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Systemic Lupus Erythematosus — is a Viral Aetiology a Credible Hypothesis?
Journal of Infection (2000) 40, 229–233 doi:10.1053/jinf.2000.0670, available online at http://www.idealibrary.com on
LEADING ARTICLE
Systemic Lupus Erythematosus – is a Viral Aetiology a Credible Hypothesis? A. M. Denman* Northwick Park and St Mark’s NHS Trust, Watford Road, Harrow, Middlesex HA1 3UJ, U.K.
Systemic lupus erythematosus (SLE) is still regarded as a paradigm for autoimmune diseases, an Everest which if conquered would allow the solution of other unsolved problems in autoimmunity. Thus, interest in this issue is not confined to physicians and medical scientists who deal directly with patients with this disorder. The seach for viral causes of SLE has continued despite many decades of largely fruitless endeavour. Indeed, interest in this possibility has been sustained mainly by the discovery of the many mechanisms in natural infections and model systems by which persistently infective viruses are able to evade elimination by host immune defences and to stimulate autoimmune phenomena or disease.1,2 Of particular relevance to the immunopathogenesis of SLE are those viral infections which induce the characteristic spectrum of anti-nuclear and related autoantibodies and also lesions related to immune complex deposition and cytokine-induced inflammation resembling those characteristic of human SLE. For example, viral infections reveal cryptic auto-antigens to the immune system, thereby allowing tolerance to these antigens to be broken.3 The inflammatory reaction accompanying many viral infections generates high local concentrations of pro-inflammatory cytokines with subsequent autoantibody production.4,5 Such findings mirror the many situations in which transgenic manipulation of constitutive cytokine production is followed by autoimmune disorders. More simplistically, in clinical medicine parvovirus B19 infection is a telling example of a situation in which an ubiquitous agent induces acute clinical features and immunological abnormalities closely resembling SLE, often over a period of many months.6 However, analogy is no substitute for firm observations and by itself cannot be expected to sustain interest in this
*Address correspondence to: M. Denman, Gaters, Old Shire Lane, Chorleywood, Hertfordshire WD3 5PW, UK. Accepted for publication 22 March 2000.
0163-4453/00/030229]04 $35.00/0
field indefinitely. Fortunately for protagonists of a viral aetiology for SLE, other observations have shown how occult infection can continue to have immunopathological consequences, often only in the long term. Thus, T cells may remain ignorant of persistent CNS infection by Borna disease virus until T cells are stimulated by peripheral expression of the viral nucleoprotein in a later, systemic infection, thereby stimulating an immune response to the previously tolerated local infection.7 This results in inflammatory disease. Viruses such as lymphocytic choriomeningitis virus (LCM) may persist in very low, barely detectable levels even in the face of intact immune defences.8 Lyme arthritis vividly illustrates the difficulty which may be encountered in detecting microbial persistence in inflammatory disease even when the causative agent is known. The spirochaete Borrelia burgdorferi responsible for this disease can rarely be isolated from the joints of patients who continue to display chronic arthritis and even spirochaetal DNA is undetectable in these joints.9 It can be argued, therefore, that absence of an obvious provoking infection or a series of negative findings do not negate the viral hypothesis. Despite these difficulties, some viruses have been specifically implicated in the immunopathogenesis of SLE. For candidate agents to be persuasive causes of the disease, they must account for its most prominent features. By convention, the diagnosis of SLE can only be established if the patient displays at least four of 11 specified criteria.10 In reality, the nosological justification for this selection may eventually prove rather arbitrary. This is especially true of the autoantibodies included in these criteria, which are for the most part encountered in many other disorders. This is an important point in attempts to associate viral infection with SLE overall. It is very likely that this diagnostic term of convenience embraces many different disorders which will not prove to have a single cause. Genetic predisposition to SLE is well recognized, of which the most striking illustration is the 25%
© 2000 The British Infection Society
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concordance in monozygotic compared with dizygotic twins.11 The most clear-cut genetic association is with the complement coding genes; at least 50% of individuals with C2 deficiency develop SLE or a closely related disorder.12 However, this deficiency is as likely to signify impaired clearance of immune complexes irrespective of their composition as a deficient host response to infectious agents. This interpretation is supported by the effects of targeted deletion of the gene encoding serum amyloid protein (SAP). This protein binds to chromatin released from dying, apoptotic cells. Mice with this deletion develop glomerulonephritis and anti-nuclear antibodies, both characteristic features of SLE.13 The significance of the association between SLE and other polymorphic genes remains uncertain, but it is probable that these gene families influence the nature of a wide range of specific immune and non-specific inflammatory responses to infectious agents. Traditional searches for a viral aetiology in SLE have depended on attempts to isolate candidate viruses from patients and on anti-viral antibody titres. Direct isolation has not produced any consistent findings. Furthermore, SLE is a disease with impaired immunity affecting T-cell function, antibody responses, and non-specific immunity. Thus, successful viral isolation may merely reflect opportunistic infection of an immunocompromised patient. This is especially true of infection by agents such as herpes viruses which persistently infect individuals with essentially normal immune defences. The interpretation of antibody titres is complicated by the polyclonal B cell activation characteristic