REVIEWS Anti-DNA antibodies — quintessential biomarkers of SLE David S. Pisetsky
Abstract | Antibodies that recognize and bind to DNA (anti-DNA antibodies) are serological hallmarks of systemic lupus erythematosus (SLE) and key markers for diagnosis and disease activity. In addition to common use in the clinic, anti-DNA antibody testing now also determines eligibility for clinical trials, raising important questions about the nature of the antibody–antigen interaction. At present, no ‘gold standard’ for serological assessment exists, and anti-DNA antibody binding can be measured with a variety of assay formats, which differ in the nature of the DNA substrates and in the conditions for binding and detection of antibodies. A mechanism called monogamous bivalency — in which high avidity results from simultaneous interaction of IgG Fab sites with a single polynucleotide chain — determines anti-DNA antibody binding; this mechanism might affect antibody detection in different assay formats. Although anti-DNA antibodies can promote pathogenesis by depositing in the kidney or driving cytokine production, they are not all alike, pathologically, and anti-DNA antibody expression does not necessarily correlate with active disease. Levels of anti-DNA antibodies in patients with SLE can vary over time, distinguishing anti-DNA antibodies from other pathogenic antinuclear antibodies. Elucidation of the binding specificities and the pathogenic roles of anti-DNA antibodies in SLE should enable improvements in the design of informative assays for both clinical and research purposes.
Medical Research Service, Durham Veterans Administration Medical Center, Box 151G, 508 Fulton Street, Durham, North Carolina 27705, USA. Correspondence to D.S.P. ([email protected]) doi:10.1038/nrrheum.2015.151 Published online 19 Nov 2015
Antibodies that recognize and bind to DNA (anti-DNA antibodies) are the serological hallmark of systemic lupus erythematosus (SLE) and quintessential biomarkers to probe the mechanisms of autoimmunity 1–3. These antibodies have been characterized with numerous state-of-the-art technologies, and more is known about their molecular properties than those of any other auto antibody. Indeed, the molecular analyses of anti-DNA antibodies rival in size and detail those of antibodies to HIV4. The extent of these efforts demonstrates the importance of anti-DNA antibodies as a paradigm to enable understanding of the basis of aberrant B‑cell expression in autoimmune disease5. The importance of anti-DNA antibodies extends beyond the laboratory to the clinical arena, wherein the assumption that they equate with SLE is a foundation of serological diagnosis. Further interest in testing results from the utilization of anti-DNA antibody positivity as a measure to determine eligibility of individuals for inclusion in clinical trials or to guide the therapeutic use of certain agents in routine clinical practice6,7. Because the interpretation of test results relating to anti-DNA antibodies can be nuanced and even perplexing 8,9, in this Review I consider the salient features of anti-DNA
antibody binding and the value of testing, especially as it moves from a diagnostic to a theranostic setting.
Antigenic properties of DNA The unexpected ability of antibodies to bind DNA was discovered in the 1950s following efforts to delineate the basis of antinuclear antibody reactivity 10–13. Prior to this discovery, little evidence existed for recognition of nucleic acids by the immune system. By contrast, evidence for the antigenicity of proteins and carbo hydrates was strong, reflecting the focus of immuno logical research on host defense mechanisms and the development of vaccines to prevent bacterial and viral disease. Subsequent results demonstrated the importance of anti-DNA antibodies in the diagnosis of SLE, and the immune role of DNA seemed to be exclusive to the setting of immunological disease1,2,9. The double helix is synonymous with DNA, and double-stranded DNA (dsDNA) consists of two polynucleotide chains wound around each other, with base pairs keeping the chains tightly bound. However, the DNA strands can come apart to form single-stranded DNA (ssDNA). Although dsDNA and ssDNA can be considered as distinct forms, in
NATURE REVIEWS | RHEUMATOLOGY
ADVANCE ONLINE PUBLICATION | 1 © 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS Key points • Antibodies that recognize DNA (anti-DNA antibodies) can bind to sites on the phosphodiester backbone of single-stranded DNA and double-stranded DNA, to nucleotide sequences or to higher-order structures such as nucleosomes • The molecular properties of anti-DNA antibodies, as well as the associated genetic properties, including variable-region somatic mutations, point to a role for antigen selection in anti-DNA antibody generation • In the absence of a ‘gold standard’, various assay formats exist for anti-DNA antibody testing, differing in the nature of DNA substrates and the conditions for binding and detection of antibodies • High-affinity binding by an anti-DNA antibody depends on monogamous bivalency, in which both Fab sites of an IgG molecule contact the same polynucleotide chain • The use of anti-DNA antibody testing as a measure of disease activity to determine clinical trial eligibility depends on clear understanding of assay differences and the role of anti-DNA antibodies in pathogenesis
fact, they comprise a continuum, both biochemically and antigenically 14. Because all naturally occurring dsDNA can exist as the classic helical B conformation, anti-DNA-antibody assays have used DNA from many sources as the test antigen, including mammalian, bacterial, viral and even protozoal DNA15. However, DNA from the protozoan Crithidia luciliae can have a bent structure, which has similarities to the conformation of nucleosomal DNA, and might only enable binding of a subgroup of anti-DNA antibodies. An important characteristic — and possible source of bias — of immunological research relates to the use of biochemically well-defined molecules to probe the molecular basis of B‑cell and T‑cell recognition. Studies have typically involved highly purified preparations of DNA to characterize its antigenicity and immuno genicity 16. However, the biochemical properties of DNA pose immediate challenges to this seemingly reason able approach. Structurally, DNA has a simple aspect in the phosphodiester backbone, but it also has complex elements in the variable nucleotide sequence and the association with nuclear proteins — in particular, histones — to form the nucleosome, the fundamental building block of chromosomal structure17.
