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Epstein-Barr virus etiology in rheumatoid synovitis
Autoimmunity Reviews 4 (2005) 106 – 110 www.elsevier.com/locate/autrev
Epstein-Barr virus etiology in rheumatoid synovitis Shigemasa Sawada*,1, Masami Takei1 Department of Rheumatology and Hematology, Nihon University School of Medicine, 30-kamimachi Oyaguchi, Tokyo, Japan Department of Medicine, Nerima Hikarigaoka Hospital, Nihon University School of Medicine, 2-11-1 Hikarigaoka Nerima-ku, Tokyo, Japan Accepted 10 August 2004 Available online 11 September 2004
Abstract The etiology of rheumatoid arthritis (RA) has remained unknown, although it has been investigated and speculated that both genetic and environmental components contribute to the cause of this disease. Epstein-Barr virus (EBV) has been a strong candidate about for over 25 years as environmental infectious agent(s). There are many circumstantial evidence for association between EBV and RA, but definite evidence is wanting. In present article, we review an increase circumstantial proof which has been investigated so far and demonstrate direct evidence for the presence of EBV in inflamed synovial cells in patients with RA and discuss on the recent finding of signaling lymphocytic-activation molecule (SLAM)-associated protein (SAP), which opened a new approach to understand on impaired function of cytotoxic T cell for EBV in patients with RA. D 2004 Elsevier B.V. All rights reserved. Keywords: Epstein-Barr virus; Rheumatoid synovitis; EBV-encoded small RNA (EBER); Signaling lymphocytic activation molecule (SLAM); SLAM-associated protein (SAP)
Contents 1. 2. 3.
Why, Epstein-Barr virus and rheumatoid synovitis . . . . . . . . . . . . . . . . . . . . . . . . . Evidence of Epstein-Barr virus in rheumatoid synovium . . . . . . . . . . . . . . . . . . . . . . Signaling lymphocytic activating molecule-associated protein (SAP) plays a crucial cytotoxic role Epstein-Barr virus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Corresponding author. Tel.: +81 3 3979 3611; fax: +81 3 3979 3868. E-mail address: [email protected] (S. Sawada). 1 Both authors equally contributed. 1568-9972/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2004.08.034
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S. Sawada, M. Takei / Autoimmunity Reviews 4 (2005) 106–110
1. Why, Epstein-Barr virus and rheumatoid synovitis The etiology of RA remains unclear, although the cause of RA has been attributed to several factors, such as genetic or environmental/infectious agents (bacteria, viruses) for over 25 years. In twin studies, Silman et al. reported that the concordance rate for RA in a nationwide study of 91 monozygotic pairs was 15%, which is lower than the 30% figure usually quoted from a study 30 years ago and sets a limit on the potential genetic role in RA etiology [1]. Furthermore, they investigated the genetic contribution to the clinical features of RA by comparing RA-concordant monozygotic twin pairs, which highlighted the importance of non-genetic factors in RA susceptibility [2]. These data indicate that genetics is not the sole component and that both genetic and environmental/infectious factor(s) play important roles in the etiopathogenesis of RA. The current theory for the cause of RA is the RA shared epitope hypothesis, which is based on the fact that RA is associated with HLA-DRh alleles containing the QKRAA amino acid sequence in their third hypervariable regions [3]. It was reported that RA patients have enhanced humoral and cellular immune responses to the EBV and to microorganisms that share the QKRAA sequence [4]. Immune reactivity to recombinant proteins encompassing the shared epitope was also seen in 22 monozygotic twin pairs discordant for RA, thus suggesting that either genetic or shared environmental/infectious agents were associated with the etiology of this disease. Antibodies against EBV increased in patients with RA [5] and antibodies against an antigen in the nucleus of EBV-transformed B cells, designated RAassociated nuclear antigen (RANA), are also present in patients [6]. RANA is identical to EBNA-1 [7]. The antibody against EBNA-1 reacts with a 62-kDa protein in the synovium of patients with RA [8], and there is substantial homology in the amino acid sequences of gp110, which is a component of the EBV capsid protein, and HLA-DR4 [9]. These results suggest that EBV has a strong etiologic role in RA. Furthermore, the number of infected peripheral B lymphocytes in RA patients tends to be higher than in normal individuals [10,11] and RA patients show an
107
impaired ability to generate EBV-specific cytotoxic T lymphocytes [11,12]. Taken together, the above results suggest that aberrant gene function and EBV may be present in patients with RA.
