic lupus erythematosus patients. Lupus 2009; 18:1129–1135.
5. Medrano San Ildefonso MM, Mauri Llerda JA. Human parvovirus B19 and juvenile systemic lupus erythematosus. Clin Exp Rheumatol 2004; 22:504–505.
6. Blank M, Shoenfeld Y, Perl A. Cross-talk of the environment with the host genome and the immune system through endogenous retroviruses in systemic lupus erythematosus. Lupus 2009; 18:1136–1143.
7. Chen CJ, Lin KH, Lin SC, Tsai WC, Yen JH, Chang SJ, et al.. High prevalence of immunoglobulin A antibody against Epstein–Barr virus capsid antigen in adult patients with lupus with disease flare: case control studies. J Rheumatol 2005; 32:44–47.
8. Esen BA, Yilmaz G, Uzun S, Ozdamar M, Aksozek A, Kamali S, et al.. Serologic response to Epstein–Barr virus antigens in patients with systemic lupus erythematosus: a controlled study. Rheumatol Int 2012; 32:79–83.
9. Harley JB, James JA. Epstein–Barr virus infection may be an environmental risk factor for systemic lupus erythematosus in children and teenagers. Arthritis Rheum 1999; 42:1782–1783.
10. Mohamed AE, Hasen AM, Mohammed GF, Elmaraghy NN. Real-time PCR of cytomegalovirus and Epstein–Barr virus in adult Egyptian patients with systemic lupus erythematosus. Int J Rheum Dis 2013; 18:452–458.
11. Lossius A, Johansen JN, Torkildsen O, Vartdal F, Holmoy T. Epstein–Barr virus in systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis-association and causation. Viruses 2012; 4:3701–3730.
12. Abramson SB, Amin AR, Clancy RM, Attur M. The role of nitric oxide in tissue destruction. Best Pract Res Clin Rheumatol 2001; 15:831–845.
13. Gao X, Tajima M, Sairenji T. Nitric oxide down-regulates Epstein–Barr virus reactivation in epithelial cell lines. Virology 1999; 258:375–381.
14. Belmont HM, Levartovsky D, Goel A, Amin A, Giorno R, Rediske J, et al.. Increased nitric oxide production accompanied by the up-regulation of inducible nitric oxide synthase in vascular endothelium from patients with systemic lupus erythematosus. Arthritis Rheum 1997; 40:1810–1816.
15. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997; 40:1725.
16. Bombardier C, Gladman DD, Urowitz MB, Caron D, Chang CH. Derivation of the SLEDAI. A disease activity index for lupus patients. The Committee on Prognosis Studies in SLE. Arthritis Rheum 1992; 35:630–640.
17. Murray PG, Young LS. Epstein–Barr virus infection: basis of malignancy and potential for therapy. Expert Rev Mol Med 2001; 3:1–20.
18. Aalto SM, Linnavuori K, Peltola H, Vuori E, Weissbrich B, Schubert J, et al.. Immunoreactivation of Epstein–Barr virus due to cytomegalovirus primary infection. J Med Virol 1998; 56:186–191.
19. Bauer G. Simplicity through complexity: immunoblot with recombinant antigens as the new gold standard in Epstein–Barr virus serology. Clin Lab 2001; 47:223–230.
20. Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 2001; 5:62–71.
21. James JA, Neas BR, Moser KL, Hall T, Bruner GR, Sestak AL, et al.. Systemic lupus erythematosus in adults is associated with previous Epstein–Barr virus exposure. Arthritis Rheum 2001; 44:1122–1126.
22. James JA, Kaufman KM, Farris AD, Taylor-Albert E, Lehman TJ, Harley JB. An increased prevalence of Epstein–Barr virus infection in young patients suggests a possible etiology for systemic lupus erythematosus. J Clin Invest 1997; 100:3019–3026.
23. Parks CG, Cooper GS, Hudson LL, Dooley MA, Treadwell EL St, Clair EW, et al.. Association of Epstein–Barr virus with systemic lupus erythematosus: effect modification by race, age, and cytotoxic T lymphocyte-associated antigen 4 genotype. Arthritis Rheum 2005; 52:1148–1159.
24. Yu SF, Wu HC, Tsai WC, Yen JH, Chiang W, Yuo CY, et al.. Detecting Epstein–Barr virus DNA from peripheral blood mononuclear cells in adult patients with systemic lupus erythematosus in Taiwan. Med Microbiol Immunol 2005; 194:115–120.
