Selective recognition of DNA antigenic determinants by murine monoclonal anti-DNA antibodies.

To assess the immune recognition of DNA in systemic lupus erythematosus, the antigenic specificity of monoclonal anti-DNA antibodies from autoimmune MRL-lpr/lpr mice was investigated Determinant specificity was assessed by ELISA in terms of binding to a panel of ssDNA antigens including calf thymus, human placenta, Escherichia coli, Clostridium perfringens, Micrococcus lysodeikticus, salmon testes, chicken blood and murine DNA. Among the monoclonal antibodies, a variety of binding patterns was observed, although for all antibodies tested murine DNA was among the most reactive antigens. Binding to other DNAs varied markedly, with some antibodies showing only low reactivity to certain antigens in the test panel. Similar results were obtained with sera of individual MRL-lpr/lpr mice. These results suggest that anti-DNA antibodies bind specific antigenic determinants variably expressed by DNAs of various species. Furthermore, the preferential binding to mouse DNA by some MRL-lpr/lpr antibodies may suggest a role of self-DNA in the in vivo selection of anti-DNA antibodies for expression.     

The binding of sera of patients with SLE to bacterial and mammalian DNA

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by antibodies to DNA (anti-DNA). Although these antibodies have features of antigen drive, the source of this DNA is not defined. To assess the potential role of foreign and self-DNA as driving antigens, the specificity of SLE sera for bacterial and mammalian DNA was evaluated. Micrococcus lysodeikticus (MC) and calf thymus (CT) DNA were tested as antigens, with absorption on CT DNA columns used to identify antibodies to antigenic sites on the two DNA. Among 9 sets of longitudinal sera tested, all showed binding to both DNA, and none showed exclusive or predominant binding to CT DNA. With absorbed sera, antibodies could be distinguished in terms of cross-reactive or selective binding to the DNAs. These findings suggest that anti-DNA antibodies vary in specificity and are consistent with a role of both foreign and self-DNA in anti-DNA induction.     

Influence of assay conditions on ELISA determinations of anti-DNA antibodies

The influence of assay conditions on anti-DNA determinations by an enzyme-linked immunosorbent assay (ELISA) was investigated to evaluate the detection of various DNA antigenic specificities. Among 4 monoclonal anti-DNA antibodies of MRL-lpr/lpr strain origin, 2 showed higher titers in 100 mM NaCl-50 mM Tris, pH 7.5 (Tris-NaCl) than in phosphate-buffered saline (PBS). The determination of 'polyspecificity' for these monoclonal products also depended on the set of conditions used for assay with inhibitory activity of polynucleotides differing in the 2 buffers. The buffer effects were not confined to the monoclonal antibodies as increases in anti-DNA titers were demonstrated for certain SLE patient sera when assayed in Tris-NaCl rather than PBS; sera of MRL-lpr/lpr mice showed an opposite effect, however, with enhancement of anti-DNA activity by PBS. These results suggest that the representation of antigenic sites on DNA may be variably affected by the conditions of assay, altering quantitative and qualitative assessment of this important serological marker.     

Specificity of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA.

To determine the specificity of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA, sera from BALB/c mice immunized with single-stranded DNA from Escherichia coli (EC) were tested for binding to a panel of synthetic DNA and RNA homopolymers as well as duplexes. Results of these studies indicate that sera from EC DNA immunized mice preferentially bind certain DNA and RNA homopolymers as well as DNA duplexes. Furthermore, the specificity of the antibodies from immunized mice resembled those of sera from autoimmune MRL-lpr/lpr mice in terms of the synthetic antigens recognized, although some differences were noted in the magnitude of the response to individual duplexes. These results suggest that anti-DNA antibodies induced by bacterial DNA bind to DNA structures dependent on both the base and the sugar phosphate moieties of the nucleic acid antigen and may resemble some anti-DNA antibodies expressed in spontaneous autoimmune disease in these binding properties.     