2. Evidence of Epstein-Barr virus in rheumatoid synovium Fox et al. demonstrated that a monoclonal antibody, selected for reactivity with the EBV-encoded antigen EBNA-1, exhibited strong reactivity with synovial lining cells in joint biopsies from 10 of 12 patients with RA as well as with the adherent cells eluted from these tissues. No staining of RA synovial membrane or eluted synovial-lining cells was observed with monoclonal antibodies directed against antigens encoded by cytomegalovirus, herpes simplex viruses or human T cell leukemia virus type I. Among 12 osteoarthritis and normal synovial biopsies, few reactive cells were noted. These results suggest that the inflamed synovial cells in patients with RA contain EBV [8]. However, evidence of EBV DNA in rheumatoid synovial cells could not be demonstrated by Southern blotting [13]. The failure to detect EBV DNA may be due to insufficient sensitivity. Using polymerase chain reaction (PCR), EBV DNA was detected and confirmed in the synovial tissue of RA, including the RNA of several latent and lytic EBV genes [14 –16] with also us, indicating that EBV is present in the RA synovial tissue. There were no differences in EBV gene expression between synovial tissues and peripheral blood when comparing RAwith osteoarthritis and other disease controls because PCR is not able to identify the source of amplified signals and is typically a qualitative, rather than quantitative, form of analysis. In order to identify of the cells in synovial tissue that EBV DNA was amplified from, we undertook the detection of EBV using in situ hybridization for the presence of EBV-encoded small RNA (EBER) in synovial cells and immunohistochemical staining for expression of CD21 molecules or latent membrane protein (LMP-1) and EBNA-2. EBER was identified in synovial cells and lymphocytes from RA patients (23.5%) but was not seen in any of the control synovial tissues (osteoarthritis and psoriasis). Interestingly, in some cases, we found that EBER was localized in the
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synovial lining cells that were located at the apex of the villus proliferating lesions. Furthermore, LMP-1 molecules were also detected in synovial cells. However, CD19, CD21 and EBNA-2 were not observed. The incidence of EBV-positive cells in synovial cells with severe lymphocyte infiltration tended to be higher than in cells with moderate infiltration [17]. This study was very carefully performed in order to confirm EBV infection in patients with RA. Considerable evidence has been accumulated regarding the presence of EBV in synovial cells and lymphocytes in patients with RA [17–19], with the exception of a study by Niedobitek et al. [20]. EBV infection of synovial cells still eludes us, because CD21, a receptor for EBV, could not be detected on the synovial membrane. Cell fusion with EBV-infected lymphocytes has been suggested to play a role in viral infection of non-lymphoid cells [21]. It has recently been proposed that pre-synovial stem cells are recruited into the joints from bone marrow. Patients with rheumatoid arthritis may therefore have EBV-infected pre-synovial stem cells in their marrow and these cells move to the synovial membranes in the joints (Fig. 1).
3. Signaling lymphocytic activating moleculeassociated protein (SAP) plays a crucial cytotoxic role in Epstein-Barr virus infection In 1996, we cloned several cDNA clones from patients with IgA nephritis. The function of those
Fig. 1. Impaired SAP function may involve EBV infection to synovial cells and synovitis in patients with RA.
