Specificity analysis of monoclonal anti-DNA antibodies.

The specificity of a panel of murine monoclonal anti-DNA antibodies for DNA antigenic determinants was evaluated by testing their relative binding to various animal and bacterial DNAs. The antibody panel consisted of six monoclonal anti-DNAs of MRL-lpr/lpr and B6-lpr/lpr origin, while the antigens tested were calf thymus (CT), salmon testes (ST), E. coli (EC) and Micrococcus (MC) DNA. While all antibodies bound to CT, ST, and EC DNA to a similar extent by direct ELISA, only one showed an equivalent level of interaction with MC DNA. The relationship of antigenic sites recognized by the antibodies was evaluated further by competition ELISA, assessing the ability of the anti-DNAs to block the interaction of a biotinylated anti-DNA with solid-phase DNA antigen. For each of the DNAs tested, two patterns of DNA interaction could be distinguished on the basis of the relative inhibitory activity of the different monoclonals. These results suggest that anti-DNA antibodies can be characterized using naturally occurring DNAs, with the observed patterns of binding suggesting recognition of unique antigenic sites, some of which are discrete and non-overlapping.     

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.     

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.     

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.     

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.     

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.     

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).     

Suppression of anti-DNA antibody production in MRL mice by treatment with anti-idiotypic antibodies.

Antibodies against idiotypic determinants carried by a monoclonal polyspecific natural autoantibody were raised in rabbits and in syngeneic BALB/c mice. These anti-idiotypic antibodies were administered to newborn and to pregnant BALB/c mice and to MRL-lpr/lpr mice. Serial measurements of the idiotypes, naturally occurring autoantibodies, and antibodies obtained after antigenic stimulation were performed in the sera of the injected mice and in the offspring of pregnant mice. No idiotypic suppression was noted in newborn injected mice. Transient suppression of idiotypes recognized by the syngeneic anti-idiotypic antibody was noted in the offspring of pregnant mice injected with the rabbit polyclonal anti-idiotypic antiserum. No changes in naturally occurring autoantibodies or in antibodies appearing after antigenic stimulation were noted in BALB/c mice. In contrast, a significant decrease of spontaneously occurring anti-DNA antibodies was found in MRL-lpr/lpr mice treated with rabbit polyclonal anti-idiotypic antiserum. Furthermore in these mice a slight decrease of anti-TNP antibodies was also observed. These results suggest that anti-idiotypic antibodies directed against natural autoantibodies may play a regulatory role in the immune system; this role is more easily appreciated in mice suffering from immune dysregulation.     

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.     

Expression of a highly conserved anti-DNA idiotype in normal and autoimmune mice

The specificity and idiotypic relationships of a monoclonal anti-DNA antibody were investigated to evaluate genetic control in this autoantibody response. 6/0 is an IgG2a monoclonal anti-DNA derived by the fusion of spleen cells from an autoimmune MRL-lpr/lpr mouse and the cell line NS1. By an enzyme-linked immunosorbent assay (ELISA) for anti-DNA, 6/0 demonstrated preference for single-stranded DNA and bound deoxyribo- and ribohomopolymers of dissimilar base composition. The control of 6/0 expression was evaluated by idiotypic analysis using a rabbit anti-6/0 antiserum made specific by absorption with the BALB/c myelomas UPC 10 (IgG2a) and MOPC 21 (IgG1). The resulting preparation was fractionated by BALB/c IgG affinity columns to provide antibodies to idiotypic determinants essentially unique to 6/0 and those commonly expressed in sera. The commonly expressed 6/0 idiotype was found in sera of ten inbred strains of mice and was not exclusive to the autoimmune strains. MRL-lpr/lpr and A/J strain mice displayed idiotype levels almost fivefold greater than other strains, with 6/0 idiotype-bearing antibodies having serum concentrations as high as 1 mg/ml. Levels of the 6/0 idiotype, however, did not correlate with anti-DNA levels among the various strains. In addition to mice, the majority of individuals of three inbred rat strains showed detectable 6/0 idiotype in their sera. These results suggest that the 6/0 idiotype, although identified using a monoclonal anti-DNA antibody, represents a framework determinant that is phylogenetically conserved. The mechanisms for the expression of this determinant may differ among the normal and autoimmune strains.     

Characterization and idiotypic analysis of an anti-RNP monoclonal antibody

To investigate mechanisms of anti-RNP antibody expression in autoimmune disease, idiotypes of a monoclonal anti-RNP of murine origin were analysed. This antibody, designated 4L1, was obtained from a MRL-lpr/lpr mouse and shown to have anti-RNP specificity by gel analysis of radiolabelled cellular RNA. An anti-idiotypic antiserum was prepared in a rabbit to 4L1 and rendered specific for idiotype by absorption with IgG from B6 mice and two BALB/c myelomas of the same chain composition as 4L1 (IgM kappa). In competition ELISA assays, this antiserum detected idiotypes commonly expressed in sera of MRL-lpr/lpr mice irrespective of the presence of anti-RNP. These idiotypes were not exclusive to this autoimmune strain, however, and could also be identified in normal mice. To identify other antibodies with this idiotype, a panel of MRL hybridomas was tested. This analysis demonstrated idiotypic cross-reactivity between 4L1 and two anti-Sm monoclonal antibodies derived from another animal. These results suggest that 4L1 belongs to a larger idiotype bearing family only some of whose members may have aberrant expression.     