Nucleosomes and chromatin The use of purified DNA for the assay substrate conforms to paradigms of antigenicity, but could actually impede understanding of the immune recognition of this molecule. In the cell, DNA is rarely — if ever — pure. Essentially, DNA is a protein-binding molecule that exists in the eukaryotic cell in intimate association with histones to form structural units known as nucleo somes17–19. In a nucleosome, a stretch of DNA of around 147 bp is wrapped around a core protein octamer comprising two molecules each of histones H2a, H2b, H3 and H4, which neutralize the negative charge of the DNA phosphodiester groups. Many other proteins, such as transcription factors, bind to DNA, and chromatin is the ensemble of nuclear macromolecules in the cell and includes DNA, histones, nonhistone proteins and even nascent RNA transcripts, depending on the preparation15.
The structure of chromatin means that DNA can be thought of as part of the nucleosome, and anti-DNA antibodies as part of a family of anti-nucleosome or anti-chromatin antibodies20–24. In this model, DNA, histones and DNA–histone complexes represent epitopes of the nucleosome25,26. The frequent coexistence of antiDNA antibodies with antibodies to histones supports the idea that the nucleosome, rather than isolated DNA, is the relevant target of autoreactivity. The presence of anti-nucleosome antibodies can be inferred from positive results in assays that use chromatin as the antigen.
In vivo release of DNA from cells DNA and nucleosomes can be released into the blood by cell death27–32. Although apoptotic cells have generally been considered the source of extracellular nuclear material, cell death can occur by various pathways that include apoptosis, necrosis, necroptosis, pyroptosis and NETosis (a form of induced cell death that involves the release of neutrophil extracellular traps (NETs))33. These pathways differ in terms of stimuli, molecular events that cause cellular demise, structure of the DNA released (FIG. 1) and the immunological properties of the deceased cells. Events during cell death can contribute to the immunogenicity of DNA by altering the extent of exposure of DNA to the extracellular environment, the molecular context and the presence of other immuno active molecules. These molecules include IL‑1, ATP and high mobility group protein B1 (HMG‑1, also known as HMGB1). The DNA-binding protein HMG‑1 is a proto typical alarmin and can act as an adjuvant to stimulate both innate and adaptive immune responses34–36. All cell-death processes can lead to the release of DNA, but the size of the DNA and the nature of associ ated molecules vary depending on the mechanism involved. In particular, DNA exiting from apoptotic cells is cleaved to form the classic ladder wherein the fragment sizes are multiples of the unit length of nucleosomal DNA29,31. By contrast, DNA released during NETosis can be present in a large mesh studded with proteins following mixing with the contents of cytoplasmic granules (FIG. 1). NETs could have a direct role in the pathogenesis of autoimmune inflammatory disease, including SLE, by stimulating immune cells or endothelium37,38. Although assays are available to measure the size and the amount of DNA in blood, few studies have been conducted to determine the origin of cell-free DNA in SLE and its effect on antigenicity and immunogenicity 39,40. DNA released during apoptotic cell death can also exist in the form of microparticles, which are small, membrane-bound vesicles. Antibodies in sera from patients with SLE and murine models, as well as murine monoclonal anti-DNA antibodies, have been shown to bind DNA in microparticles41,42. Microparticles have been identified in immune complexes in the plasma of patients with SLE41,42. Whereas serological studies on SLE have utilized purified DNA as the antigenic sub strate, DNA is evidently present in vivo in alternative antigenic moieties, including complexes of DNA with other macromolecules — as in nucleosomes — as well as higher-order structures such as microparticles.
