Elevated Antibody Titers to Epstein-Barr Virus and Cytomegalovirus in Patients with Drug-Induced Lupus.

Systemic lupus erythematosus (SLE) is an autoimmune disease, which has been associated with Epstein-Barr virus (EBV) and Cytomegalovirus (CMV) infection. Drug-induced lupus (DIL) is a lupus-like disease caused by the intake of therapeutic drugs, which has been estimated to cause approximately 10-15% of lupus-like cases. Although SLE and DIL share common clinical symptoms, there are some fundamental differences between DIL and SLE onset. Moreover, it remains to be examined whether environmental factors, such as EBV and CMV infections, may contribute to the development of DIL. This study focused on examining the possible association between DIL and EBV and CMV infections, by examining IgG titers to EBV and CMV antigens in serum samples by enzyme-linked immunosorbent assays. Antibody titers to EBV early antigen-diffuse and CMV pp52 were found to be significantly elevated in both SLE and DIL patients compared to healthy controls, although no correlation was found for antibodies to the two virus antigens in the respective disease groups. Moreover, total IgG titers were reduced in SLE and DIL serum samples, which may reflect a general lymphocytopenia, which commonly is associated with SLE. The current findings support that EBV and CMV infections may contribute to the development of DIL and that onset of both diseases are related.

Human herpesvirus infections and circulating microvesicles expressing galectin-3 binding protein in patients with systemic lupus erythematosus.

OBJECTIVES: Circulating microvesicles (MVs) expressing the type 1 interferon-inducible protein galectin-3 binding protein (G3BP) are potentially major sources of autoantigens in systemic lupus erythematosus (SLE). In this study, we explore if plasma concentrations of G3BP-expressing MVs correlate with signs of various active human herpesvirus (HHV) infections in SLE patients, suggesting a virus-induced mechanism for the generation of these vesicles. METHODS: In 49 SLE patients, the plasma levels of immunoglobulin G (IgG) against cytomegalovirus (CMV) pp52, Epstein-Barr virus (EBV) early antigen diffuse (EA/D), and HHV6 p41 were measured by ELISAs and used as humoral markers of ongoing/recently active viral infection. MVs in platelet-poor plasma were quantified and characterised by flow cytometry, with regard to the binding of Annexin V (AnxV) and the expression of G3BP. Spearman's rho and the Wilcoxon rank-sum test were applied for associative evaluation of virus serology with MV subsets, and clinical and demographic data. RESULTS: The CMV pp52-directed antibodies correlated positively with the high G3BP-expressing MVs; either low (rho=0.4, p-value=0.005) or high (rho=0.37, p-value=0.01) in AnxV-expression. Furthermore, these MV subsets were higher in individuals with high and low IgG levels against CMV pp52 and EBV EA/D, respectively, relative to subjects with low and high IgG levels against these HHV antigens. Importantly, none of the associations were explained by immunosuppressants or antimalarials. CONCLUSIONS: Ongoing/recently active CMV infection is associated with circulating MVs expressing G3BP in SLE patients, supporting a link between specific viral infections and potentially pathogenic MVs in SLE.

Altered Antibody Response to Epstein-Barr Virus in Patients With Rheumatoid Arthritis and Healthy Subjects Predisposed to the Disease. A Twin Study.