cloned gene has since been unclear. It was recently reported that Signaling lymphocytic activating molecule (SLAM) is a cell surface molecule (CD150) to which SAP binds, thus initiating a new T-cell signal-transduction pathway via the coreceptor molecule SLAM. One of these cDNA clones from patients with IgA was identified as SAP. In T cells expressing SAP, SLAM–SLAM interactions trigger selective up-regulation of IL-4 secretion in response to antigen receptor stimulation [22]. Because defective SAP protein produced by a mutated SAP gene plays a crucial role in the pathogenesis of the inherited immunodeficiency X-linked lymphoproliferative syndrome (XLP) [23,24], we attempted to identify genes causing disturbances in the function of EBV cytotoxic T cells in RA patients, focusing particularly on the SAP encoded by the XLP gene. Using quantitative real-time PCR, the expression levels of SAP transcripts in peripheral T lymphocytes or leukocytes were examined. SAP transcript levels in peripheral leukocytes of RA patients were significantly lower than those of normal individuals, those of inactive systemic lupus erythematosus patients, and those of chronic renal disease patients [25]. Decreased SAP transcript levels in patients with RA were also observed in peripheral CD2 T lymphocytes when compared with normal individuals. Furthermore, we found that the nucleotide sequence of SAP cDNA did not possess any mutations or deletions in the coding region when compared with wild-type SAP cDNA [25]. The role of this decrease in SAP transcripts is uncertain. Genomic polymorphism in the promoter or enhancer region might be present in RA patients or certain cytokines may inhibit SAP mRNA expression. Because SAP primarily exists in T cells, disturbances in SAP mRNA expression would affect SLAM-induced signal-transduction events in T cells. RA patients have impaired IFN-g production by T cells, which indicative of Th2 type responses [26]. SLAM production during T cell activation induces IFN-g production and redirects Th2 type cells to Th1 type cells, and an inadequate response in RA patients would result from impaired function of the SLAM/ SAP pathway. An impaired SAP pathway may contribute to the failure to eliminate EBV-infected
S. Sawada, M. Takei / Autoimmunity Reviews 4 (2005) 106–110
cells by cytotoxic T lymphocytes or NK cells in patients with RA (Fig. 1). This hypothesis is supported by the results of abnormally elevated EBV-infected B cells [10,11] and defective EBV-suppressive T cells in patients with RA [11,12]. The role of decreased SAP transcripts in patients with RA is unclear. Abnormal promoter or enhancer genes may contribute to this event in patients with RA. We investigated nucleotide mutations in the SAP promoter region in patients with RA, and identified a single nucleotide mutation (manuscript in preparation). This single nucleotide mutation lead to the failure of the immune system to eliminate Epstein-Barr virus infected synovial cells.
4. Summary We are encouraged by Ollier, who stated that a causal link between EBV and RA cannot yet be supported, but it does seem increasingly likely that viruses, such as EBV, have a role in the progression or exacerbation of inflammatory responses within the RA joint, and if treatments can be developed that can limit or prevent reactivation of EBV, these may be beneficial for RA [27]. We believe that we are very close to identifying a link between EBV and RA. Take-home messages !
!
! !
!
Antibodies against Epstein-Barr virus (EBV) increased in patients with rheumatoid arthritis (RA). Number of infected peripheral B lymphocytes in RA patients tends to be higher than in normal individuals. Specific cytotoxic T lymphocytes against EBV show an impairment in patients with RA. EBV DNA was detected and confirmed in the synovial tissue in patients with RA by polymerase chain reaction. EBV gene and latent membrane protein were demonstrated and confirmed in synovial cells and lymphocytes in patients with RA by the methods of in situ hybridization and immunohistochemical staining.
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Decreased SAP transcript levels in patients with RA were observed in peripheral T lymphocytes when compared with normal individuals. An impaired SAP pathway may contribute to the failure to eliminate EBV-infected cells by cytotoxic T lymphocytes or NK cells in patients with RA.