of this disorder. Thus, raised anti-microbial antibody titres do not necessarily indicate a response to conventional infection. Analysis of V gene sequences encoding the equally characteristic autoantibodies shows rearrangement and hypermutation in response to persistent antigen stimulation. As yet, similar studies on the genes encoding anti-viral antibodies have not been reported, which might in principle suggest that some raised anti-viral antibody titres in SLE are driven by continued viral stimulation. However, this would be a difficult undertaking given the ubiquity of the viruses in question and the need for appropriate controls. Nevertheless, it should not be automatically assumed that agents encountered in patients with disturbed immunity are invariably aetiologically irrelevant. There are many such ambiguous situations which may prove relevant to SLE. A pertinent example is EBV infection, which is almost universally acquired, usually causes trivial or self-limited illness after primary infection, but can also cause lymphoproliferative disease in genetically susceptible individuals.14 Even seemingly harmless viruses such as
the TT found in 50% of some populations may eventually prove to have disease associations.15 The situation is more complicated when a virus present in a high percentage of normal individuals is found in higher copy numbers in diseased tissues. An intriguing example is the ubiquitous polyoma virus JC virus, which is specifically associated with colo-rectal cancer cells.16 Although such observations can readily be dismissed by assuming that passenger viruses or their genes will predictably be found more frequently in malignant than in normal cells, one has to be more circumspect when the virus in question, in this instance JC virus, can cause chromosomal changes characteristic of the malignant cells. There is reasonable evidence that certain specific viral infections may be relevant in SLE. Furthermore, there is additional evidence that the infections could account for at least some of the defining characteristics of the disease. One such virus is polyoma virus. Bredholt and colleagues detected antibodies to the large T antigen in 55% of SLE sera compared with only one of the 20 control sera which were tested. Moreover, there was a strong correlation between anti-T antigen antibodies and autoantibodies to single-stranded DNA. The authors suggest that T antigen bound to nucleosomes may act as a hapten and thereby trigger anti-DNA autoantibodies.17 The remarkable association between hepatitis C virus (HCV) infection and mixed cryoglobulinaemia, often in association with vasculitis and with other systemic autoimmune diseases,18 has encouraged the suspicion that infection by this agent might also account for SLE at least in some patients. However, this has proved to be an unlikely explanation. The incidence of anti-HCV antibodies is not increased in SLE patients.19 Claimed increases in the incidence of anti-HCV antibodies are misleading because of the frequency of false positive results. In the majority of SLE patients who appear to display these antibodies, HCV viraemia cannot be detected by polymerase chain reaction (PCR).20 Despite these negative findings, the ability of such a widely prevalent virus to cause autoimmune disease is a fillip for investigators who regard this as a model with broader implications. Firstly, the association between HCV infection and mixed cryoglobulinaemia emerged by screening patients for this infection; hitherto it had not been detected by even the most sophisticated immunochemical analysis of the complexes. Secondly, there is experimental evidence that an HCVderived core protein epitope induced cytotoxic T-cells which cross-react with cytochrome P450.21 This autoreactivity may contribute to the immunopathogenesis of chronic active hepatitis in persistently infected individuals.22 Indeed, this property of HCV is persuasive support
Systemic Lupus Erythematosus for the general theory that viral infections induce autoimmune disease through molecular mimicry.23 There are further hints that common viral infections may be relevant to the aetiology of SLE. The determining role of cytomegalovirus (CMV) infection in the development of chronic graft versus host (GvH) disease in recipients of bone marrow transplants has long been recognized. This disease has many features in common with SLE and related autoimmune disorders, and indeed animal models of chronic GvH disease are good models of spontaneous SLE. Patients with chronic GvH disease, especially in severe form, develop antibodies to the CD13 molecule expressed on the surface of many populations of peripheral blood mononuclear cells. This molecule is incorporated into the surface envelope of newly synthesized CMV virions and may thereby form an autoantigenic target.24 Analysis of the antigen specificity of the anti-La autoantibodies found in SLE and other systemic autoimmune diseases shows that these antibodies react with protein sequences related to human herpes viruses, providing another possible example of molecular mimicry.25 Even influenza viruses, arguably the commonest cause of virus infections, cannot be exonerated, since influenza viral haemagglutinins are potent B cell activators and hence able to induce autoantibody production.26 For valid reasons the possible aetiological importance of retroviruses in SLE has attracted increasing interest. Firstly, known retroviral infections induce autoantibodies or overt autoimmune disease. These may be associated with immunodeficiency disease (HIV infections) or lymphoproliferative diseases (HTLV-1 infections). Secondly, even if conventional person-to-person (horizontal) infection by retroviruses proves irrelevant, endogenous retroviruses may still prove to be important in the disease process. Moreover, genetically transmitted (vertical) retroviral infection in this form would be consistent with the epidemiological evidence which emphasises the importance of genetic factors. Finally, the ability of endogenous retroviral sequences to influence the expression of unrelated, contiguous or distant genes provides a plausible explanation for the mixed picture of inappropriate immunological activation and suppression so characteristic of SLE. There are few reports identifying complete, potentially infectious retroviral particles in SLE patients. However, syncytial cells and retroviral particles were encountered in the bronchoalveolar lavage fluid of a patient with interstitial pneumonia complicating SLE.27 Clinical and laboratory abnormalities characteristic of SLE are encountered in only a small percentage of patients with unequivocal HTLV-1 infection. Nevertheless, the immunopathological abnormalities encountered in these patients are sufficiently
