dsDNA Autoantibodies

CHAPTER

dsDNA Autoantibodies

22 Dörte Hamann and Ruud J.T. Smeenk Sanquin Blood Supply, Amsterdam, the Netherlands

Historical notes Components in the serum of systemic lupus erythematosus (SLE) patients reactive with deoxyribonucleic acid (DNA) were first described in 1957 [1]. Following the identification of these components as antibodies, many different techniques have been developed for the detection, characterization, and quantitation of anti-DNA. Initially developed methods showed a strong association of anti-DNA with SLE [2]. Later, more sensitive methods were developed, with which anti-DNA was also found to occur in other clinical syndromes. The specificity for SLE of anti-DNA was regained when reaction conditions were more carefully controlled and purely double-stranded DNA (dsDNA) was used as a s­ ubstrate. More recently, it was shown that nucleosomes most probably are the antigen that triggers development of antibodies to DNA.

Autoantigen Definition DNA as antigen may be either double stranded (dsDNA) or single stranded (ssDNA). In vivo, DNA will almost always occur in the form of nucleosomes, that is, double stranded and closely associated with histones. Because the epitopes situated on DNA are—at least in part—based on the repetitive negative charge of the molecule, synthetic polynucleotides are often also recognized by anti-DNA antibodies. DNA is found in all prokaryotic and nucleated eukaryotic cells of all species. DNA from various species may differ in antigenicity; recently, it was suggested that for human anti-DNA, human DNA forms the best recognized antigen [3]. Yet, while studying species specificity of anti-DNA, it was also found that (human) serum antibodies as well as monoclonal (murine) anti-DNA antibodies bound DNA of all species tested, though not to the same extent.

Biological function DNA is the carrier of the genetic information of the individual and as such is present in every nucleated cell. In the cell, DNA is encapsulated in nucleosomes. These nucleosomes form the basic structure of chromatin and have an important function in the compaction of DNA in the nucleus of the cell. A nucleosome consists of dimers of the four core histones, H2A, H2B, H3, and H4, which together form Autoantibodies. http://dx.doi.org/10.1016/B978-0-444-56378-1.00022-8 Copyright © 2014 Elsevier B.V. All rights reserved.

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a histone-octamer around which 146 base pairs of DNA are wrapped twice. Two nucleosome subunits are connected via a stretch of linker DNA to which histone H1 is bound.

Origin/sources DNA occurs in all living beings. It can be purified for use in specific assays from various species. For detection of anti-DNA, frequently used sources include calf thymus, micro-organisms (bacteriophages such as PM2 or bacteria such as Escherichia coli (E. coli)), and various plasmids (such as pUC18). It is used unpurified in the Crithidia luciliae immunofluorescence test (IFT). Regarding the source of antigen, it is difficult to decide which antigen preparation would be preferred. However, some general remarks can be made.   1. DNA may be contaminated by proteins, especially histones. This is often the case with commercially available DNA (such as calf thymus DNA). It is absolutely necessary to avoid such contaminations, since otherwise measurement of anti-DNA is disturbed by detection of other antibodies or even immune complexes. 2. dsDNA is preferred above ssDNA, since it has often been reported that anti-dsDNA is more specific to SLE than anti-ssDNA. 3. The DNA should be large enough to present antigenic epitopes correctly. The latter also implies that synthetic polynucleotides may not always present (all of) the relevant epitopes.  

Methods of purification For use in anti-DNA assays, DNA can be purified (through standard DNA purification protocols) from tissue (e.g., calf thymus), (eukaryotic) cells, bacteria, or bacteriophages. In particular, the DNA from bacteriophage PM2 (which can easily be grown on its host, the bacterium Pseudomonas BAL31) has been shown to be very useful, since it can be radiolabeled in vivo and easily isolated in a purely doublestranded form. Plasmid DNA (e.g., from the vector pUC18) forms a suitable alternative; this DNA is easily iodinated after isolation. DNA can also be obtained commercially; in particular, calf thymus DNA has often been used in anti-DNA assays. Care should be taken to avoid protein contamination of the employed preparation. A different approach is to make use of the hemoflagellate Crithidia luciliae for the measurement of anti-DNA: Crithidia luciliae contains a giant mitochondrion, the kinetoplast, composed of pure dsDNA not “contaminated” with proteins.