Objectives: To study Epstein-Barr virus (EBV) antibody patterns in twin individuals with rheumatoid arthritis (RA) and their healthy co-twins, and to determine the heritability of antibody responses against the EBV encoded EBNA1 protein. Methods: Isotypes of EBNA1 antibodies were measured in 137 RA affected- and 150 healthy twin pairs. We estimated the effect of RA and RA predisposition, anti-citrullinated antibodies (ACPA), IgM rheumatoid factor (RF), the shared epitope (SE) and the PTPN22-T allele (PTPN22) on the level of EBNA1 antibodies. We also determined the heritability of EBNA1 antibody levels. Results: IgA-EBNA1 antibody levels were increased in twins from RA discordant twin pairs irrespective of RA, ACPA or IgM-RF status. The IgG-EBNA1 antibody level was elevated in healthy co-twins from RA discordant twin pairs but not in RA affected twins. The IgM-EBNA1 antibody level was elevated in both RA twins and their healthy co-twins. The effect of RA on the IgA-EBNA1 antibody level was reversed when SE was present and with no effect of PTPN22. The heritability of IgA-, IgG- and IgM-EBNA1 antibody level was 40.6, 65.5, and 54.3%, with no effect of environment shared by the twins. Conclusion: EBNA1 antibody levels are distinctively different between patients with RA and healthy subjects but also between relatives of RA strongly predisposed to RA and healthy subjects. The high level of IgA EBNA1 antibodies associated with RA and a family predisposition to RA is attributable to both genetics incl. the shared epitope and environmental variation.

Increased antibody levels to stage-specific Epstein-Barr virus antigens in systemic autoimmune diseases reveal a common pathology.

The immune responses to antigens from different stages of the Epstein-Barr virus (EBV) life cycle were investigated in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren's syndrome (SS), and systemic sclerosis (SSc) to gain knowledge of EBV's involvement in the etiology of systemic autoimmune diseases (SADs) and for an overview of the humoral immune responses against EBV. Investigations were performed by the use of ELISA. IgM, IgA, and IgG antibody binding to 11 EBV antigens: EBNA1, EBNA2, BALF5, EAD, BALF2, EA/R, VCA p18, VCA p23, gB, gp350, and gp42 were examined in serum pools from SAD patients and healthy controls (HCs). Increased antibody levels against the 11 EBV antigens in the SAD pools were seen compared to the HC pool. Specifically, SLE was characterized by strongly increased IgA to EAD both compared to HCs and other SADs, and RA was characterized by increased IgM levels to several EBV antigens. The SADs may be partly distinguished by their differential immune responses to various antigens in the EBV life cycle. All together, these findings support an association between EBV infection and SADs.

Immune responses to an early lytic cytomegalovirus antigen in systemic lupus erythematosus patients: T-cell responses, cytokine secretions and antibody status.

We investigated immune responses to a lytic cytomegalovirus antigen (CMVpp52), and to a lytic human herpes virus (HHV) 6 antigen (HHV6p41), in systemic lupus erythematosus (SLE) patients and healthy controls (HCs), in order to clarify if the previously established impaired responses to Epstein-Barr virus (EBV) in SLE patients is a general defect in their responses against (all) HHVs. Multiplex Luminex technology results showed a normal induction of five quantified cytokines (interferon γ, interleukin(IL)12, IL17, IL10, and tumor necrosis factor α) in SLE patients compared to HCs upon stimulation with CMVpp52 and HHV6p41. However, flow cytometric results showed a reduced upregulation of the activation marker CD69 on T-cells from SLE patients (n = 17) compared to HCs (n = 17) upon stimulation with CMVpp52, indicating limited or defective CMVpp52-specific T-cells and/or poor antigen-presentation in SLE patients, and thereby possibly decreased control of the CMV infection. In conclusion, the dysfunctional immune response against EBV previously established in SLE patients does not seem to apply to the same degree regarding the immune responses against CMV or HHV6. Results designate that the main contributing HHV agent in development or exacerbation of SLE (in genetically predisposed individuals) is the previously determined uncontrolled EBV infection, and to a lesser extent CMV infection, and probably with no involvement of HHV6 infection.

Impaired Cytokine Responses to Epstein-Barr Virus Antigens in Systemic Lupus Erythematosus Patients.