231
impressive to encourage the belief that a closely related agent could be operating in other SLE patients.28 Human retrovirus 5 (HRV-5) was found in the peripheral blood cells of 15% of patients with SLE and also 53% of synovial membrane samples obtained from arthritic joints. It was not found in blood samples from patients with other autoimmune diseases, and in only one of 200 control tissues. The invariable presence of an open reading frame and the extent of sequence variation in different samples suggest that HRV-5 in these patients was an actively replicating virus and not an endogenous retroviral sequence.29 The relatively low incidence of positive findings in the SLE patients studied in this report does not necessarily indicate that HRV-5 is a passenger virus of unlikely relevance to the pathogenesis of the disease. It can be rationally argued that a range of agents including different retroviruses rather than a single agent is more likely to account for so pleomorphic a disorder. Most of the arguments favouring a role for endogenous retroviruses (HERV) depend on the potential effects of these sequences and on the findings in animal models of autoimmune disease.30,31 However, many groups have obtained serological data showing that sera from a high percentage of SLE patients react with protein sequences related to HERV. Initial studies revealed an increased frequency of antibodies to two peptides encoded by the env gene of the endogenous retroviruses ERV-9 and HERV-H in SLE sera compared with sera from normal controls.32 Similar studies showed that nearly 50% of SLE sera react with a p30 gag protein derived from HERV. These experiments used recombinant proteins as well as synthetic peptide sequences as the target antigens.33 A recent investigation found that one-third of sera from SLE patients reacted with the p24 core protein antigen of HIV even though there was no other evidence that these patients were HIV-infected.34 These results are suggestive but still problematical in technical and interpretative terms. In vitro antibody studies dependent on linear peptide antigens take no account of in vivo protein conformation. As yet there is little information about the physical properties of anti-HERV antibodies, and in particular information concerning the strength of their binding affinity. There is also a hint that anti-HERV titres in some patients vary with disease activity, but it is not known whether antibody affinity increases with disease duration, as one would expect if HERV sequences were a major source of antigenic stimulation. Inhibition studies with gp120 peptides indicate that the p24 driven response in some autoimmune diseases may be an oligoclonal response against a more non-specific polyclonal background35: this observation increases the likelihood that the response is specifically
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anti-HERV rather than a chance cross-reaction, but detailed information is lacking. The observed linkage between polymorphic genotypes of the HERV HRES-136 and susceptibility to SLE introduces a further complexity, since there may be linkage disequilibrium between a limited number of HERV genotypes and immune response genes. If retroviruses are involved in the immunopathogenesis of SLE, the evidence to date favours a role for HERV rather than horizontal infection by fully infectious retroviruses. However, this conclusion introduces further difficulties in interpretation. It is possible that HERV sequences are peculiarly vulnerable to inappropriate activation or mutation induced by agents such as viral infection or UV irradiation. There is an intriguing proposal that inappropriate presentation of retroviral antigens by HLA antigens after excessive cell damage is a major stimulus to autoimmune disease.37 However, this special pleading for the particular importance of HERV in such circumstances is still speculative. Should the classic hypothesis be correct that SLE results from a fundamental and generalized loss of tolerance, there would be no reason to suppose that HERV encoded antigens would be any more or less likely to stimulate autoantibody responses than other autoantigens. Currently there is much circumstantial evidence implicating a range of viral infections in the aetiology of SLE in genetically susceptible individuals. The link with complement deficiency and other defects in handling immune complexes gives the second part of this statement scientific credibility. Indeed, it is possible that the primary insult is increased apoptosis secondary to a variety of viral infections and that SLE results from autoantibodies induced by the excessive burden of nucleosomes released by dying cells in individuals with such defects.38 If a more specific aetiological role for one or more viruses is to be more firmly established, it is clear that new diagnostic criteria will have to be devised in this as in other chronic diseases of unknown cause. It would take unprecedented good fortune to isolate such agents in fully replicating form or to identify virus-derived proteins and diagnostic anti-viral antibodies in the time honoured manner. Any successful approach is likely to be a painstaking exercise which would need to generate three crucially important pieces of information. Firstly, the detection of viral sequences in the normal host cell genome with the potential to influence normal genes encoding immune responses and inflammatory reactions. Secondly, an analysis of the proteins synthesised by the affected cells, namely an application of the formidably complicated study of normal cells newly defined as proteomics.39 Finally, a detailed analysis of the immune response to the relevant gene sequences and the products these encode. Alas, “to ask the hard question is simple”40; finding the answers in this field is a quest scarcely begun.