Autoantibody Definition Antibodies to DNA by definition react with DNA, either pure or complexed with proteins such as histones. Sequential as well as backbone determinants of DNA can be the targets of anti-DNA recognition. Backbone determinants on either ssDNA or dsDNA are short regions of DNA helix or short nucleotide sequences. The interaction between B-cell paratope and dsDNA epitope is predominantly based on electrostatic interactions and, as such, is extremely sensitive to salt concentration and pH. Yet,

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especially in the case of high-avidity anti-DNA, secondary hydrogen bonding also plays a role. Most likely, such dsDNA epitopes are constituted by the sugar-phosphate backbone of the DNA. Specificity of autoantibodies for such epitopes might also explain why anti-DNA reacts with DNA of all species: SLE serum antibodies react with DNA of animal, bacterial, viral, and plant origin. Apart from backbone recognition there is also selective recognition of DNA epitopes variably expressed on different DNA molecules. Such a binding seems more pronounced in the case of ssDNA and is presumably based on recognition of defined nucleotide sequences. Although anti-DNA specific for ssDNA may exist as a separate entity, most of what is generally called anti-ssDNA reactivity actually is anti-dsDNA of a low avidity. When dsDNA is denatured and ssDNA is formed, the strands of DNA become more flexible. Upon cooling, internal duplex formation over short stretches of DNA occur. Reactivity of anti-dsDNA with ssDNA is mainly due to this kind of internal duplex formation. Epitopes formed by these internal duplexes are exposed completely different than in dsDNA. The difference lies in the flexibility of the DNA backbone, which is of extreme importance in terms of allowing multipoint attachment (and thus high-avidity binding) of antibodies to DNA. Therefore, the greater flexibility of ssDNA will lead to higher avidity binding. The actual binding site of an anti-DNA autoantibody encompasses only about six nucleotides, but most anti-DNA antibodies require DNA fragments from 40 to several hundreds of base pairs in length for stable interaction. The size dependency, however, differs very much among antibodies. These findings suggest that both antibody fragments (Fabs) of an anti-DNA antibody need to be bound for a stable interaction via (monogamous) bivalent interactions with antigenic sites distributed along the DNA molecule.

Pathogenic role Antibodies to DNA have always been claimed to play an important role in the pathogenesis of SLE [4]. Traditionally, SLE is considered an immune complex disease. In this concept, anti-DNA binds DNA and the resulting immune complexes are deposited in the tissues. This binding of DNA by antibodies may occur in the circulation, but it may also happen in situ. At the site of deposition, subsequent complement activation then leads to inflammation and the characteristic disease features of SLE. In later years, this concept of the pathophysiology of SLE has been challenged, and a modified hypothesis has been proposed. This concept is based on studies that have shown that anti-DNA may interact via nucleosomes with tissue structures such as heparin sulfate (HS), the major glycosaminoglycan side chain of the glomerular basement membrane (GBM) [5]. Increased levels of nucleosomes have indeed been demonstrated in the plasma of SLE patients, especially preceding peaks in anti-DNA levels. These nucleosomes seem to originate from apoptotic cells that are not cleared efficiently enough in SLE patients. A third hypothesis claims cross reaction of anti-DNA with structures in the tissues, leading to local binding and inflammation [6]. In all of these theories, however, anti-DNA plays a key role in mediating disease phenomena.

Genetics Although SLE is generally considered not to be an inherited disease, estimates of twin concordance in monozygotic twins vary between 24% and 69%. So at least, there seems to be an inherited factor predisposing to susceptibility for SLE. Another finding pointing in this direction is the observation that family members of SLE patients often show increased incidences of antinuclear and anti-DNA antibodies.