2 | ADVANCE ONLINE PUBLICATION
www.nature.com/nrrheum © 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS a
Apoptotic bodies
b
Apoptosis
Microparticles
Free lowmolecular-weight DNA
Necrosis
Free highmolecular-weight DNA
c
NETosis
NET
Figure 1 | The release of DNA from dead and dying cells. Any cell type can undergo apoptosis or necrosis, but NETosis Nature Reviews | Rheumatology involves only immune cells, most prominently neutrophils. a | In apoptosis, the cell shrinks and the nucleus condenses. DNA undergoes cleavage by nucleases, resulting in a ladder of low-molecular-weight fragments corresponding to multiples of the length of DNA in the nucleosome. Apoptosis results in extracellular DNA in the form of apoptotic bodies (collapsed remains of cells or large fragments thereof), membrane-bound microparticles or free DNA. b | In necrosis, as the cell lyses, high-molecular-weight DNA is released in the absence of intracellular cleavage. c | In NETosis of neutrophils, DNA mixes with enzymes in neutrophil granules and is then extruded in the form of a mesh or neutrophil extracellular trap (NET) in which DNA is coated with anti-bacterial proteins such as myeloperoxidase and neutrophil elastase.
Assays for anti-DNA antibodies Anti-DNA-antibody assays vary in the source of DNA for the antigenic substrate and in the principle that is employed to assess binding. In essence, all antiDNA-antibody assays measure the formation of immune complexes of anti-DNA antibodies and DNA. The physical principle (such as radioimmunoassay, fluorescence immunoassay and ELISA) as well as assay conditions (such as salt concentration) for detection of such complexes vary between assays, influencing the type of anti-DNA antibodies measured and the levels that are considered significant15 (BOX 1). Notably, in the criteria of the Systemic Lupus International Collaborating Clinics (SLICC) group for disease classification, anti-dsDNA- antibody positivity is defined as an assay result above the laboratory reference range, except for ELISA, where a value of twice the laboratory reference range is considered positive18. However, these criteria do not specify which assay should be used, nor the required levels of sensitivity and specificity 43,44. Given the differences in the various testing platforms, the serum of a patient can give positive results in one assay and negative results in another 15,45–47. This situation can create confusion in the clinic and raise questions about the use of anti-DNA antibodies as a theranostic measure. In the presence of so many different assays, a fair question is whether a gold standard exists, especially for clinical decision-making. To some investigators, the Farr assay has that status because it detects high-affinity
antibodies, which are the indication of a mature antibody response. Detection is limited to high-affinity antibodies because of the high-salt conditions (saturated ammonium sulphate) that are used to precipitate the immune complex of DNA and anti-DNA antibodies48,49. Although this assay has high specificity for SLE, it does not have the highest sensitivity of the available assays. Compared with the Farr assay, an ELISA has higher sensitivity, because it detects both high-affinity and low-affinity anti-DNA antibodies15. Depending on the concentration of DNA and the manner in which it is attached to plates (for example, using positively charged molecules as a ‘pre-coat’), an ELISA can produce a false-positive detection of anti-DNA antibodies. Because an ELISA is a sensitive assay, positive results can sometimes occur in patients with conditions that are not SLE, or in patients who have features of SLE, but who do not yet meet the criteria for this disease; this situation can also occur with other assays, but the importance of these apparently false-positive results is not known. Furthermore, the relative contribution of high-affinity and low-affinity antibodies to the pathogenesis in SLE has not yet been determined. In routine testing, the choice of assay is usually made by the clinical or reference laboratory on the basis of considerations that can include cost and ease-of-performance. However, if suspicion is high for the presence of anti-DNA antibodies, or a serological marker is needed to confirm diagnosis, the attending