References 01. Whitton JL, Fujinami RS. Viruses as triggers of autoimmunity: facts and fantasies. Curr Opin Microbiol 1999; 2: 392–397. 02. Aichele P, Bachmann MF, Hengartner H, Zinkernagel RM. Immunopathology or organ-specific autoimmunity as a consequence of virus infection. Immunologic Rev 1996; 152: 21–45. 03. Barnaba V. Viruses, hidden self-epitopes, and autoimmunity. Immunologic Rev 1996; 152: 47–66. 04. Gianani R, Sarvetnick N. Viruses, cytokines, antigens, and autoimmunity. Proc Natl Acad Sci USA 1996; 93: 2257–2259. 05. Horwitz MS, Sarvetnick N. Viruses, host responses, and autoimmunity. Immunologic Rev 1999; 169: 241–253. 06. Naides SJ. Viral arthritis. In: Maddison PJ, Isenberg DA, Woo P, Glass DN (eds). Oxford Textbook of Rheumatology 2nd edition. Oxford: Oxford University Press, 1998, pp. 896–906. 07. Hausmann J, Hallensleben W, de la Torre JC et al. T cell ignorance in mice to Borna disease virus can be overcome by peripheral expression of the viral nucleoprotein. Proc Natl Acad Sci USA 1999; 96: 9769–9774. 08. Ciurea A, Klenerman P, Hunziker L et al. Persistence of lymphocytic choriomeningitis virus at very low levels in immune mice. Proc Nat Acad Sci USA 1999; 96: 11964–11969. 09. Carlson D, Hernandez J, Bloom BJ et al. Lack of Borrelia Burgdorfeeri DNA in synovial samples from patients with antibiotic treatment resistant Lyme arthritis. Arthritis Rheumatism 1999; 42: 2705–2709. 10. Tan EM et al. The 1982 revised criteria for the classification of systemic lupus erythematosus (SLE). Arthritis Rheumatism 1982; 25: 1271–1277. 11. Cooper GS, Dooley MA, Treadwell EL et al. Hormonal, environmental, and infectious risk factors for developing systemic lupus erythematosus. Arthritis Rheumatism 1998; 41: 1714–1724. 12. Walport MJ, Davies KA, Morley BJ, Botto M. Complement deficiency and autoimmunity. Ann New York Acad Sci 1997; 815: 267–281. 13. Bickerstaff MCM, Botto M, Hutchinson WL et al. Serum amyloid P component controls chromatin degradation and prevents antinuclear autoimmunity. Nature Med 1999; 5: 694–697. 14. Maia DM, Garwacki CP X-linked lymphoproliferative disease: pathology and diagnosis. Pediat Develop Pathol 1999; 2: 72–77. 15. Maggi F, Fornai C, Morrica A et al. High prevalence of TT virus viremia in Italian patients regardless of age, clinical diagnosis, and previous interferon treatment. J Infect Dis 1999; 180: 838–842. 16. Laghi L, Randolph AE, Chauhan DP et al. JC virus DNA is present in the mucosa of the human colon and in colorectal cancers. Proc Natl Acad Sci USA 1999; 96: 7464–7489. 17. Bredholt G, Olaussen E, Moens U, Rekvig OP. Linked production of antibodies to mammalian DNA and to human polyomavirus large T antigen. Arthritis Rheumatism 1999; 42: 2583–2592. 18. Buskila D, Sikuler E, Shoenfeld Y. Hepatitis C virus, autoimmunity and rheumatic disease. Lupus 1997; 6: 685–689. 19. Abu-Shakra M, El-Sana S, Margalith M et al. Hepatitis B and C viruses serology in patients with SLE. Lupus 1997; 6: 543–544. 20. Kowdley KV, Subler DE, Scheffel J et al. Hepatitis C virus antibodies in systemic lupus erythematosus J Clin Gastroenterol 1997; 25: 437–439. 21. Kammer AR, van der Burg SH, Grabscheid B et al. Molecular mimicry of human cytochrome P450 by hepatitis C virus at the level of cytotoxic T-cell recognition. J Experiment Med 1999; 190: 169–176. 22. Parez N, Herzog D, Jacqz-Aigrain E et al. Study of the B cell response to cytochrome P450IID6 in sera from chronic hepatitis C patients. Clin Exp Immunol 1996; 106: 336–343. 23. Albert LJ, Inman RD. Molecular mimicry and autoimmunity. New Engl J Med 1999; 341: 2068–2074. 24. Naucler CS, Larsson S, Moller E. A novel mechanism for virus-induced autoimmunity in humans. Immunologic Rev 1996; 152: 175–192. 25. Haaheim LR, Halse AK, Kvakestad R et al. Serum antibodies from patients with primary Sjogren’s syndrome and systemic lupus
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26. 27. 28. 29. 30. 31. 32.