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Susceptibility to SLE is associated with certain major histocompatibility complex (MHC)-encoded genes. HLA-DR2, DQw1, and the rare allele DQβ1.AZH confer high relative risk (RR = 14) for lupus nephritis. DR4 is significantly decreased in patients with lupus nephritis. Of the patients with lupus nephritis, 50% have the DQβ1.1, the DQβ1.AZH, or the DQβ1.9 allele. These alleles, therefore, seem to have a direct role in the predisposition to lupus nephritis. Studies related to VH and VL gene usage have pointed out that both V chains are necessary for DNA reactivity of an anti-DNA antibody, and that no unique V, D, or J gene segments are used to construct the antibody. Genetic studies have further suggested that anti-DNA is produced by a process of somatic mutation and clonal expansion favoring sequences with accumulated positively charged amino acids in the complementarity-determining regions. The antigens that trigger this process are not known, but nucleosomes most probably are implicated. Indeed, in MRL/lpr mice, initially, the autoantibody response is directed to nucleosomes, with anti-DNA antibodies appearing later in the disease (epitope spreading).

Methods of detection A multitude of assays has been developed to detect the presence of antibodies to DNA. Currently, the most used assays are enzyme-linked immunosorbent assays (ELISAs) in various forms, IFT on Crithidia luciliae (CLIFT), and radioimmunoassays (RIA) (e.g., Farr assay). More recently, multiplex and array technologies have also been employed for anti-DNA detection. These methods can either be obtained in kit form or employed as inhouse assays. In ELISA systems, DNA has to be coated to plastic. Though ssDNA can easily be coated directly, dsDNA is mostly coated via intermediates such as poly-L-lysine, protamine, or methylated bovine serum albumin (BSA). Such precoats introduce problems related to the binding of immune complexes and/or aspecific immunoglobulins (Ig) to the plates (via the intermediate molecule). A better alternative is to make use of biotinylated DNA and coat this via streptavidin to the plates. The latter has also been used in a modified technique (SPADE), where anti-DNA is first allowed to react with biotinylated DNA in solution and thereafter is captured on streptavidin-coated plates [7]. Recently, it has been suggested that coating of purified dsDNA via histon-1-free nucleosomes increases the sensitivity for SLE with a specificity comparable to conventional anti-dsDNA assays [8]. In fact, the assay also detects anti-nucleosome antibodies and should be regarded as a screening test for SLE rather than a strict anti-dsDNA assay. CLIFT is a method that specifically detects dsDNA and thus couples high disease specificity to good sensitivity. Therefore, it is one of the preferred methods. However, CLIFT requires specific expertise of laboratory personnel. Automated indirect immunofluorescence (IIF) systems that include automated analysis of slides are becoming available. In RIAs, the choice of antigen again is of great importance. The DNA employed has to be bigger than 105 kDa but smaller than 107 kDa. Furthermore, the DNA must be double stranded and, to allow quantitation of antibody reactivity, monodisperse in size. This indicates that circular double-stranded bacteriophage DNA (such as from PM2) or plasmids (such as pUC18) are to be preferred. Different assay systems are not always comparable, for the following reasons:   1. the source of antigen differs: DNA may be from eukaryotic or prokaryotic origin, be double stranded or single stranded, be polydisperse in size or homogeneous etc.; 2. presentation of the antigen to the antibody differs: in RIAs it is generally in solution, in ELISAs it is coated to plastic; in the CLIFT test it is presented intact in cells;

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3. r eaction conditions are different: for example, due to the employed ammonium sulfate precipitation step used in the Farr assay, anti-dsDNA of low avidity is missed with this method; in second antibody techniques such as IFT and ELISA the choice of conjugated antibody is of importance; often, only IgG anti-DNA is measured with these techniques.   From the above considerations it follows that standardization will be difficult if not impossible because a given standard will react differently in the various test systems. Since absolute values of anti-dsDNA are not directly associated with severity or activity of SLE, but in individual patients rises in antidsDNA can precede exacerbations, it is important to follow a patient with the same quantitative assay for monitoring of disease activity.

Clinical utility Disease association Anti-DNA antibodies are thought to play a pivotal role in the development of SLE disease features. Flares of SLE are generally preceded by a rise in anti-DNA levels, followed by a steep drop during the exacerbation. In particular, lupus nephritis is correlated with anti-DNA of high avidity. Studies in murine models of SLE have shown that an initial IgM anti-DNA response is followed in time with an IgG response (and affinity maturation of the antibodies). Only after the development of IgG anti-DNA does nephritis occur in these mice. In general, IgG antibodies seem to be of greater relevance to the disease than IgM antibodies, because the latter may also more often be found in non-SLE patients.