NATURE REVIEWS | RHEUMATOLOGY
ADVANCE ONLINE PUBLICATION | 3 © 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS Box 1 | Assays for anti-DNA antibodies15 Immunodiffusion assay Classic Ouchterlony assay leading to visible precipitate of immune complexes Complement-fixation assay Fixation of complement by immune complexes formed by combination of DNA with anti-DNA antibodies Farr assay Incubation of radiolabelled DNA antigen with serum followed by addition of saturated ammonium sulphate to precipitate immune complexes; the proportion of the initial radiolabel that is precipitated gives an indication of the amount of anti-DNA antibody in the serum sample PEG-precipitation assay Similar to the Farr assay, but with precipitation by polyethylene glycol (PEG), which is less stringent than precipitation by ammonium sulphate; the proportion of the initial radiolabel that is precipitated gives an indication of the amount of anti-DNA antibody in the serum sample Filter-binding assay Incubation of radiolabelled DNA with serum followed by filtration through nitrocellulose to retain immune complexes; assessment of the proportion of the radiolabel that is retained on the filter gives an indication of the amount of anti-DNA antibody in the serum sample Crithidia luciliae immunofluorescence test (CLIFT) Semi-quantitative indirect immunofluorescence assay with the kinetoplast of C. luciliae as the source of naked double-stranded DNA; serial dilution of serum samples enables determination of the maximum dilution that gives a positive immunofluorescence reaction ELISA Indirect assay in which DNA is bound to a solid-phase support, exposed to serum, and anti-DNA antibody binding detected with enzyme-labelled antibodies to human IgG; quantitation is achieved by comparison of colour development in sample wells with that in wells treated with dilutions of a known standard Multiplex assay Immunofluorescence assay in which multiple antigens are bound to solid-phase supports (beads); after incubation with serum, followed by fluorescence-labelled secondary antibody, antigen binding is detected by measurement of fluorescence associated with individual beads
Box 2 | Monogamous bivalency50–52 Monogamous bivalency is the term for a mode of antibody binding in which both Fab sites of an IgG molecule interact with contiguous sites on the same antigen. The binding affinity of each Fab site is low, and high functional affinity (or avidity) depends upon the binding of both sites. In this process, interaction of the first Fab site with a determinant on a multivalent antigen increases the likelihood of the second Fab site binding; similarly, dissociation of one site from the antigen is likely to lead to re-association if the antigen remains bound by the other site. In essence, the local concentration of antigen rises. Monogamous bivalency can be indicated by discrepancies in the affinities measured in different assay formats, including the magnitude of binding to antigens in solution or attached to solid-phase supports, or to intact antigens compared with smaller antigenic fragments, or of proteolytic Fab fragments relative to whole IgG. The prevalence of monogamous bivalency either with or without autoimmune responses is unknown, because of the use of single assay formats with intact antibody, so its influence on inter-assay variability has not yet been determined.
physician can consider requesting another type of antiDNA assay. In this regard, the ability of the ELISA to detect a broad range of antibodies might make it a good choice for screening. Multiplex assays are increasingly being used for routine serological testing, including quantitative determination of levels of anti-DNA antibodies. These
assays employ antigen-coated beads, and are a form of solid-phase assay, with the DNA bound to a surface. Such assays are convenient and have the advantage of simultaneously providing data on multiple antibody specificities. The performance of multiplex assays, with regard to the detection of anti-DNA, is comparable to that of other assay formats. Because the diagnosis of SLE depends upon the clinical context and the fulfillment of other clinical and laboratory criteria, no one test will suffice, and a sensitive assay for anti-DNA antibodies will help to ensure that the diagnosis of SLE is not missed.