References [1] MacGregor AJ, Bamber S, Carthy D, Venocovsky J, Mageed RA, Ollier WE, et al. Heterogeneity of disease phenotype in monozygotic twins concordant for rheumatoid arthritis. Br J Rheumatol 1995;34(3):215 – 20. [2] Silman AJ, MacGregor AJ, Thompson W, Holligan S, Carthy D, Farhan A, et al. Twin concordance rates for rheumatoid arthritis: results from nationwide study. Br J Rheumatol 1993;32(10):903 – 7. [3] Gregersen P, Silver J, Winchester R. The shared epitope hypothesis. An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum 1987;30:1205 – 13. [4] La Cava A, Lee Nelson J, Ollier WER, MacGregor A, Keystone EC, Thorne JC, et al. Genetic bias in immune responses to a cassette shared by different microorganisms in patients with rheumatoid arthritis. J Clin Invest 1977;100(3): 658 – 63. [5] Alspaugh MA, Henle G, Lennette ET, Henle W. Elevated levels of antibodies to Epstein-Barr virus antigens in sera and synovial fluids of patients with rheumatoid arthritis. J Clin Invest 1981;67(4):1134 – 40. [6] Billings PB, Hoch SO, White PJ, Carson DA, Vaughan JH. Antibodies to the Epstein-Barr virus nuclear antigen and to rheumatoid arthritis nuclear antigen identify the same polypeptide. Proc Natl Acad Sci U S A 1983;80(23): 7104 – 08. [7] Venables PJ, Pawlowski T, Mumford PA, Brown C, Crawford DH, Maini RN. Reaction of antibodies to rheumatoid arthritis nuclear antigen with a synthetic peptide corresponding to part of Epstein-Barr nuclear antigen. Ann Rheum Dis 1988;47(4):270 – 9. [8] Fox R, Sportsman R, Rhodes G, Luka J, Pearson G, Vaughan J. Rheumatoid arthritis synovial membrane contains a 62000molecular weight protein that shares an antigenic epitope with the Epstein-Barr virus encoded associated nuclear antigen. J Clin Invest 1986;77(5):1539 – 47. [9] Roudier J, Rhodes G, Petersen J, Vaughan JH, Carson DA. The Epstein-Barr virus glycoprotein gp110, a molecular link between HLA DR4. HLADR1 and rheumatoid arthritis. Scand J Immunol 1988;27(4):367 – 71. [10] Tosato G, Steinberg AD, Yarchoan R, Heilman CA, Pike SE, De-Seau V, et al. Abnormally elevated frequency of EpsteinBarr virus-infected B cells in the blood of patients with rheumatoid arthritis. J Clin Invest 1984;73(6):1789 – 95.
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[11] Balandraud N, Roudier J, Roudier C. Epstein-Barr virus and rheumatoid arthritis. Autoimmun Rev 2004;3(5):362 – 7. [12] Tosato G, Steinberg AD, Blaese RM. Defective EBV-specific suppressor T-cell function in rheumatoid arthritis. N Engl J Med 1981;305(21):1238 – 43. [13] Fox RI, Chilton T, Rhodes G, Vaughan JH. Lack of reactivity of rheumatoid arthritis synovial membrane DNA with cloned Epstein Barr virus DNA probes. J Immunol 1986;137(2): 498 – 501. [14] Newkirk MM, Watanabe DKH, Leclerc J, Lambert N, Shiroky JB. Detection of cytomegalovirus, Epstein-Barr virus and Herpes virus-6 in patients with rheumatoid arthritis with or without Sjogren’s syndrome. Br J Rheumatol 1994;33(4): 317 – 22. [15] Edinger JW, Bonneville M, Scotet E, Houssaint E, Schumacher HR, Posnett DN. EBV gene expression not altered in rheumatoid synovia despite the presence of EBV antigenspecific T cell clones. J Immunol 1999;162(6):3694 – 701. [16] Saal JG, Krimmel M, Steidle M, Gerneth F, Wagner S, Fritz P, et al. Synovial Epstein-Barr virus infection increases the risk of rheumatoid arthritis in individuals with the shared HLADR4 epitope. Arthritis Rheum 1999;42(7):1485 – 96. [17] Takei M, Mitamura K, Fujiwara S, Horie T, Tyu J, Osaka S, et al. Detection of Epstein-Barr virus encoded small RNA 1 and latent membrane protein 1 in synovial lining cells from rheumatoid arthritis patients. Int Immunol 1997;9(5):739 – 43. [18] Blaschke S, Schwarz G, Monke D, Binder L, Muller G, Reuss-Borst M. Epstein-Barr virus infection in peripheral blood mononuclear cells, synovial fluid cells and synovial membranes of patients with rheumatoid arthritis. J Rheumatol 2000;27(4):866 – 73. [19] Takeda T, Mizugaki Y, Matsubara L, Imai S, Koike T, Takada K. Lytic Epstein-Barr virus infection in the synovial tissue of