Disease prevalence SLE occurs mainly in young people, aged between 20 and 40 years, and is 9 to 15 times more common in women than in men. Prevalence is estimated at 50 females and 3.6 males per 100,000 individuals. People of African, American Indian, and Asian origin appear to have a higher incidence than Caucasians. Given an anti-DNA assay with a high sensitivity, 99% of the SLE patients will be found to have anti-DNA at some time during the course of their disease.

Diagnostic value There are two important but different ways in which anti-DNA assays may be used: either as an aid to the diagnosis of SLE or as a tool to follow the clinical course of a defined SLE patient. Toward the first aim, it is advisable that the assay used has a high specificity toward SLE. Since SLE specificity appears to be inversely related to the sensitivity of the assay, often reflected in the avidity spectrum detected, it is not surprising to find that the Farr assay and the CLIFT have the highest specificity for SLE. If screening for the presence of anti-DNA is done by an assay that is not selective for high-avidity anti-DNA, then a positive assay result does not always indicate that the patient has SLE: anti-DNA of lower avidity occurs in other diseases than SLE as well. Indeed, Haugbro et al. published the presence of anti-DNA in up to 30% of antinuclear antibodies (ANA)-positive patients without SLE, if measured by ELISA [7]. Therefore, such a screening should be followed by a more SLE-specific method.

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With respect to the diagnostic value of anti-DNA, Arbuckle et al. published the presence of a­ nti-DNA in 55% of cases prior to SLE diagnosis. The onset of anti-DNA ranged from 1 month to 9.3 years (mean 2.7 years) before the diagnosis [9]. Earlier, we evaluated the diagnostic value of anti-DNA in a group of 441 non-SLE patients with Farr-assay detectable anti-DNA. It was found that more than 85% of these patients developed SLE within 5 years of the first Farr-positive assay result [10]. If it is expected that dsDNA autoantibodies are directly involved in the pathophysiology of SLE the question remains when and why this antibody response switches from clinical irrelevance to pathogenicity. In the past, a causal relationship has been proposed between SLE nephritis and complement fixing ability of anti-DNA. However, it is more likely that complement fixing titers are a direct reflection of anti-DNA titers. Patients with nephritis generally have higher titers of anti-DNA than patients without nephritis, and, therefore, more complement fixing anti-DNA will be found in sera of patients with nephritis. Therapeutic regimens used in the treatment of patients with SLE have varying influences on antidsDNA levels. Immunosuppressive therapy indeed suppresses production of anti-DNA and, because anti-DNA is implicated in the development of lupus nephritis, may reduce kidney injury. Although plasmapheresis initially dramatically reduces anti-dsDNA levels, no differences in the final outcome of the disease are reached. Anti-DNA may cross the placenta and is associated with neonatal lupus syndromes. However, disease features, which include rashes and cytopenia, are transient; with the disappearance of anti-DNA from the circulation disease features subside. The only permanent injury inflicted upon neonates is congenital heart block, which is associated with anti-Ro/SS-A and anti-La/SS-B antibodies but not with anti-DNA. Treatment of patients with rheumatoid arthritis with tumor necrosis factor (TNF) blockers may induce anti-DNA production in up to 10% of the patients. Mostly, this anti-DNA is of IgM isotype only and no clinical effects are noticed. Charles et al. published that production of IgG anti-DNA in such a patient led to symptoms of SLE [11]. After termination of the anti-TNF therapy, both the anti-DNA antibodies and the SLE-like symptoms in these patients disappear again.