Immune recognition of DNA Monogamous bivalency Although anti-DNA antibodies can display high affinity (befitting the products of antigen selection), high affinity in this context reflects functional affinity, or avidity, which involves a form of antigen binding known as monogamous bivalency 50–52. This binding is a type of cross-linking in which the two Fab sites on each anti body contact antigenic determinants on the same DNA molecule, resulting in a stable interaction (BOX 2). The binding site of each Fab region could accommo date 3–5 nucleotides. In general, oligonucleotides are not effective antigens, and longer DNA fragments are necessary for stable interactions. Depending on the isotype and segmental flexibility, the distance between the Fab sites of an intact IgG molecule is around 100 Å53. The minimum size of a helical DNA antigen for antigenicity should be around 30 bp, although longer molecules are more-effective antigens54–56 (FIG. 2). Monogamous bivalent interaction means that immune complexes are not likely to form as a result of one antibody binding simultaneously to two different polynucleotide chains. Instead, complex formation results from many antibodies binding to the same piece of DNA, so that the length of the DNA that is bound will influence lattice formation57,58. Various lengths of DNA are found in blood, but they are generally
Abstract | Antibodies that recognize and bind to DNA (anti-DNA antibodies) are serological hallmarks of systemic lupus erythematosus (SLE) and key markers for diagnosis and disease activity. In addition to common use in the clinic, anti-DNA antibody testing now also determines eligibility for clinical trials, raising important questions about the nature of the antibody–antigen interaction. At present, no ‘gold standard’ for serological assessment exists, and anti-DNA antibody binding can be measured with a variety of assay formats, which differ in the nature of the DNA substrates and in the conditions for binding and detection of antibodies. A mechanism called monogamous bivalency — in which high avidity results from simultaneous interaction of IgG Fab sites with a single polynucleotide chain — determines anti-DNA antibody binding; this mechanism might affect antibody detection in different assay formats. Although anti-DNA antibodies can promote pathogenesis by depositing in the kidney or driving cytokine production, they are not all alike, pathologically, and anti-DNA antibody expression does not necessarily correlate with active disease. Levels of anti-DNA antibodies in patients with SLE can vary over time, distinguishing anti-DNA antibodies from other pathogenic antinuclear antibodies. Elucidation of the binding specificities and the pathogenic roles of anti-DNA antibodies in SLE should enable improvements in the design of informative assays for both clinical and research purposes.
Medical Research Service, Durham Veterans Administration Medical Center, Box 151G, 508 Fulton Street, Durham, North Carolina 27705, USA. Correspondence to D.S.P. ([email protected]) doi:10.1038/nrrheum.2015.151 Published online 19 Nov 2015
Antibodies that recognize and bind to DNA (anti-DNA antibodies) are the serological hallmark of systemic lupus erythematosus (SLE) and quintessential biomarkers to probe the mechanisms of autoimmunity 1–3. These antibodies have been characterized with numerous state-of-the-art technologies, and more is known about their molecular properties than those of any other auto antibody. Indeed, the molecular analyses of anti-DNA antibodies rival in size and detail those of antibodies to HIV4. The extent of these efforts demonstrates the importance of anti-DNA antibodies as a paradigm to enable understanding of the basis of aberrant B‑cell expression in autoimmune disease5. The importance of anti-DNA antibodies extends beyond the laboratory to the clinical arena, wherein the assumption that they equate with SLE is a foundation of serological diagnosis. Further interest in testing results from the utilization of anti-DNA antibody positivity as a measure to determine eligibility of individuals for inclusion in clinical trials or to guide the therapeutic use of certain agents in routine clinical practice6,7. Because the interpretation of test results relating to anti-DNA antibodies can be nuanced and even perplexing 8,9, in this Review I consider the salient features of anti-DNA
antibody binding and the value of testing, especially as it moves from a diagnostic to a theranostic setting.
Antigenic properties of DNA The unexpected ability of antibodies to bind DNA was discovered in the 1950s following efforts to delineate the basis of antinuclear antibody reactivity 10–13. Prior to this discovery, little evidence existed for recognition of nucleic acids by the immune system. By contrast, evidence for the antigenicity of proteins and carbo hydrates was strong, reflecting the focus of immuno logical research on host defense mechanisms and the development of vaccines to prevent bacterial and viral disease. Subsequent results demonstrated the importance of anti-DNA antibodies in the diagnosis of SLE, and the immune role of DNA seemed to be exclusive to the setting of immunological disease1,2,9. The double helix is synonymous with DNA, and double-stranded DNA (dsDNA) consists of two polynucleotide chains wound around each other, with base pairs keeping the chains tightly bound. However, the DNA strands can come apart to form single-stranded DNA (ssDNA). Although dsDNA and ssDNA can be considered as distinct forms, in
NATURE REVIEWS | RHEUMATOLOGY
ADVANCE ONLINE PUBLICATION | 1 © 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS Key points • Antibodies that recognize DNA (anti-DNA antibodies) can bind to sites on the phosphodiester backbone of single-stranded DNA and double-stranded DNA, to nucleotide sequences or to higher-order structures such as nucleosomes • The molecular properties of anti-DNA antibodies, as well as the associated genetic properties, including variable-region somatic mutations, point to a role for antigen selection in anti-DNA antibody generation • In the absence of a ‘gold standard’, various assay formats exist for anti-DNA antibody testing, differing in the nature of DNA substrates and the conditions for binding and detection of antibodies • High-affinity binding by an anti-DNA antibody depends on monogamous bivalency, in which both Fab sites of an IgG molecule contact the same polynucleotide chain • The use of anti-DNA antibody testing as a measure of disease activity to determine clinical trial eligibility depends on clear understanding of assay differences and the role of anti-DNA antibodies in pathogenesis
fact, they comprise a continuum, both biochemically and antigenically 14. Because all naturally occurring dsDNA can exist as the classic helical B conformation, anti-DNA-antibody assays have used DNA from many sources as the test antigen, including mammalian, bacterial, viral and even protozoal DNA15. However, DNA from the protozoan Crithidia luciliae can have a bent structure, which has similarities to the conformation of nucleosomal DNA, and might only enable binding of a subgroup of anti-DNA antibodies. An important characteristic — and possible source of bias — of immunological research relates to the use of biochemically well-defined molecules to probe the molecular basis of B‑cell and T‑cell recognition. Studies have typically involved highly purified preparations of DNA to characterize its antigenicity and immuno genicity 16. However, the biochemical properties of DNA pose immediate challenges to this seemingly reason able approach. Structurally, DNA has a simple aspect in the phosphodiester backbone, but it also has complex elements in the variable nucleotide sequence and the association with nuclear proteins — in particular, histones — to form the nucleosome, the fundamental building block of chromosomal structure17.