We analyzed cytokine responses against latent and lytic Epstein-Barr virus (EBV) antigens in systemic lupus erythematosus (SLE) patients and healthy controls (HCs) to obtain an overview of the distinctive immune regulatory response in SLE patients and to expand the previously determined impaired EBV-directed T-cell response. The concentrations of 14 cytokines (IL2, IL4, IL5, IL6, IL10, IL12, IL17, IL18, IL1ß, IFNγ, TNFα, TNFß, TGFß, and GM-CSF) were quantified upon stimulation of whole blood with latent state antigen EBNA1, lytic cycle antigen EBV-EA/D, and the superantigen SEB. To avoid results affected by lack of lymphocytes, we focused on SLE patients with normal levels. Decreased induction of IL12, IFNγ, IL17, and IL6 upon EBNA1 stimulation and that of IFNγ, IL6, TNFß, IL1ß, and GM-CSF upon EBV-EA/D stimulation were detected in SLE patients compared to HCs. IFNγ responses, especially, were shown to be reduced. Induction of several cytokines was furthermore impaired in SLE patients upon SEB stimulation, but no difference was observed in basic levels. Results substantiate the previously proposed impaired regulation of the immune response against latent and lytic cycle EBV infection in SLE patients without lymphopenia. Furthermore, results indicate general dysfunction of leukocytes and their cytokine regulations in SLE patients.

How compelling are the data for Epstein-Barr virus being a trigger for systemic lupus and other autoimmune diseases?

PURPOSE OF REVIEW: Systemic lupus erythematosus (SLE) is caused by a combination of genetic and acquired immunodeficiencies and environmental factors including infections. An association with Epstein-Barr virus (EBV) has been established by numerous studies over the past decades. Here, we review recent experimental studies on EBV, and present our integrated theory of SLE development. RECENT FINDINGS: SLE patients have dysfunctional control of EBV infection resulting in frequent reactivations and disease progression. These comprise impaired functions of EBV-specific T-cells with an inverse correlation to disease activity and elevated serum levels of antibodies against lytic cycle EBV antigens. The presence of EBV proteins in renal tissue from SLE patients with nephritis suggests direct involvement of EBV in SLE development. As expected for patients with immunodeficiencies, studies reveal that SLE patients show dysfunctional responses to other viruses as well. An association with EBV infection has also been demonstrated for other autoimmune diseases, including Sjögren's syndrome, rheumatoid arthritis, and multiple sclerosis. SUMMARY: Collectively, the interplay between an impaired immune system and the cumulative effects of EBV and other viruses results in frequent reactivation of EBV and enhanced cell death, causing development of SLE and concomitant autoreactivities.

Elevated Concentrations of Serum Immunoglobulin Free Light Chains in Systemic Lupus Erythematosus Patients in Relation to Disease Activity, Inflammatory Status, B Cell Activity and Epstein-Barr Virus Antibodies.

OBJECTIVE: In this study, we examined the concentration of serum immunoglobulin free light chains (FLCs) in systemic lupus erythematosus (SLE) patients and investigated its association with various disease parameters in order to evaluate the role of FLCs as a potential biomarker in SLE. Furthermore, FLCs' association with Epstein-Barr virus (EBV) antibodies was examined. METHODS: Using a nephelometric assay, κFLC and λFLC concentrations were quantified in sera from 45 SLE patients and 40 healthy controls. SLE patients with renal insufficiency were excluded in order to preclude high concentrations of serum FLCs due to decreased clearance. RESULTS: Serum FLC concentrations were significantly elevated in SLE patients compared to healthy controls (p<0.0001) also after adjusting for Ig levels (p<0.0001). The concentration of serum FLCs correlated with a global disease activity (SLE disease activity index (SLEDAI)) score of the SLE patients (r = 0.399, p = 0.007). Furthermore, concentrations of FLCs correlated with titers of dsDNA antibodies (r = 0.383, p = 0.009), and FLC levels and SLEDAI scores correlated in the anti-dsDNA-positive SLE patients, but not in anti-dsDNA-negative SLE patients. Total immunoglobulin (IgG and IgA) concentrations correlated with FLC concentrations and elevated FLC levels were additionally shown to associate with the inflammatory marker C-reactive protein and also with complement consumption determined by low C4 in SLE patients. Collectively, results indicated that elevated serum FLCs reflects increased B cell activity in relation to inflammation. SLE patients had an increased seropositivity of EBV-directed antibodies that did not associate with elevated FLC concentrations. An explanation for this could be that serum FLC concentrations reflect the current EBV activity (reactivation) whereas EBV-directed antibodies reflect the extent of previous infection/reactivations. CONCLUSION: SLE patients have elevated concentrations of serum FLCs that correlate with global disease activity scores and especially serologic markers for active disease. These findings are suggestive of circulating FLCs having potential as a new supplementary serologic biomarker in SLE.