The effect of polyamines on the binding of anti-DNA antibodies from patients with SLE and normal human subjects

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE). To elucidate specificity further, the effect of polyamines on the binding of anti-DNA antibodies from patients with lupus was tested by ELISA to calf thymus (CT) DNA; we also assessed the binding of plasmas of patients and normal human subjects (NHS) to Micrococcus luteus (MC) DNA. As these studies showed, spermine can dose-dependently inhibit SLE anti-DNA binding to CT DNA and can promote dissociation of preformed immune complexes. With MC DNA as antigen, spermine failed to inhibit the NHS anti-DNA binding. Studies using plasmas adsorbed to a CT DNA cellulose affinity indicated that SLE plasmas are mixtures of anti-DNA that differ in inhibition by spermine and binding to conserved and non-conserved determinants. Together, these studies demonstrate that spermine can influence the binding of anti-DNA autoantibodies and may contribute to the antigenicity of DNA.     

Characterization of monoclonal antibodies with specificity for DNA and the synthetic polypeptide antigen (T,G)-A-L

Because of evidence for structural similarity of variable region genes of anti-DNA and anti-(T,G)-A-L antibodies, polyspecific interactions of monoclonal anti-DNA and anti-(T,G)-A-L antibodies were investigated. Of 20 monoclonal antibodies from C57BL/10 mice with (T,G)-A-L binding, two bound DNA as determined by ELISA. In contrast, two of five anti-DNA monoclonal antibodies from MRL-lpr/lpr mice bound (T,G)-A-L. For both sets of antibodies, antigen binding was shown to be the activity of the same antibody by cross-inhibition studies. To determine whether such polyspecific antibodies were expressed during autoimmune disease, sera of autoimmune MRL-lpr/lpr mice were tested for anti-(T,G)-A-L activity. This analysis demonstrated minimal elevations of anti-(T,G)-A-L in comparison to BALB/c controls. These studies thus confirm predictions about the binding activity of anti-(T,G)-A-L and anti-DNA antibodies based on structural analysis of variable region genes. They further indicate, that while anti-(T,G)-A-L and anti-DNA antibodies may have overlapping specificity, polyspecific antibodies of this kind are not preferentially expressed during autoimmunity.     

IgG binding of monoclonal anti-nuclear antibodies from MRL-lpr/lpr mice

To assess the specificity of anti-nuclear antibodies with cross-reactive rheumatoid factor (RF) activity, monoclonal anti-DNA and anti-Sm antibodies from MRL-lpr/lpr mice were tested for binding to a variety of IgG antigens. These antibodies had all been previously identified as binding heterologous IgG. By ELISA, antibodies in this panel all bound BALB/c myeloma proteins representing the different IgG subclasses, indicating broad reactivity with murine IgG as well as heterologous IgG. The determinant recognized by these antibodies was further investigated using the Fab, F(ab')2 and Fc fragments of both human as well as rabbit origin. All antibodies bound well to fragments as well as intact IgG antigens. These antibodies were further analysed by Western blotting, demonstrating that most bound to both heavy and light chains of human origin. Together, these observations suggest that some anti-nuclear antibodies bind a conserved antigenic determinant present widely on immunoglobulin chains. This determinant may represent a common sequence important in immunoglobulin domain structure.     

Murine monoclonal antibodies to DNA. A comparison of MRL/lpr NZB/W and chronically graft-versus-host-diseased mice

Hybridomas producing monoclonal antibodies to DNA were prepared from NZB/W F1 (n = 20), MRL/lpr (n = 13), mice with a chronical graft versus-host-disease (GVHD) (n = 8) and polyclonally stimulated mice (n = 9). Screening was performed by means of an anti-DNA ELISA. Reaction patterns in four different anti-DNA assays (anti-DNA ELISA, indirect immunofluorescence on Crithidia luciliae, PEG assay and Farr assay) as well as avidity and cross-reactivity of these monoclonals were studied in relation to anti-DNA (sub)class and murine origin of the clones. It was found that monoclonal anti-DNA derived from mice with chronic GVHD did not differ from monoclonal anti-DNA derived from NZB/W F1 or MRL/lpr mice, with respect to isotype distribution, avidity towards DNA, cross-reactivity and assay behaviour in the anti-DNA assays mentioned before. In contrast, monoclonal anti-DNA obtained from polyclonally stimulated mice were all of the IgM isotype and displayed a stronger cross-reactive behaviour than the other three models. Altogether, these results exclude the possibility that anti-DNA in the GVHD mice originates from the non-specific pool of natural autoantibodies and further emphasize the relevance of chronic GVHD as a murine model of systemic lupus erythematosus.     