Nucleosomes and chromatin The use of purified DNA for the assay substrate conforms to paradigms of antigenicity, but could actually impede understanding of the immune recognition of this molecule. In the cell, DNA is rarely — if ever — pure. Essentially, DNA is a protein-binding molecule that exists in the eukaryotic cell in intimate association with histones to form structural units known as nucleo somes17–19. In a nucleosome, a stretch of DNA of around 147 bp is wrapped around a core protein octamer comprising two molecules each of histones H2a, H2b, H3 and H4, which neutralize the negative charge of the DNA phosphodiester groups. Many other proteins, such as transcription factors, bind to DNA, and chromatin is the ensemble of nuclear macromolecules in the cell and includes DNA, histones, nonhistone proteins and even nascent RNA transcripts, depending on the preparation15.
The structure of chromatin means that DNA can be thought of as part of the nucleosome, and anti-DNA antibodies as part of a family of anti-nucleosome or anti-chromatin antibodies20–24. In this model, DNA, histones and DNA–histone complexes represent epitopes of the nucleosome25,26. The frequent coexistence of antiDNA antibodies with antibodies to histones supports the idea that the nucleosome, rather than isolated DNA, is the relevant target of autoreactivity. The presence of anti-nucleosome antibodies can be inferred from positive results in assays that use chromatin as the antigen.
In vivo release of DNA from cells DNA and nucleosomes can be released into the blood by cell death27–32. Although apoptotic cells have generally been considered the source of extracellular nuclear material, cell death can occur by various pathways that include apoptosis, necrosis, necroptosis, pyroptosis and NETosis (a form of induced cell death that involves the release of neutrophil extracellular traps (NETs))33. These pathways differ in terms of stimuli, molecular events that cause cellular demise, structure of the DNA released (FIG. 1) and the immunological properties of the deceased cells. Events during cell death can contribute to the immunogenicity of DNA by altering the extent of exposure of DNA to the extracellular environment, the molecular context and the presence of other immuno active molecules. These molecules include IL‑1, ATP and high mobility group protein B1 (HMG‑1, also known as HMGB1). The DNA-binding protein HMG‑1 is a proto typical alarmin and can act as an adjuvant to stimulate both innate and adaptive immune responses34–36. All cell-death processes can lead to the release of DNA, but the size of the DNA and the nature of associ ated molecules vary depending on the mechanism involved. In particular, DNA exiting from apoptotic cells is cleaved to form the classic ladder wherein the fragment sizes are multiples of the unit length of nucleosomal DNA29,31. By contrast, DNA released during NETosis can be present in a large mesh studded with proteins following mixing with the contents of cytoplasmic granules (FIG. 1). NETs could have a direct role in the pathogenesis of autoimmune inflammatory disease, including SLE, by stimulating immune cells or endothelium37,38. Although assays are available to measure the size and the amount of DNA in blood, few studies have been conducted to determine the origin of cell-free DNA in SLE and its effect on antigenicity and immunogenicity 39,40. DNA released during apoptotic cell death can also exist in the form of microparticles, which are small, membrane-bound vesicles. Antibodies in sera from patients with SLE and murine models, as well as murine monoclonal anti-DNA antibodies, have been shown to bind DNA in microparticles41,42. Microparticles have been identified in immune complexes in the plasma of patients with SLE41,42. Whereas serological studies on SLE have utilized purified DNA as the antigenic sub strate, DNA is evidently present in vivo in alternative antigenic moieties, including complexes of DNA with other macromolecules — as in nucleosomes — as well as higher-order structures such as microparticles.