erythematosus recognise multiple epitopes on the La (SS-B) autoantigen resembling viral protein sequences. Scand J Immunology 1996; 43: 115–121. Cash E, Charreire J, Rott O. B-cell activation by superstimulatory influenza virus haemagglutinin: a pathogenesis for autoimmunity? Immunologic Rev 1996; 152: 67–88. Tamura N, Sekigawa I, Hashimoto H et al. Syncytial cell formation in vivo by type C retroviral particles in the systemic lupus erythematosus (SLE) lung. Clin Exp Immunol 1997; 107: 474–479. Inchi S, Osame M. Human T-cell lymphotropic virus type I and systemic lupus erythematosus. Intern Med 1999; 38: 459. Griffiths DJ, Cooke SP, Herve C et al. Detection of human retrovirus 5 in patients with arthritis and systemic lupus erythematosus, Arthritis Rheumatism 1999; 42: 448–454. Herrmann M, Hagenhofer M, Kalden JR. Retroviruses and systemic lupus erythematosus. Immnunologic Rev 1996; 152: 145–156. Nakagawa K, Harrison LC. The potential role of endogenous retroviruses in autoimmunity. Immunologic Rev 1996; 152: 193–236. Bengtsson A, Blomberg J, Nived O et al. Selective antibody reactivity with peptides from human endogenous retroviruses and nonviral poly (amino acids) in patients with systemic lupus erythematosus. Arthritis Rheumatism 1996; 39: 1654–1663.
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33. Hishikawa T, Ogasawara H, Kaneko H et al. Detection of antibodies to a recombinant gag protein derived from human endogenous retrovirus clone 4-1 in autoimmune diseases. Viral Immunol 1997; 10: 132–147. 34. Deas JE, Liu LG, Thompson JJ et al. Reactivity of sera from systemic lupus erythematosus and Sjogren’s syndrome patients with peptides derived from human imnunodeficieney virus p24 capsid antigen. Clin Diagnostic Lab Immunol 1998; 5: 181–185. 35. Fraziano M, Momtesano C, Lombardi VRM et al. Epitope specificity of anti-HIV antibodies in human and murine autoimmune diseases. AIDS Res Human Retrovirus 1996; 12: 491–496. 36. Magistrell C, Samoilova E, Agarwal RK et al. Polymorphic genotypes of the HRES-1 human endogenous retrovirus locus correlate with systemic lupus erythematosus and autoreactivity Immunogenetics 1999; 49: 829–834. 37. Boyd GW. An evolution-based hypothesis on the origin and mechanisms of autoimmune disease. Immunol Cell Biol 1997; 75: 503–507. 38. Amoura Z, Piette J-C, Bach J-F, Koutouzov S. The key role of nucleosomes in lupus. Arthritis Rheumatism 1999; 42: 833–843. 39. Abbott A. A post-genomic challenge: learning to read patterns of protein sysntheseis. Nature 1999; 402: 715–720. 40. Auden WH. Poems No. 27, 1933.
LEADING ARTICLE
Systemic Lupus Erythematosus – is a Viral Aetiology a Credible Hypothesis? A. M. Denman* Northwick Park and St Mark’s NHS Trust, Watford Road, Harrow, Middlesex HA1 3UJ, U.K.