Specific binding to methylated polynucleotides in monoclonal anti-poly(dT) antibodies from autoimmune mice

Six monoclonal antibodies (mAbs) reactive to synthetic polynucleotide, poly(dT), were established from spontaneous autoimmune MRL/MpJ-lpr/lpr mice and male BXSB mice by spleen cell hybridization method, and were analyzed for cross-reactivity with polydeoxy-5-methylcytidylic acid [poly(dmC)] in comparison with its unmethylated counterpart poly(dC). By direct binding tests, these mAbs, all of which had preponderant binding activity to poly(dT) relative to poly(dU), were divided into two groups: (i) four mAbs showing reactivity to poly(dmC) as well as to natural DNA preparations and (ii) two mAbs with limited reactivity to poly(dT) but no binding to poly(dmC) or natural DNAs. Inhibition binding tests with these synthetic polynucleotides demonstrated that one mAb (TP-A9) in the first group reacted specifically to poly(dmC), as well as to poly(dT). In the second group, one mAb (TP-B5) showed highly specific reactivity to poly(dT) that could not be inhibited by poly(dU), poly(dC), or poly(dmC). However, another mAb (TP-C8) in the second group showed reactivity to poly(dT) that could be inhibited by poly(dU) as well as by poly(dT). Thus, these findings indicate that monoclonal anti-poly(dT) antibodies can cross-react with poly(dmC) with different specificities and suggest that the methylated base may be one of the major antigenic sites of DNA molecules recognized by anti-DNA antibodies spontaneously produced in autoimmune mice.     

Murine lupus anti-DNA antibodies cross-react with the hapten (4-hydroxy-5-iodo-3-nitrophenyl)acetyl, but immunization-induced anti-DNA antibodies do not

The antigen-binding selectivity of 2 sets of anti-DNA antibodies from autoimmune mice and from normal mice was examined. Eighteen affinity-purified anti-DNA auto-antibodies from MRL-lpr/lpr mice were examined for binding to the haptens azobenzenearsonate, phosphorylcholine, (4-hydroxy-3-nitrophenyl)acetyl and (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP). Five of these autoantibodies bound to NIP-protein conjugates. In contrast, none of 12 monoclonal antibodies to single-stranded DNA or left-handed Z-DNA induced by immunization of BALB/c and C57BL/6 mice with nucleic acid antigens reacted with the tested haptens. In a reciprocal test of the relationship between anti-DNA and anti-NIP binding, we examined 24 monoclonal antibodies to NIP, from various strains of mice, for binding to DNA. One such antibody from a BALB/c mouse also bound to DNA. These results are discussed in the context of the mechanisms underlying autoantibody hyperproduction.     

Specificity and immunochemical properties of anti-DNA antibodies induced in normal mice by immunization with mammalian DNA with a CpG oligonucleotide as adjuvant

To elucidate the role of DNA antigen drive in the anti-DNA response, the specificity and immunochemical properties of anti-DNA antibodies induced in normal mice by immunization with double stranded (ds) mammalian DNA with a CpG oligonucleotide (ODN) adjuvant were characterized. Like spontaneous anti-DNA from MRL/lpr mice, the induced anti-DNA bound cross-reactively to DNA from five different species by ELISA. The induced antibodies displayed a predominance of IgG2a and had much lower amount of IgG3 than spontaneous antibodies. Surface plasmon resonance indicated that the induced and spontaneous anti-DNA antibodies have a similar range of avidity and binding kinetics. While sera from the MRL/lpr mice had substantial binding to histones and nucleosomes, the immunized mice had antibody levels to these antigens similar to those of mice treated only with incomplete Freund's adjuvant. Together, these results indicate that normal mice can produce autoantibodies to dsDNA, with a CpG ODN allowing the generation of antibodies resembling those in spontaneous autoimmunity.     

Cross-reactive binding patterns of monoclonal antibodies to DNA are often caused by DNA/anti-DNA immune complexes