2 | ADVANCE ONLINE PUBLICATION
www.nature.com/nrrheum © 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS a
Apoptotic bodies
b
Apoptosis
Microparticles
Free lowmolecular-weight DNA
Necrosis
Free highmolecular-weight DNA
c
NETosis
NET
Figure 1 | The release of DNA from dead and dying cells. Any cell type can undergo apoptosis or necrosis, but NETosis Nature Reviews | Rheumatology involves only immune cells, most prominently neutrophils. a | In apoptosis, the cell shrinks and the nucleus condenses. DNA undergoes cleavage by nucleases, resulting in a ladder of low-molecular-weight fragments corresponding to multiples of the length of DNA in the nucleosome. Apoptosis results in extracellular DNA in the form of apoptotic bodies (collapsed remains of cells or large fragments thereof), membrane-bound microparticles or free DNA. b | In necrosis, as the cell lyses, high-molecular-weight DNA is released in the absence of intracellular cleavage. c | In NETosis of neutrophils, DNA mixes with enzymes in neutrophil granules and is then extruded in the form of a mesh or neutrophil extracellular trap (NET) in which DNA is coated with anti-bacterial proteins such as myeloperoxidase and neutrophil elastase.
Assays for anti-DNA antibodies Anti-DNA-antibody assays vary in the source of DNA for the antigenic substrate and in the principle that is employed to assess binding. In essence, all antiDNA-antibody assays measure the formation of immune complexes of anti-DNA antibodies and DNA. The physical principle (such as radioimmunoassay, fluorescence immunoassay and ELISA) as well as assay conditions (such as salt concentration) for detection of such complexes vary between assays, influencing the type of anti-DNA antibodies measured and the levels that are considered significant15 (BOX 1). Notably, in the criteria of the Systemic Lupus International Collaborating Clinics (SLICC) group for disease classification, anti-dsDNA- antibody positivity is defined as an assay result above the laboratory reference range, except for ELISA, where a value of twice the laboratory reference range is considered positive18. However, these criteria do not specify which assay should be used, nor the required levels of sensitivity and specificity 43,44. Given the differences in the various testing platforms, the serum of a patient can give positive results in one assay and negative results in another 15,45–47. This situation can create confusion in the clinic and raise questions about the use of anti-DNA antibodies as a theranostic measure. In the presence of so many different assays, a fair question is whether a gold standard exists, especially for clinical decision-making. To some investigators, the Farr assay has that status because it detects high-affinity
antibodies, which are the indication of a mature antibody response. Detection is limited to high-affinity antibodies because of the high-salt conditions (saturated ammonium sulphate) that are used to precipitate the immune complex of DNA and anti-DNA antibodies48,49. Although this assay has high specificity for SLE, it does not have the highest sensitivity of the available assays. Compared with the Farr assay, an ELISA has higher sensitivity, because it detects both high-affinity and low-affinity anti-DNA antibodies15. Depending on the concentration of DNA and the manner in which it is attached to plates (for example, using positively charged molecules as a ‘pre-coat’), an ELISA can produce a false-positive detection of anti-DNA antibodies. Because an ELISA is a sensitive assay, positive results can sometimes occur in patients with conditions that are not SLE, or in patients who have features of SLE, but who do not yet meet the criteria for this disease; this situation can also occur with other assays, but the importance of these apparently false-positive results is not known. Furthermore, the relative contribution of high-affinity and low-affinity antibodies to the pathogenesis in SLE has not yet been determined. In routine testing, the choice of assay is usually made by the clinical or reference laboratory on the basis of considerations that can include cost and ease-of-performance. However, if suspicion is high for the presence of anti-DNA antibodies, or a serological marker is needed to confirm diagnosis, the attending
NATURE REVIEWS | RHEUMATOLOGY