Systemic lupus erythematosus (SLE) is still regarded as a paradigm for autoimmune diseases, an Everest which if conquered would allow the solution of other unsolved problems in autoimmunity. Thus, interest in this issue is not confined to physicians and medical scientists who deal directly with patients with this disorder. The seach for viral causes of SLE has continued despite many decades of largely fruitless endeavour. Indeed, interest in this possibility has been sustained mainly by the discovery of the many mechanisms in natural infections and model systems by which persistently infective viruses are able to evade elimination by host immune defences and to stimulate autoimmune phenomena or disease.1,2 Of particular relevance to the immunopathogenesis of SLE are those viral infections which induce the characteristic spectrum of anti-nuclear and related autoantibodies and also lesions related to immune complex deposition and cytokine-induced inflammation resembling those characteristic of human SLE. For example, viral infections reveal cryptic auto-antigens to the immune system, thereby allowing tolerance to these antigens to be broken.3 The inflammatory reaction accompanying many viral infections generates high local concentrations of pro-inflammatory cytokines with subsequent autoantibody production.4,5 Such findings mirror the many situations in which transgenic manipulation of constitutive cytokine production is followed by autoimmune disorders. More simplistically, in clinical medicine parvovirus B19 infection is a telling example of a situation in which an ubiquitous agent induces acute clinical features and immunological abnormalities closely resembling SLE, often over a period of many months.6 However, analogy is no substitute for firm observations and by itself cannot be expected to sustain interest in this
*Address correspondence to: M. Denman, Gaters, Old Shire Lane, Chorleywood, Hertfordshire WD3 5PW, UK. Accepted for publication 22 March 2000.
0163-4453/00/030229]04 $35.00/0
field indefinitely. Fortunately for protagonists of a viral aetiology for SLE, other observations have shown how occult infection can continue to have immunopathological consequences, often only in the long term. Thus, T cells may remain ignorant of persistent CNS infection by Borna disease virus until T cells are stimulated by peripheral expression of the viral nucleoprotein in a later, systemic infection, thereby stimulating an immune response to the previously tolerated local infection.7 This results in inflammatory disease. Viruses such as lymphocytic choriomeningitis virus (LCM) may persist in very low, barely detectable levels even in the face of intact immune defences.8 Lyme arthritis vividly illustrates the difficulty which may be encountered in detecting microbial persistence in inflammatory disease even when the causative agent is known. The spirochaete Borrelia burgdorferi responsible for this disease can rarely be isolated from the joints of patients who continue to display chronic arthritis and even spirochaetal DNA is undetectable in these joints.9 It can be argued, therefore, that absence of an obvious provoking infection or a series of negative findings do not negate the viral hypothesis. Despite these difficulties, some viruses have been specifically implicated in the immunopathogenesis of SLE. For candidate agents to be persuasive causes of the disease, they must account for its most prominent features. By convention, the diagnosis of SLE can only be established if the patient displays at least four of 11 specified criteria.10 In reality, the nosological justification for this selection may eventually prove rather arbitrary. This is especially true of the autoantibodies included in these criteria, which are for the most part encountered in many other disorders. This is an important point in attempts to associate viral infection with SLE overall. It is very likely that this diagnostic term of convenience embraces many different disorders which will not prove to have a single cause. Genetic predisposition to SLE is well recognized, of which the most striking illustration is the 25%
© 2000 The British Infection Society
230
A. M. Denman
concordance in monozygotic compared with dizygotic twins.11 The most clear-cut genetic association is with the complement coding genes; at least 50% of individuals with C2 deficiency develop SLE or a closely related disorder.12 However, this deficiency is as likely to signify impaired clearance of immune complexes irrespective of their composition as a deficient host response to infectious agents. This interpretation is supported by the effects of targeted deletion of the gene encoding serum amyloid protein (SAP). This protein binds to chromatin released from dying, apoptotic cells. Mice with this deletion develop glomerulonephritis and anti-nuclear antibodies, both characteristic features of SLE.13 The significance of the association between SLE and other polymorphic genes remains uncertain, but it is probable that these gene families influence the nature of a wide range of specific immune and non-specific inflammatory responses to infectious agents. Traditional searches for a viral aetiology in SLE have depended on attempts to isolate candidate viruses from patients and on anti-viral antibody titres. Direct isolation has not produced any consistent findings. Furthermore, SLE is a disease with impaired immunity affecting T-cell function, antibody responses, and non-specific immunity. Thus, successful viral isolation may merely reflect opportunistic infection of an immunocompromised patient. This is especially true of infection by agents such as herpes viruses which persistently infect individuals with essentially normal immune defences. The interpretation of antibody titres is complicated by the polyclonal B cell activation characteristic of this disorder. Thus, raised anti-microbial antibody titres do not necessarily indicate a response to conventional infection. Analysis of V gene sequences encoding the equally characteristic autoantibodies shows rearrangement and hypermutation in response to persistent antigen stimulation. As yet, similar studies on the genes encoding anti-viral antibodies have not been reported, which might in principle suggest that some raised anti-viral antibody titres in SLE are driven by continued viral stimulation. However, this would be a difficult undertaking given the ubiquity of the viruses in question and the need for appropriate controls. Nevertheless, it should not be automatically assumed that agents encountered in patients with disturbed immunity are invariably aetiologically irrelevant. There are many such ambiguous situations which may prove relevant to SLE. A pertinent example is EBV infection, which is almost universally acquired, usually causes trivial or self-limited illness after primary infection, but can also cause lymphoproliferative disease in genetically susceptible individuals.14 Even seemingly harmless viruses such as