25. Kasapcopur O, Ergul Y, Kutlug S, Candan C, Camcioglu Y, Arisoy N. Systemic lupus erythematosus due to Epstein–Barr virus or Epstein–Barr virus infection provoking acute exacerbation of systemic lupus erythematosus? Rheumatol Int 2006; 26:765–767.
26. Barzilai O, Sherer Y, Ram M, Izhaky D, Anaya JM, Shoenfeld Y. Epstein–Barr virus and cytomegalovirus in autoimmune diseases: are they truly notorious? A preliminary report. Ann N Y Acad Sci 2007; 1108:567–577.
27. Kang I, Quan T, Nolasco H, Park SH, Hong MS, Crouch J, et al.. Defective control of latent Epstein–Barr virus infection in systemic lupus erythematosus. J Immunol 2004; 172:1287–1294.
28. Maurmann S, Fricke L, Wagner HJ, Schlenke P, Hennig H, Steinhoff J, Jabs WJ. Molecular parameters for precise diagnosis of asymptomatic Epstein–Barr virus reactivation in healthy carriers. J Clin Microbiol 2003; 41:5419–5428.
29. Kimura H, Morita M, Yabuta Y, Kuzushima K, Kato K, Kojima S, et al.. Quantitative analysis of Epstein–Barr virus load by using a real-time PCR assay. J Clin Microbiol 1999; 37:132–136.
30. Ning S. Innate immune modulation in EBV infection. Herpesviridae 2011; 2:1.
31. Gross AJ, Hochberg D, Rand WM, Thorley-Lawson DA. EBV and systemic lupus erythematosus: a new perspective. J Immunol 2005; 174:6599–6607.
32. Lu JJ, Chen DY, Hsieh CW, Lan JL, Lin FJ, Lin SH. Association of Epstein–Barr virus infection with systemic lupus erythematosus in Taiwan. Lupus 2007; 16:168–175.
33. Moon UY, Park SJ, Oh ST, Kim WU, Park SH, Lee SH, et al.. Patients with systemic lupus erythematosus have abnormally elevated Epstein–Barr virus load in blood. Arthritis Res Ther 2004; 6:R295–R302.
34. Zandman-Goddard G, Berkun Y, Barzilai O, Boaz M, Blank M, Ram M, et al.. Exposure to Epstein–Barr virus infection is associated with mild systemic lupus erythematosus disease. Ann N Y Acad Sci 2009; 1173:658–663.
35. McClain MT, Poole BD, Bruner BF, Kaufman KM, Harley JB, James JA. An altered immune response to Epstein–Barr nuclear antigen 1 in pediatric systemic lupus erythematosus. Arthritis Rheum 2006; 54:360–368.
36. Poole BD, Templeton AK, Guthridge JM, Brown EJ, Harley JB, James JA. Aberrant Epstein–Barr viral infection in systemic lupus erythematosus. Autoimmun Rev 2009; 8:337–342.
37. Babcock GJ, Decker LL, Freeman RB, Thorley-Lawson DA. Epstein–Barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients. J Exp Med 1999; 190:567–576.
38. Ho CY, Wong CK, Li EK, Tam LS, Lam CW. Elevated plasma concentrations of nitric oxide, soluble thrombomodulin and soluble vascular cell adhesion molecule-1 in patients with systemic lupus erythematosus. Rheumatology (Oxford) 2003; 42:117–122.
39. Gilkeson G, Cannon C, Oates J, Reilly C, Goldman D, Petri M. Correlation of serum measures of nitric oxide production with lupus disease activity. J Rheumatol 1999; 26:318–324.
40. Oates JC, Shaftman SR, Self SE, Gilkeson GS. Association of serum nitrate and nitrite levels with longitudinal assessments of disease activity and damage in systemic lupus erythematosus and lupus nephritis. Arthritis Rheum 2008; 58:263–272.
41. Nagy G, Koncz A, Telarico T, Fernandez D, Ersek B, Buzas E, Perl A. Central role of nitric oxide in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus. Arthritis Res Ther 2010; 12:210.
42. Ahmad R, Ahsan H. Role of peroxynitrite-modified biomolecules in the etiopathogenesis of systemic lupus erythematosus. Clin Exp Med 2014; 14:1–11.
Keywords:

Epstein–Barr virus; nitric oxide; real-time PCR; systemic lupus erythematosus

© 2016 Egyptian Women's Dermatologic Society