Recently, the role of antibodies to DNA in the pathogenesis of systemic lupus erythematosus (SLE) has been reevaluated, since observed cross-reactive binding of anti-DNA to tissue-related antigens might substantially contribute to the inflammatory process of the disease. Evidence of this cross-reactivity has, in part, been obtained from studies with monoclonal anti-DNA. However, we now report that the presence of DNA/anti-DNA immune complexes in monoclonal antibody preparations may be the cause of the observed cross-reactive binding patterns. Studying a panel of anti-DNA producing hybridomas (n = 63), we detected such immune complexes in 76% of the obtained culture supernatants by using an anti-protamine sulphate (PS) ELISA; complexes formed with 1 microgram/ml DNA or more were traced in this assay. In cultures of anti-DNA-producing hybridomas, complexes were detected from day 3 on. Treatment of supernatants with DNase reduced the anti-PS reactivity to an average of only 20% of the original reactivity. Contaminating DNA/anti-DNA immune complexes were found to play no role in the cross-reactivity of anti-DNA antibodies with cardiolipin, a minor role in cross-reactivity with dextran sulphate, but a substantial role in the cross-reactivity with heparan sulphate, histones and other nuclear or cytoplasmic antigens. Our results clearly demonstrate that exclusion of the presence of immune complexes in antibody preparations is a prerequisite when cross-reactivity patterns of anti-DNA antibodies are studied.     

Specificity of mouse hybridoma antibodies to DNA. II. Phospholipid reactivity and biological false positive serological test for syphilis

Using mouse hybridoma monoclonal antibodies to DNA from MRL/lpr and NZB X NZW (B/W F1) mice, the reactivity of anti-DNA antibodies to several phospholipids was analysed. The anti-DNA antibody which reacted with the common antigenic determinants on the phosphate-sugar backbone of nucleic acid could bind to the cardiolipin, but failed to bind to other phospholipids, including VDRL antigen. We tentatively conclude that the anti-cardiolipin antibody is identical with the anti-DNA antibody, but differs from the BFP reactor.     

A polyspecific monoclonal anti-DNA autoantibody also binds to cell-surface protein(s)

A monoclonal anti-DNA autoantibody (EM85) produced in an autoimmune MRL/lpr/lpr mouse was studied. Its antigenic specificity was demonstrated to be directed against single-stranded DNA, double-stranded DNA, and also a variety of polynucleotides. We have recently reported that a murine monoclonal anti-DNA autoantibody produced in autoimmune B/W mice, with specificity for double-stranded DNA, also binds to cell-surface protein(s). We show here that EM85, which recognizes a variety of polynucleotides, also binds to protein(s) on the surface of Raji cells. These data indicate that the antigenic determinant, recognized by this monoclonal anti-DNA antibody, is common to a variety of polynucleotides and cell-surface protein(s).     

The role of antigen specificity in the binding of murine monoclonal anti-DNA antibodies to microparticles from apoptotic cells

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and markers of underlying immune system disturbances. These antibodies bind to both single-stranded and double-stranded DNA, mediating pathogenesis by forming immune complexes. As shown recently, DNA in blood exists in both free and particulate forms, with DNA representing an important component of microparticles. Microparticles are membrane-bound vesicles containing nuclear molecules, released by membrane blebbing during cell death and activation. A panel of monoclonal NZB/NZW F1 anti-DNA antibodies was tested for binding to microparticles generated from apoptotic THP-1 and Jurkat cells. These studies showed that only certain anti-DNA antibodies in the panel, specific for double-stranded DNA, bound to microparticles. Binding to particles was reduced by soluble DNA or DNase treatment. Together, these results indicate that particle binding is a feature of only certain anti-DNA antibodies, reflecting immunochemical properties of the antibodies and the nature of the exposed DNA antigens.     

Preferential Binding of Cross-Reactive Group a Streptococcal Anti-DNA Monoclonal Antibodies to Decondensed Human Sperm DNA

Four murine anti-streptococcal monoclonal antibodies (mAbs) that cross-react with human DNA were evaluated by immunofluorescence flow cytometry for their reactivity with condensed and decondensed human sperm nuclei. All 4 anti-DNA mAbs reacted with condensed sperm nuclei. The reactivity of 3 of these mAbs with decondensed sperm nuclei was 13 to 177 times higher than that found with condensed nuclei. Under identical conditions, mAbs to cytoskeletal/cytocontractile proteins lacked reactivity with decondensed sperm nuclei. Binding of monoclonal anti-DNA antibodies to decondensed sperm nuclei was abolished by preincubation with double-stranded DNA. The preferential binding of anti-DNA antibodies to decondensed sperm DNA suggests the utility of decondensed sperm nuclei as the antigenic substrate for screening anti-DNA antibodies by flow cytometry.     