the TT found in 50% of some populations may eventually prove to have disease associations.15 The situation is more complicated when a virus present in a high percentage of normal individuals is found in higher copy numbers in diseased tissues. An intriguing example is the ubiquitous polyoma virus JC virus, which is specifically associated with colo-rectal cancer cells.16 Although such observations can readily be dismissed by assuming that passenger viruses or their genes will predictably be found more frequently in malignant than in normal cells, one has to be more circumspect when the virus in question, in this instance JC virus, can cause chromosomal changes characteristic of the malignant cells. There is reasonable evidence that certain specific viral infections may be relevant in SLE. Furthermore, there is additional evidence that the infections could account for at least some of the defining characteristics of the disease. One such virus is polyoma virus. Bredholt and colleagues detected antibodies to the large T antigen in 55% of SLE sera compared with only one of the 20 control sera which were tested. Moreover, there was a strong correlation between anti-T antigen antibodies and autoantibodies to single-stranded DNA. The authors suggest that T antigen bound to nucleosomes may act as a hapten and thereby trigger anti-DNA autoantibodies.17 The remarkable association between hepatitis C virus (HCV) infection and mixed cryoglobulinaemia, often in association with vasculitis and with other systemic autoimmune diseases,18 has encouraged the suspicion that infection by this agent might also account for SLE at least in some patients. However, this has proved to be an unlikely explanation. The incidence of anti-HCV antibodies is not increased in SLE patients.19 Claimed increases in the incidence of anti-HCV antibodies are misleading because of the frequency of false positive results. In the majority of SLE patients who appear to display these antibodies, HCV viraemia cannot be detected by polymerase chain reaction (PCR).20 Despite these negative findings, the ability of such a widely prevalent virus to cause autoimmune disease is a fillip for investigators who regard this as a model with broader implications. Firstly, the association between HCV infection and mixed cryoglobulinaemia emerged by screening patients for this infection; hitherto it had not been detected by even the most sophisticated immunochemical analysis of the complexes. Secondly, there is experimental evidence that an HCVderived core protein epitope induced cytotoxic T-cells which cross-react with cytochrome P450.21 This autoreactivity may contribute to the immunopathogenesis of chronic active hepatitis in persistently infected individuals.22 Indeed, this property of HCV is persuasive support
Systemic Lupus Erythematosus for the general theory that viral infections induce autoimmune disease through molecular mimicry.23 There are further hints that common viral infections may be relevant to the aetiology of SLE. The determining role of cytomegalovirus (CMV) infection in the development of chronic graft versus host (GvH) disease in recipients of bone marrow transplants has long been recognized. This disease has many features in common with SLE and related autoimmune disorders, and indeed animal models of chronic GvH disease are good models of spontaneous SLE. Patients with chronic GvH disease, especially in severe form, develop antibodies to the CD13 molecule expressed on the surface of many populations of peripheral blood mononuclear cells. This molecule is incorporated into the surface envelope of newly synthesized CMV virions and may thereby form an autoantigenic target.24 Analysis of the antigen specificity of the anti-La autoantibodies found in SLE and other systemic autoimmune diseases shows that these antibodies react with protein sequences related to human herpes viruses, providing another possible example of molecular mimicry.25 Even influenza viruses, arguably the commonest cause of virus infections, cannot be exonerated, since influenza viral haemagglutinins are potent B cell activators and hence able to induce autoantibody production.26 For valid reasons the possible aetiological importance of retroviruses in SLE has attracted increasing interest. Firstly, known retroviral infections induce autoantibodies or overt autoimmune disease. These may be associated with immunodeficiency disease (HIV infections) or lymphoproliferative diseases (HTLV-1 infections). Secondly, even if conventional person-to-person (horizontal) infection by retroviruses proves irrelevant, endogenous retroviruses may still prove to be important in the disease process. Moreover, genetically transmitted (vertical) retroviral infection in this form would be consistent with the epidemiological evidence which emphasises the importance of genetic factors. Finally, the ability of endogenous retroviral sequences to influence the expression of unrelated, contiguous or distant genes provides a plausible explanation for the mixed picture of inappropriate immunological activation and suppression so characteristic of SLE. There are few reports identifying complete, potentially infectious retroviral particles in SLE patients. However, syncytial cells and retroviral particles were encountered in the bronchoalveolar lavage fluid of a patient with interstitial pneumonia complicating SLE.27 Clinical and laboratory abnormalities characteristic of SLE are encountered in only a small percentage of patients with unequivocal HTLV-1 infection. Nevertheless, the immunopathological abnormalities encountered in these patients are sufficiently