Isotypes of Epstein-Barr virus antibodies in rheumatoid arthritis: association with rheumatoid factors and citrulline-dependent antibodies.

In order to study the humoral immune response against Epstein-Barr virus (EBV) in patients with rheumatoid arthritis (RA) and to compare it with the two major autoantibody types in RA, plasma samples from 77 RA patients, 28 patients with systemic lupus erythematosus (SLE), and 28 healthy controls (HCs) were investigated by enzyme-linked immunosorbent assays (ELISA). Increased percentages of positives and concentrations of IgG/IgA/IgM antibodies against the latent EBV nuclear antigen-1 (EBNA-1) were observed in RA patients compared to SLE patients and HCs. Increased concentrations and percentages of positives of IgG/IgA/IgM against the early lytic EBV antigen diffuse (EAD) were also found in RA patients compared to HCs but were highest in SLE patients. Furthermore, associations between the elevated EBNA-1 IgA and EBNA-1 IgM levels and the presence of IgM and IgA rheumatoid factors (RFs) and anti-citrullinated protein antibodies (ACPAs, IgG) and between elevated IgA concentrations against EAD and the presence of RFs and ACPAs in RA patients were found. Thus, RA patients had elevated antibodies of all isotypes characteristic of latent EBV infection (whereas SLE patients had elevated antibodies characteristic of lytic EBV infection). Notably, for IgM and IgA (but not IgG), these were associated with the presence of characteristic RA autoantibodies.

Epstein-Barr virus in systemic autoimmune diseases.

Systemic autoimmune diseases (SADs) are a group of connective tissue diseases with diverse, yet overlapping, symptoms and autoantibody development. The etiology behind SADs is not fully elucidated, but a number of genetic and environmental factors are known to influence the incidence of SADs. Recent findings link dysregulation of Epstein-Barr virus (EBV) with SAD development. EBV causes a persistent infection with a tight latency programme in memory B-cells, which enables evasion of the immune defence. A number of immune escape mechanisms and immune-modulating proteins have been described for EBV. These immune modulating functions make EBV a good candidate for initiation of autoimmune diseases and exacerbation of disease progression. This review focuses on systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjögren's syndrome (SS) and sum up the existing data linking EBV with these diseases including elevated titres of EBV antibodies, reduced T-cell defence against EBV, and elevated EBV viral load. Together, these data suggest that uncontrolled EBV infection can develop diverse autoreactivities in genetic susceptible individuals with different manifestations depending on the genetic background and the site of reactivation.

Epstein-Barr virus and systemic lupus erythematosus.

The etiology of SLE is not fully established. SLE is a disease with periods of waning disease activity and intermittent flares. This fits well in theory to a latent virus infection, which occasionally switches to lytic cycle, and EBV infection has for long been suspected to be involved. This paper reviews EBV immunobiology and how this is related to SLE pathogenesis by illustrating uncontrolled reactivation of EBV as a disease mechanism for SLE. Studies on EBV in SLE patients show enlarged viral load, abnormal expression of viral lytic genes, impaired EBV-specific T-cell response, and increased levels of EBV-directed antibodies. These results suggest a role for reactivation of EBV infection in SLE. The increased level of EBV antibodies especially comprises an elevated titre of IgA antibodies, and the total number of EBV-reacting antibody isotypes is also enlarged. As EBV is known to be controlled by cell-mediated immunity, the reduced EBV-specific T-cell response in SLE patients may result in defective control of EBV causing frequent reactivation and expression of lytic cycle antigens. This gives rise to enhanced apoptosis and amplified cellular waste load resulting in activation of an immune response and development of EBV-directed antibodies and autoantibodies to cellular antigens.