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
patients with rheumatoid arthritis. Arthritis Rheum 2000; 43(6):1218 – 25. Niedobitek G, Lisner R, Swoboda B, Rooney N, Fassbender HG, Kirchner T, et al. Lack of evidence for an involvement of Epstein-Barr virus infection of synovial membranes in the pathogenesis of rheumatoid arthritis. Arthritis Rheum 2000; 43(1):151 – 4. Timens W, Boes A, Vos H, Poppema S. Tissue distribution of the C3d/EBV-receptor: CD21 monoclonal antibodies reactive with a variety of epithelial cells, medullary thymocytes, and peripheral T-cells. Histochemistry 1991;95(6):605 – 11. Veillette A. SLAM family receptors regulate immunity with and without AP-related adaptors. J Exp Med 2004;199(9): 1175 – 78. Sayos J, Wu C, Morra M, Wang N, Zhang X, Allen D, et al. The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM. Nature 1998;395:462. Coffey AJ, Brooksbank RA, Brandau O, Oohashi T, Howell JM, Bye JM, et al. Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH-2domain encoding gene. Nat Genet 1998;20:129. Takei M, Ishwata T, Mitamura K, Fujiwara S, Sasaki K, Nishi T, et al. Decreased expression of signaling lymphocyticactivation molecule-associated protein (SAP) transcripts in T cells from patients with rheumatoid arthritis. Int Immunol 2001;13(4):559 – 65. Loubet-Lescoulie P, Constantin A, Mazieres B, Tkaxzuk J, de Prival C, Cantagrel A. Decreased peripheral blood T cell cytokine gene expression in rheumatid arthritis. Scand J Rheumatol 1999;28(4):244 – 51. Ollier W. Rheumatoid arthritis and Epsten-Barr virus: a case of living with the enemy? Ann Rheum Dis 2000;59(7):497 – 503.
Anti-peroxiredoxin I and IV autoantibodies Anti-oxidative enzymes are protective from various oxidative stresses. Karasawa et al. (Microbiol Immunol 2005;49:57) investigated whether autoimmunity to the anti-oxidative peroxiredoxin enzymes exists in patients having systemic autoimmune diseases. The authors found that 33% of 92 patients with autoimmune diseases had autoantibodies to peroxiredoxin I: 57% of systemic lupus erythematosus patients, 19% of rheumatoid arthritis, 5% of Behcet disease, and 46% of primary systemic vasculitides. Nonetheless, autoantibodies to peroxiredoxin IV were detected in only 17% of these patients. The presence of anti-peroxiredoxin I autoantibodies correlated with lower serum levels of CH50, C3, and C4. Based on their findings the authors suggest that anti- peroxiredoxin autoantibodies may be involved in the pathophysiology of systemic autoimmune diseases.
CD226 expression deficiency and apoptosis in NK T cells from lupus patients Tao et al. (J Immunol 2005;174:1281) report that NK T cells from active lupus patients are highly sensitive to anti-CD95-induced apoptosis compared with those from normal subjects and inactive lupus patients. They present data supporting that deficient expression of CD226 and survivin in NK T cells from active lupus patients is a molecular base of high sensitivity of the cells to anti-CD95-induced apoptosis.