Specificity and sensitivity The specificity and sensitivity for SLE of anti-DNA antibodies depends on the assay used for its detection and the patient population studied and varies widely (Table 22.1; compiled from [7,12–15]). In one meta-analysis, ELISA, CLIFT, and Farr assay were compared [16]. CLIFT had the highest specificity Table 22.1  Specificity and Sensitivity of Different Methods of Detection Assay

Specificity

Sensitivity

ELISA FEIA Farr CLIFT

71–97% 84–94% 95–99% 99–100%

44–79% 40–73% 32–85% 13–47%

CLIFT: Crithidia luciliae immunofluorescence test; FEIA: fluorescence enzyme immunoassay; ELISA: enzyme-linked immunosorbent assay.

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(97.2%) and the lowest sensitivity (33.6%) (five studies). ELISA showed an intermediate sensitivity (55.8%) and the lowest specificity (92.5%) (20 studies). Farr had the highest sensitivity with a slightly lower specificity (96.7%) than CLIFT (seven studies). Individual studies show a great variety of sensitivities and specificities.

Prognostic value Disease activity During prognostic follow-up of SLE patients, it is very worthwhile to be informed of the fluctuations in the anti-DNA level as well as of the relative avidity of the anti-DNA present. Repeated serum sampling of individual patients (every 4–6 weeks) may be very informative about the clinical course of the disease, since a relation exists between anti-DNA levels and disease activity (in particular, nephritis). Exacerbations of the disease are often preceded by rises in anti-dsDNA titer but not every rise in anti-dsDNA titer is followed by a clinical exacerbation. Therefore, a rise in titer without clinical exacerbation should lead to awareness of the clinician but as such is not sufficient to modify treatment.

Organ involvement/damage Characteristics of anti-DNA may be related to the damage inflicted by these antibodies. As mentioned before, there is a correlation between anti-DNA avidity and organ involvement: high-avidity anti-DNA is related to kidney involvement. A relationship with IgG/IgM ratio of anti-DNA has also been published: a continuous ratio under 0.8 was associated with absence of renal involvement [17]. Even more intriguing, renal damage could be prevented in NZB/W F1 mice by treating these mice with IgM antiDNA [18]. Clustering anti-DNA with one or more other autoantibodies may help predict clinical subsets and damage in SLE. To and Petri observed that in SLE patients with anti-DNA, lupus anticoagulant and anticardiolipin showed higher incidences of thrombosis and livedo reticularis than patients with antiDNA and anti-Ro plus anti-La [19]. The latter patients comprised a significantly higher incidence of patients with secondary Sjögren syndrome.

anti-DNA avidity HIGH

LOW ELISA

CLIFT

Farr assay

FIGURE 22.1 Anti-DNA avidity and assay behavior.

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Clinical utility of the different methods of detection For the purposes of screening patients for the presence of anti-DNA, a method of high sensitivity should be employed. The presence of anti-DNA should then be confirmed using a method with high disease specificity. For the follow-up of defined SLE patients, any method that allows quantitation of anti-DNA can be used. However, the Farr assay is preferred, particularly because Farr assay-detectable anti-DNA fluctuations correlate with exacerbations of SLE, in particular nephritis.

Take-home messages • • • • • • •

 igh-avidity anti-DNA is very specific for anti-DNA. H Lower-avidity anti-DNA also occurs in autoimmune diseases other than SLE. Anti-DNA may be present years before SLE becomes overt. Frequent measurement of anti-DNA may help predict upcoming exacerbations. High-avidity anti-DNA correlates with SLE nephritis. Anti-DNA is directly involved in induction of nephritis. Anti-DNA and antinucleosome are in part the same antibodies. 