ADVANCE ONLINE PUBLICATION | 3 © 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS Box 1 | Assays for anti-DNA antibodies15 Immunodiffusion assay Classic Ouchterlony assay leading to visible precipitate of immune complexes Complement-fixation assay Fixation of complement by immune complexes formed by combination of DNA with anti-DNA antibodies Farr assay Incubation of radiolabelled DNA antigen with serum followed by addition of saturated ammonium sulphate to precipitate immune complexes; the proportion of the initial radiolabel that is precipitated gives an indication of the amount of anti-DNA antibody in the serum sample PEG-precipitation assay Similar to the Farr assay, but with precipitation by polyethylene glycol (PEG), which is less stringent than precipitation by ammonium sulphate; the proportion of the initial radiolabel that is precipitated gives an indication of the amount of anti-DNA antibody in the serum sample Filter-binding assay Incubation of radiolabelled DNA with serum followed by filtration through nitrocellulose to retain immune complexes; assessment of the proportion of the radiolabel that is retained on the filter gives an indication of the amount of anti-DNA antibody in the serum sample Crithidia luciliae immunofluorescence test (CLIFT) Semi-quantitative indirect immunofluorescence assay with the kinetoplast of C. luciliae as the source of naked double-stranded DNA; serial dilution of serum samples enables determination of the maximum dilution that gives a positive immunofluorescence reaction ELISA Indirect assay in which DNA is bound to a solid-phase support, exposed to serum, and anti-DNA antibody binding detected with enzyme-labelled antibodies to human IgG; quantitation is achieved by comparison of colour development in sample wells with that in wells treated with dilutions of a known standard Multiplex assay Immunofluorescence assay in which multiple antigens are bound to solid-phase supports (beads); after incubation with serum, followed by fluorescence-labelled secondary antibody, antigen binding is detected by measurement of fluorescence associated with individual beads
Box 2 | Monogamous bivalency50–52 Monogamous bivalency is the term for a mode of antibody binding in which both Fab sites of an IgG molecule interact with contiguous sites on the same antigen. The binding affinity of each Fab site is low, and high functional affinity (or avidity) depends upon the binding of both sites. In this process, interaction of the first Fab site with a determinant on a multivalent antigen increases the likelihood of the second Fab site binding; similarly, dissociation of one site from the antigen is likely to lead to re-association if the antigen remains bound by the other site. In essence, the local concentration of antigen rises. Monogamous bivalency can be indicated by discrepancies in the affinities measured in different assay formats, including the magnitude of binding to antigens in solution or attached to solid-phase supports, or to intact antigens compared with smaller antigenic fragments, or of proteolytic Fab fragments relative to whole IgG. The prevalence of monogamous bivalency either with or without autoimmune responses is unknown, because of the use of single assay formats with intact antibody, so its influence on inter-assay variability has not yet been determined.
physician can consider requesting another type of antiDNA assay. In this regard, the ability of the ELISA to detect a broad range of antibodies might make it a good choice for screening. Multiplex assays are increasingly being used for routine serological testing, including quantitative determination of levels of anti-DNA antibodies. These
assays employ antigen-coated beads, and are a form of solid-phase assay, with the DNA bound to a surface. Such assays are convenient and have the advantage of simultaneously providing data on multiple antibody specificities. The performance of multiplex assays, with regard to the detection of anti-DNA, is comparable to that of other assay formats. Because the diagnosis of SLE depends upon the clinical context and the fulfillment of other clinical and laboratory criteria, no one test will suffice, and a sensitive assay for anti-DNA antibodies will help to ensure that the diagnosis of SLE is not missed.
Immune recognition of DNA Monogamous bivalency Although anti-DNA antibodies can display high affinity (befitting the products of antigen selection), high affinity in this context reflects functional affinity, or avidity, which involves a form of antigen binding known as monogamous bivalency 50–52. This binding is a type of cross-linking in which the two Fab sites on each anti body contact antigenic determinants on the same DNA molecule, resulting in a stable interaction (BOX 2). The binding site of each Fab region could accommo date 3–5 nucleotides. In general, oligonucleotides are not effective antigens, and longer DNA fragments are necessary for stable interactions. Depending on the isotype and segmental flexibility, the distance between the Fab sites of an intact IgG molecule is around 100 Å53. The minimum size of a helical DNA antigen for antigenicity should be around 30 bp, although longer molecules are more-effective antigens54–56 (FIG. 2). Monogamous bivalent interaction means that immune complexes are not likely to form as a result of one antibody binding simultaneously to two different polynucleotide chains. Instead, complex formation results from many antibodies binding to the same piece of DNA, so that the length of the DNA that is bound will influence lattice formation57,58. Various lengths of DNA are found in blood, but they are generally