Epstein-Barr virus etiology in rheumatoid synovitis Shigemasa Sawada*,1, Masami Takei1 Department of Rheumatology and Hematology, Nihon University School of Medicine, 30-kamimachi Oyaguchi, Tokyo, Japan Department of Medicine, Nerima Hikarigaoka Hospital, Nihon University School of Medicine, 2-11-1 Hikarigaoka Nerima-ku, Tokyo, Japan Accepted 10 August 2004 Available online 11 September 2004
Abstract The etiology of rheumatoid arthritis (RA) has remained unknown, although it has been investigated and speculated that both genetic and environmental components contribute to the cause of this disease. Epstein-Barr virus (EBV) has been a strong candidate about for over 25 years as environmental infectious agent(s). There are many circumstantial evidence for association between EBV and RA, but definite evidence is wanting. In present article, we review an increase circumstantial proof which has been investigated so far and demonstrate direct evidence for the presence of EBV in inflamed synovial cells in patients with RA and discuss on the recent finding of signaling lymphocytic-activation molecule (SLAM)-associated protein (SAP), which opened a new approach to understand on impaired function of cytotoxic T cell for EBV in patients with RA. D 2004 Elsevier B.V. All rights reserved. Keywords: Epstein-Barr virus; Rheumatoid synovitis; EBV-encoded small RNA (EBER); Signaling lymphocytic activation molecule (SLAM); SLAM-associated protein (SAP)
Contents 1. 2. 3.
Why, Epstein-Barr virus and rheumatoid synovitis . . . . . . . . . . . . . . . . . . . . . . . . . Evidence of Epstein-Barr virus in rheumatoid synovium . . . . . . . . . . . . . . . . . . . . . . Signaling lymphocytic activating molecule-associated protein (SAP) plays a crucial cytotoxic role Epstein-Barr virus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Corresponding author. Tel.: +81 3 3979 3611; fax: +81 3 3979 3868. E-mail address: [email protected] (S. Sawada). 1 Both authors equally contributed. 1568-9972/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2004.08.034
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S. Sawada, M. Takei / Autoimmunity Reviews 4 (2005) 106–110
1. Why, Epstein-Barr virus and rheumatoid synovitis The etiology of RA remains unclear, although the cause of RA has been attributed to several factors, such as genetic or environmental/infectious agents (bacteria, viruses) for over 25 years. In twin studies, Silman et al. reported that the concordance rate for RA in a nationwide study of 91 monozygotic pairs was 15%, which is lower than the 30% figure usually quoted from a study 30 years ago and sets a limit on the potential genetic role in RA etiology [1]. Furthermore, they investigated the genetic contribution to the clinical features of RA by comparing RA-concordant monozygotic twin pairs, which highlighted the importance of non-genetic factors in RA susceptibility [2]. These data indicate that genetics is not the sole component and that both genetic and environmental/infectious factor(s) play important roles in the etiopathogenesis of RA. The current theory for the cause of RA is the RA shared epitope hypothesis, which is based on the fact that RA is associated with HLA-DRh alleles containing the QKRAA amino acid sequence in their third hypervariable regions [3]. It was reported that RA patients have enhanced humoral and cellular immune responses to the EBV and to microorganisms that share the QKRAA sequence [4]. Immune reactivity to recombinant proteins encompassing the shared epitope was also seen in 22 monozygotic twin pairs discordant for RA, thus suggesting that either genetic or shared environmental/infectious agents were associated with the etiology of this disease. Antibodies against EBV increased in patients with RA [5] and antibodies against an antigen in the nucleus of EBV-transformed B cells, designated RAassociated nuclear antigen (RANA), are also present in patients [6]. RANA is identical to EBNA-1 [7]. The antibody against EBNA-1 reacts with a 62-kDa protein in the synovium of patients with RA [8], and there is substantial homology in the amino acid sequences of gp110, which is a component of the EBV capsid protein, and HLA-DR4 [9]. These results suggest that EBV has a strong etiologic role in RA. Furthermore, the number of infected peripheral B lymphocytes in RA patients tends to be higher than in normal individuals [10,11] and RA patients show an
107
impaired ability to generate EBV-specific cytotoxic T lymphocytes [11,12]. Taken together, the above results suggest that aberrant gene function and EBV may be present in patients with RA.