Preferential Binding of Cross-Reactive Group a Streptococcal Anti-DNA Monoclonal Antibodies to Decondensed Human Sperm DNA

Four murine anti-streptococcal monoclonal antibodies (mAbs) that cross-react with human DNA were evaluated by immunofluorescence flow cytometry for their reactivity with condensed and decondensed human sperm nuclei. All 4 anti-DNA mAbs reacted with condensed sperm nuclei. The reactivity of 3 of these mAbs with decondensed sperm nuclei was 13 to 177 times higher than that found with condensed nuclei. Under identical conditions, mAbs to cytoskeletal/cytocontractile proteins lacked reactivity with decondensed sperm nuclei. Binding of monoclonal anti-DNA antibodies to decondensed sperm nuclei was abolished by preincubation with double-stranded DNA. The preferential binding of anti-DNA antibodies to decondensed sperm DNA suggests the utility of decondensed sperm nuclei as the antigenic substrate for screening anti-DNA antibodies by flow cytometry.     

Microparticles as antigenic targets of antibodies to DNA and nucleosomes in systemic lupus erythematosus

Systemic lupus erythematosus is a prototypic autoimmune disease characterized by antibodies to DNA and other nuclear molecules. While these antibodies can form immune complexes, the mechanisms generating the bound nuclear antigens are not known. These studies investigated whether microparticles can form complexes with anti-DNA and other anti-nucleosomal antibodies. Microparticles are small membrane-bound vesicles released from dead and dying cells; these particles contain a variety of cellular components, including DNA. To assess antigenicity, microparticles generated in vitro from apoptotic cell lines were tested using murine monoclonal anti-DNA and anti-nucleosomal antibodies as well as plasma from lupus patients. Antibody binding was assessed by flow cytometry. As these studies showed, some but not all of the monoclonal antibodies bound to microparticles prepared from apoptotic HL-60, THP-1 and Jurkat cells. For HL-60 cells, both staurosporine and UV radiation led to the production of antigenically active particles. For the anti-DNA antibody with high particle binding, prior treatment of DNase reduced activity. With plasma from patients with SLE, antibody binding to microparticles was present although a clear relationship with anti-DNA antibody levels was not observed. To determine whether lupus plasma contains immune complexes with particle properties, particle preparations were tested for bound IgG by flow cytometry. These studies indicated that lupus plasma contains particles with IgG binding, with numbers correlated with anti-DNA levels. Together, these findings indicate that microparticles display DNA and nucleosomal molecules in an antigenic form and could represent a source of immune complexes in SLE.     

Anti-DNA IgA autoantibodies are spontaneously generated in mouse Peyer's patches.

IgA antibodies in the mucosal immune system are produced specifically to environmental antigens such as virus and bacteria, and possibly to some food components, which will provide a potential luminal antigen, DNA. To study the immune response to DNA in the gut, we established B-cell hybridomas producing IgA monoclonal antibodies (mAb) from Peyer's patches (PP) of non-immunized, non-autoimmune, specific pathogen-free BALB/c mice, and examined their specificity by enzyme-linked immunosorbent assay (ELISA). Three mAb out of 18 bound strongly to self, bacterial and synthetic DNA, with Kd of about 10-7 m. One of the three mAb also reacted with the histone component and another reacted with some mouse food component. The VH genes of these three mAb have not previously been reported to have anti-DNA specificity, and carry putative somatically mutated sites favouring DNA binding in CDR. The features resemble those of anti-DNA antibodies found in human and murine models of systemic lupus erythmatosus (SLE), and are indicative of an antigen-driven selection process. Our findings suggest that even in normal healthy animals, anti-DNA antibodies of IgA isotype can be produced in certain peripheral environments such as in PP by spontaneous antigenic stimulation.     

Monoclonal anti-cardiolipin antibodies bind to DNA

BALB/c mice immunized with the phospholipid, cardiolipin, produced anti-cardiolipin and anti-DNA antibodies. Seven hybridomas derived from spleen cells of the cardiolipin-immunized mice produced cardiolipin-binding monoclonal antibodies that also bound to the polynucleotides DNA, poly(dT), and poly(I). The seven cardiolipin-induced monoclonal antibodies shared idiotypic determinants with a high frequency idiotypic marker of spontaneously expressed anti-DNA autoantibodies of lupus-prone MRL-lpr/lpr mice. The monoclonal antibodies presumably bound to phosphodiester phosphate groups that occur in both polynucleotides and phospholipids. The results imply that production of anti-DNA autoantibodies does not require immunization by DNA.