231
impressive to encourage the belief that a closely related agent could be operating in other SLE patients.28 Human retrovirus 5 (HRV-5) was found in the peripheral blood cells of 15% of patients with SLE and also 53% of synovial membrane samples obtained from arthritic joints. It was not found in blood samples from patients with other autoimmune diseases, and in only one of 200 control tissues. The invariable presence of an open reading frame and the extent of sequence variation in different samples suggest that HRV-5 in these patients was an actively replicating virus and not an endogenous retroviral sequence.29 The relatively low incidence of positive findings in the SLE patients studied in this report does not necessarily indicate that HRV-5 is a passenger virus of unlikely relevance to the pathogenesis of the disease. It can be rationally argued that a range of agents including different retroviruses rather than a single agent is more likely to account for so pleomorphic a disorder. Most of the arguments favouring a role for endogenous retroviruses (HERV) depend on the potential effects of these sequences and on the findings in animal models of autoimmune disease.30,31 However, many groups have obtained serological data showing that sera from a high percentage of SLE patients react with protein sequences related to HERV. Initial studies revealed an increased frequency of antibodies to two peptides encoded by the env gene of the endogenous retroviruses ERV-9 and HERV-H in SLE sera compared with sera from normal controls.32 Similar studies showed that nearly 50% of SLE sera react with a p30 gag protein derived from HERV. These experiments used recombinant proteins as well as synthetic peptide sequences as the target antigens.33 A recent investigation found that one-third of sera from SLE patients reacted with the p24 core protein antigen of HIV even though there was no other evidence that these patients were HIV-infected.34 These results are suggestive but still problematical in technical and interpretative terms. In vitro antibody studies dependent on linear peptide antigens take no account of in vivo protein conformation. As yet there is little information about the physical properties of anti-HERV antibodies, and in particular information concerning the strength of their binding affinity. There is also a hint that anti-HERV titres in some patients vary with disease activity, but it is not known whether antibody affinity increases with disease duration, as one would expect if HERV sequences were a major source of antigenic stimulation. Inhibition studies with gp120 peptides indicate that the p24 driven response in some autoimmune diseases may be an oligoclonal response against a more non-specific polyclonal background35: this observation increases the likelihood that the response is specifically
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anti-HERV rather than a chance cross-reaction, but detailed information is lacking. The observed linkage between polymorphic genotypes of the HERV HRES-136 and susceptibility to SLE introduces a further complexity, since there may be linkage disequilibrium between a limited number of HERV genotypes and immune response genes. If retroviruses are involved in the immunopathogenesis of SLE, the evidence to date favours a role for HERV rather than horizontal infection by fully infectious retroviruses. However, this conclusion introduces further difficulties in interpretation. It is possible that HERV sequences are peculiarly vulnerable to inappropriate activation or mutation induced by agents such as viral infection or UV irradiation. There is an intriguing proposal that inappropriate presentation of retroviral antigens by HLA antigens after excessive cell damage is a major stimulus to autoimmune disease.37 However, this special pleading for the particular importance of HERV in such circumstances is still speculative. Should the classic hypothesis be correct that SLE results from a fundamental and generalized loss of tolerance, there would be no reason to suppose that HERV encoded antigens would be any more or less likely to stimulate autoantibody responses than other autoantigens. Currently there is much circumstantial evidence implicating a range of viral infections in the aetiology of SLE in genetically susceptible individuals. The link with complement deficiency and other defects in handling immune complexes gives the second part of this statement scientific credibility. Indeed, it is possible that the primary insult is increased apoptosis secondary to a variety of viral infections and that SLE results from autoantibodies induced by the excessive burden of nucleosomes released by dying cells in individuals with such defects.38 If a more specific aetiological role for one or more viruses is to be more firmly established, it is clear that new diagnostic criteria will have to be devised in this as in other chronic diseases of unknown cause. It would take unprecedented good fortune to isolate such agents in fully replicating form or to identify virus-derived proteins and diagnostic anti-viral antibodies in the time honoured manner. Any successful approach is likely to be a painstaking exercise which would need to generate three crucially important pieces of information. Firstly, the detection of viral sequences in the normal host cell genome with the potential to influence normal genes encoding immune responses and inflammatory reactions. Secondly, an analysis of the proteins synthesised by the affected cells, namely an application of the formidably complicated study of normal cells newly defined as proteomics.39 Finally, a detailed analysis of the immune response to the relevant gene sequences and the products these encode. Alas, “to ask the hard question is simple”40; finding the answers in this field is a quest scarcely begun.
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