2. Evidence of Epstein-Barr virus in rheumatoid synovium Fox et al. demonstrated that a monoclonal antibody, selected for reactivity with the EBV-encoded antigen EBNA-1, exhibited strong reactivity with synovial lining cells in joint biopsies from 10 of 12 patients with RA as well as with the adherent cells eluted from these tissues. No staining of RA synovial membrane or eluted synovial-lining cells was observed with monoclonal antibodies directed against antigens encoded by cytomegalovirus, herpes simplex viruses or human T cell leukemia virus type I. Among 12 osteoarthritis and normal synovial biopsies, few reactive cells were noted. These results suggest that the inflamed synovial cells in patients with RA contain EBV [8]. However, evidence of EBV DNA in rheumatoid synovial cells could not be demonstrated by Southern blotting [13]. The failure to detect EBV DNA may be due to insufficient sensitivity. Using polymerase chain reaction (PCR), EBV DNA was detected and confirmed in the synovial tissue of RA, including the RNA of several latent and lytic EBV genes [14 –16] with also us, indicating that EBV is present in the RA synovial tissue. There were no differences in EBV gene expression between synovial tissues and peripheral blood when comparing RAwith osteoarthritis and other disease controls because PCR is not able to identify the source of amplified signals and is typically a qualitative, rather than quantitative, form of analysis. In order to identify of the cells in synovial tissue that EBV DNA was amplified from, we undertook the detection of EBV using in situ hybridization for the presence of EBV-encoded small RNA (EBER) in synovial cells and immunohistochemical staining for expression of CD21 molecules or latent membrane protein (LMP-1) and EBNA-2. EBER was identified in synovial cells and lymphocytes from RA patients (23.5%) but was not seen in any of the control synovial tissues (osteoarthritis and psoriasis). Interestingly, in some cases, we found that EBER was localized in the
108
S. Sawada, M. Takei / Autoimmunity Reviews 4 (2005) 106–110
synovial lining cells that were located at the apex of the villus proliferating lesions. Furthermore, LMP-1 molecules were also detected in synovial cells. However, CD19, CD21 and EBNA-2 were not observed. The incidence of EBV-positive cells in synovial cells with severe lymphocyte infiltration tended to be higher than in cells with moderate infiltration [17]. This study was very carefully performed in order to confirm EBV infection in patients with RA. Considerable evidence has been accumulated regarding the presence of EBV in synovial cells and lymphocytes in patients with RA [17–19], with the exception of a study by Niedobitek et al. [20]. EBV infection of synovial cells still eludes us, because CD21, a receptor for EBV, could not be detected on the synovial membrane. Cell fusion with EBV-infected lymphocytes has been suggested to play a role in viral infection of non-lymphoid cells [21]. It has recently been proposed that pre-synovial stem cells are recruited into the joints from bone marrow. Patients with rheumatoid arthritis may therefore have EBV-infected pre-synovial stem cells in their marrow and these cells move to the synovial membranes in the joints (Fig. 1).
3. Signaling lymphocytic activating moleculeassociated protein (SAP) plays a crucial cytotoxic role in Epstein-Barr virus infection In 1996, we cloned several cDNA clones from patients with IgA nephritis. The function of those
Fig. 1. Impaired SAP function may involve EBV infection to synovial cells and synovitis in patients with RA.