The clearance of a monoclonal anti-DNA antibody following administration of DNA in normal and autoimmune mice

To study the assembly of DNA-anti-DNA complexes in vivo, we have measured the clearance from blood and organ localization of a murine IgG2a monoclonal anti-DNA antibody, called 6/0, following the infusion of DNA intravenously or intraperitoneally. Intraperitoneal DNA caused a profound acceleration of 6/0 anti-DNA clearance that was dose dependent and demonstrable after the infusion of as little as 1.9 microgram per gram of body weight of single-stranded DNA. The antibody was cleared primarily in the liver without increased deposition in the kidney. Intraperitoneal infusions of DNA also accelerated the clearance of 6/0 in autoimmune MRL-lpr/lpr mice. In contrast, intravenous DNA given in comparable doses caused only a slight increase in 6/0 antibody clearance; this accelerated clearance was seen only at low antigen doses and only during the first 10 min following DNA infusion. Using double-radiolabeling techniques, 6/0 and Cl.18, an IgG2ak myeloma protein without anti-DNA activity, were found to disappear from blood at a comparable rate in both B6D2 mice and MRL-lpr/lpr mice. These results suggest that the DNA-anti-DNA immune complexes can form in vivo but that this process is profoundly affected by the manner in which DNA enters the circulation. In addition, the results suggest that DNA-dependent clearance is not a major pathway for anti-DNA metabolism in normal or at least one strain of autoimmune mice.     

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.     

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.     

A multidot immunobinding assay for autoimmunity and the demonstration of novel antibodies against retroviral antigens in the sera of MRL mice

A dot immunobinding assay procedure has been developed for autoantibodies, and has been applied to the sera of MRL lpr/lpr mice. The profiles thus obtained include assays for circulating immune complexes, and antibodies against single-stranded DNA, double-stranded DNA, ribosomes, soluble nuclear deoxyribonucleoprotein, and retroviral antigens. Part of the data was compared with ELISA results. The anti-DNA assays were specific, as some individual sera show exclusively anti-double-stranded DNA specificity. The finding of anti-ribosomal antibodies in these mice extended the analogy between the murine disease and human systemic lupus erythematosus. The specificity of the anti-retroviral antibodies was examined following electrophoretic separation of the antigens and blotting on nitrocellulose. Previously undescribed classes of anti-retroviral antibodies were found. Circulating anti-retroviral protein p30 was found in all sera having high anti-retroviral titers.     

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.     

The anti-La response of a single MRL/Mp-lpr/lpr mouse: Specificity for DNA and VH gene usage

Autoantibodies to ribonucleoproteins (RNP) occur prominently in human systemic lupus erythematosus and murine lupus models. In previous studies we demonstrated a relationship in MRL/Mp-lpr/lpr (MRL/lpr) mice between antibodies to Sm, an RNP autoantigen, and antibodies to DNA. Thus, many anti-Sm monoclonals bound DNA and expressed the same V region genes as anti-DNA. In addition, many had multiple V_HCDR3 Arg residues suggestive of selection by DNA, and some had somatic mutations suggesting selection for mutant B cells by DNA. To determine whether autoantibodies to other RNP antigens are also associated with the anti-DNA response, we have analyzed the response to the La RNP. Six anti-La B cell hybridomas were generated from a single MRL/lpr mouse. Southern blot analysis of Ig V gene rearrangements and V gene sequences indicated two clonally related pairs, suggesting an oligoclonal response. Antibodies from all six hybridomas bound single-stranded DNA, while antibodies from five hybridomas bound double-stranded DNA. Two hybridomas expressed a V_H7183 gene which is used by members of two previously reported anti-DNA clones and two anti-Sm/DNA clones of MRL/lpr origin. These data demonstrate an association between the anti-La and anti-DNA responses in MRL/lpr mice, suggesting that cross-reactive anti-RNP and anti-DNA responses are a general feature of autoimmunity in this lupus model.     

Binding of cytoskeletal proteins by monoclonal anti-DNA lupus autoantibodies

Monoclonal anti-DNA antibodies produced by hybridomas derived from MRL-lpr/lpr mice and human lupus patients were found to bind to the cytoskeleton of mink lung cells. When tested by indirect immunofluorescence, 17/29 human monoclonal anti-DNA antibodies reacted with the cytoskeleton; 4 of the 29 also produce antinuclear reactions with epithelial cells. The cytoskeletal staining was not inhibited by prior treatment of the cells with DNase, but it was completely blocked by prior incubation of the monoclonal antibodies with DNA and other nucleic acids. The ability of the polynucleotides to inhibit the cytoskeletal staining corresponded to their ability to bind to the antibodies in competitive immunoassays. An (Fab')2 preparation of a monoclonal antibody bound to the cytoskeleton as well as the whole immunoglobulin. The effect of colcemid on the staining pattern, the blocking effect of a monoclonal antivimentin antibody, and results with nitrocellulose blots of cellular proteins indicated that the cytoskeletal protein to which the antibodies bound was vimentin.     

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.     

Administration of monoclonal anti-Sm antibody prolongs the survival and renal function of MRL-lpr/lpr mice.

The repeated administration of a monoclonal anti-Sm antibody (KSml) resulted in a significant prolongation of life in MRL-lpr/lpr lupus mice with a 50% mortality of 36 weeks compared with 18-24 weeks in the control groups. Control animals injected with APC11 (a myeloma protein of the same isotype) lived no longer than an untreated group. In addition the renal function as assessed by blood urea levels was less impaired in the KSml-injected mice than in the controls. All KSml-injected mice showed the presence of circulating anti-Sm antibodies which had a different Sm polypeptide binding specificity from that of the injected monoclonal antibody; the increased prevalence of these antibodies compared to the control mice (10-30%) suggested that the anti-Sm antibody response had been induced. The increased longevity in the KSml-treated animals was not associated with alterations in the anti-dsDNA antibody response. The data suggest that administration of anti-Sm antibodies modifies the course of murine lupus.