References [1]  Ceppelini R, Polli E, Celada F. A DNA-reacting factor in serum of a patient with lupus erythematosus ­diffusus. Proc Soc Exp Biol Med 1957;96:572–4. [2]  Stollar BD. The specificity and applications of antibodies to helical nucleic acids. CRC Crit Rev Biochem 1975;3:45–69. [3]  Janyapoon K, Jivakanont P, Surbrsing R, Siriprapapan W, Tachawuttiwat T, Korbsrisate S. Detection of antidsDNA by ELISA using different sources of antigens. Pathology 2005;37(1):63–8. [4]  Tan EM. Autoantibodies to nuclear antigens (ANA): their immunobiology and medicine. Adv Immunol 1982;33:167–240. [5]  Bruggen MCJ, Walgreen B, Rijke T, Tamboer W, Kramers K, Smeenk RJT, et al. Antigen specificity of antinuclear antibodies complexed to nucleosomes determines glomerular basement membrane binding in vivo. Eur J Immunol 1997;27:1564–9. [6]  Rice JS, Kowal C, Volpe BT, DeGiorgio LA, Diamond B. Molecular mimicry: anti-DNA antibodies bind microbial and nonnucleic acid self-antigens. Curr Top Microbiol Immunol 2005;296:137–51. [7]  Haugbro K, Nossent JC, Winkler T, Figenschau Y, Rekvig OP. Anti-dsDNA antibodies and disease classification in antinuclear antibody positive patients: the role of analytical diversity. Ann Rheum Dis 2004;63(4):386–94. [8]  Biesen R, Dähnrich C, Rosemann A, Barkhudarova F, Rose T, Jakob O, et al. Anti-dsDNA-NcX ELISA: dsDNA-loaded nucleosomes improve diagnosis and monitoring of disease activity in systemic lupus erythematosus. Arthritis Res Ther 2011;13(1):R26. [9]  Arbuckle MR, James JA, Kohlhase KF, Rubertone MV, Dennis GJ, Harley JB. Development of anti-dsDNA autoantibodies prior to clinical diagnosis of systemic lupus erythematosus. Scand J Immunol 2001;54(1–2):211–9. [10] Swaak AJG, Smeenk R. Detection of anti-dsDNA as a diagnostic tool: a prospective study in 441 non systemic lupus erythematosus (SLE) patients with anti-dsDNA antibody (anti-dsDNA). Ann Rheum Dis 1985;44:245–51.

References

193

[11] Charles PJ, Smeenk RJT, Jong JD, Feldmann M, Maini RN. Assessment of antibodies to double-stranded DNA induced in rheumatoid arthritis patients following treatment with infliximab, a monoclonal antibody to tumor necrosis factor α. Arthritis Rheum 2000;43(11):2383–90. [12] Wasmuth JC, Grun B, Terjung B, Homrighausen A, Spengler U. ROC analysis comparison of three assays for the detection of antibodies against double-stranded DNA in serum for the diagnosis of systemic lupus erythematosus. Clin Chem 2004;50(11):2169–71. [13] Hernando M, Gonzalez C, Sanchez A, Guevara P, Navajo JA, Papisch W, et al. Clinical evaluation of a new automated anti-dsDNA fluorescent immunoassay. Clin Chem Lab Med 2002;40(10):1056–60. [14] Riboldi P, Gerosa M, Moroni G, Radice A, Allegri F, Sinico A, et al. Anti-DNA antibodies: a diagnostic and prognostic tool for systemic lupus erythematosus? Autoimmunity 2005;38(1):39–45. [15] Derksen RH, Bast EJ, Strooisma T, Jacobs JW. A comparison between the Farr radioimmunoassay and a new automated fluorescence immunoassay for the detection of antibodies against double stranded DNA in serum. Ann Rheum Dis 2002;61(12):1099–102. [16] Bizzaro N, Villalta D, Giavarina D, Tozzoli R. Are anti-nucleosome antibodies a better diagnostic marker than anti-dsDNA antibodies for systemic lupus erythematosus? A systematic review and a study of metanalysis. Autoimmun Rev 2012;12(2):97–106. [17] Forger F, Matthias T, Oppermann M, Becker H, Helmke K. Clinical significance of anti-dsDNA antibody isotypes: IgG/IgM ratio of anti-dsDNA antibodies as a prognostic marker for lupus nephritis. Lupus 2004;13(1):36–44. [18] Werwitzke S, Trick D, Kamino K, Matthias T, Kniesch K, Schlegelberger B, et al. Inhibition of lupus disease by anti-double-stranded DNA antibodies of the IgM isotype in the (NZB x NZW)F1 mouse. Arthritis Rheum 2005;52(11):3629–38. [19] To CH, Petri M. Is antibody clustering predictive of clinical subsets and damage in systemic lupus erythematosus? Arthritis Rheum 2005;52(12):4003–10.