cloned gene has since been unclear. It was recently reported that Signaling lymphocytic activating molecule (SLAM) is a cell surface molecule (CD150) to which SAP binds, thus initiating a new T-cell signal-transduction pathway via the coreceptor molecule SLAM. One of these cDNA clones from patients with IgA was identified as SAP. In T cells expressing SAP, SLAM–SLAM interactions trigger selective up-regulation of IL-4 secretion in response to antigen receptor stimulation [22]. Because defective SAP protein produced by a mutated SAP gene plays a crucial role in the pathogenesis of the inherited immunodeficiency X-linked lymphoproliferative syndrome (XLP) [23,24], we attempted to identify genes causing disturbances in the function of EBV cytotoxic T cells in RA patients, focusing particularly on the SAP encoded by the XLP gene. Using quantitative real-time PCR, the expression levels of SAP transcripts in peripheral T lymphocytes or leukocytes were examined. SAP transcript levels in peripheral leukocytes of RA patients were significantly lower than those of normal individuals, those of inactive systemic lupus erythematosus patients, and those of chronic renal disease patients [25]. Decreased SAP transcript levels in patients with RA were also observed in peripheral CD2 T lymphocytes when compared with normal individuals. Furthermore, we found that the nucleotide sequence of SAP cDNA did not possess any mutations or deletions in the coding region when compared with wild-type SAP cDNA [25]. The role of this decrease in SAP transcripts is uncertain. Genomic polymorphism in the promoter or enhancer region might be present in RA patients or certain cytokines may inhibit SAP mRNA expression. Because SAP primarily exists in T cells, disturbances in SAP mRNA expression would affect SLAM-induced signal-transduction events in T cells. RA patients have impaired IFN-g production by T cells, which indicative of Th2 type responses [26]. SLAM production during T cell activation induces IFN-g production and redirects Th2 type cells to Th1 type cells, and an inadequate response in RA patients would result from impaired function of the SLAM/ SAP pathway. An impaired SAP pathway may contribute to the failure to eliminate EBV-infected
S. Sawada, M. Takei / Autoimmunity Reviews 4 (2005) 106–110
cells by cytotoxic T lymphocytes or NK cells in patients with RA (Fig. 1). This hypothesis is supported by the results of abnormally elevated EBV-infected B cells [10,11] and defective EBV-suppressive T cells in patients with RA [11,12]. The role of decreased SAP transcripts in patients with RA is unclear. Abnormal promoter or enhancer genes may contribute to this event in patients with RA. We investigated nucleotide mutations in the SAP promoter region in patients with RA, and identified a single nucleotide mutation (manuscript in preparation). This single nucleotide mutation lead to the failure of the immune system to eliminate Epstein-Barr virus infected synovial cells.
4. Summary We are encouraged by Ollier, who stated that a causal link between EBV and RA cannot yet be supported, but it does seem increasingly likely that viruses, such as EBV, have a role in the progression or exacerbation of inflammatory responses within the RA joint, and if treatments can be developed that can limit or prevent reactivation of EBV, these may be beneficial for RA [27]. We believe that we are very close to identifying a link between EBV and RA. Take-home messages !
!
! !
!
Antibodies against Epstein-Barr virus (EBV) increased in patients with rheumatoid arthritis (RA). Number of infected peripheral B lymphocytes in RA patients tends to be higher than in normal individuals. Specific cytotoxic T lymphocytes against EBV show an impairment in patients with RA. EBV DNA was detected and confirmed in the synovial tissue in patients with RA by polymerase chain reaction. EBV gene and latent membrane protein were demonstrated and confirmed in synovial cells and lymphocytes in patients with RA by the methods of in situ hybridization and immunohistochemical staining.
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Decreased SAP transcript levels in patients with RA were observed in peripheral T lymphocytes when compared with normal individuals. An impaired SAP pathway may contribute to the failure to eliminate EBV-infected cells by cytotoxic T lymphocytes or NK cells in patients with RA.
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Anti-peroxiredoxin I and IV autoantibodies Anti-oxidative enzymes are protective from various oxidative stresses. Karasawa et al. (Microbiol Immunol 2005;49:57) investigated whether autoimmunity to the anti-oxidative peroxiredoxin enzymes exists in patients having systemic autoimmune diseases. The authors found that 33% of 92 patients with autoimmune diseases had autoantibodies to peroxiredoxin I: 57% of systemic lupus erythematosus patients, 19% of rheumatoid arthritis, 5% of Behcet disease, and 46% of primary systemic vasculitides. Nonetheless, autoantibodies to peroxiredoxin IV were detected in only 17% of these patients. The presence of anti-peroxiredoxin I autoantibodies correlated with lower serum levels of CH50, C3, and C4. Based on their findings the authors suggest that anti- peroxiredoxin autoantibodies may be involved in the pathophysiology of systemic autoimmune diseases.
CD226 expression deficiency and apoptosis in NK T cells from lupus patients Tao et al. (J Immunol 2005;174:1281) report that NK T cells from active lupus patients are highly sensitive to anti-CD95-induced apoptosis compared with those from normal subjects and inactive lupus patients. They present data supporting that deficient expression of CD226 and survivin in NK T cells from active lupus patients is a molecular base of high sensitivity of the cells to anti-CD95-induced apoptosis.