Anti-DNA antibodies in systemic lupus erythematosus and their induction in mice

Abstract

Autoantibodies capable of binding to DNA are present in significantly high levels in patients with systemic lupus erythematosus (SLE). The origin of these autoantibodies is still unresolved, but it is generally accepted that DNA-anti DNA immune complexes may play a significant role in the immunopathogenesis of SLE. Further, these autoantibodies are important serological markers of SLE and are of immense value in diagnosis and in follow up of disease activity. Anti DNA antibodies are also present in the sera of some autoimmune, SLE prone mouse strains and are implicated in disease pathogenesis, mainly glomerulonephritis. Studies using monoclonal antibodies from SLE patients and SLE prone mice (NZB × NZW, MRL/lpr) have shown that these anti DNA antibodies are polyspecific and bind to phospholipids, cytoskeletal proteins, and membrane proteins. In view of the poor immune response to DNA in experimental animals, earlier workers have suggested that the putative antigen in SLE may actually be protein(s). Antibodies against bacterial proteins are known to cross react with DNA. It is speculated that anti DNA antibodies may develop as a consequence of molecular mimicry, where DNA shares identical epitopes with certain bacterial or viral proteins or phospholipids. Further, abnormalities in the idiotype-anti idiotype network may also contribute to the spontaneous production of anti DNA antibodies. The available evidence suggests that DNA per se may not be the antigen responsible for induction of anti DNA autoantibodies that contribute to the pathogenesis of SLE and related autoimmune diseases. Hence, studies were carried out to determine the subclass distribution of anti DNA antibodies of the IgG isotype and to induce anti DNA antibodies in normal mice. The following is a synopsis of these investigations. ________________________________________ Development of AB microELISA The avidin-biotin microELISA (AB microELISA), originally developed to measure antigen specific IgE, was adapted for quantitation of IgG antibodies to nucleic acids (native DNA, denatured DNA, double stranded DNA, and RNA) in sera of SLE patients. The AB microELISA enables quantitation of nucleic acid specific IgG in less than 4 hours. IgG antibodies to nDNA, dnDNA, dsDNA and RNA were detected in 85.2%, 92.6%, 85.2% and 77.8% of SLE sera, respectively. The assay is highly specific and more sensitive than other reported quantitative immunoassays. An in house serum reference standard (pooled from 10 SLE patients) was used to express antibody titres in SLE units. The method is suitable for routine clinical screening. ________________________________________ Presence of DNA specific IgG4 antibodies DNA specific IgG4 antibodies were demonstrated in SLE sera using microtiter solid phase radioimmunoassay. One patient, seronegative for IgG anti DNA antibodies by conventional assays, showed exclusive presence of IgG4 anti DNA autoantibodies. These antibodies, directed against dsDNA conformation, cross reacted with chondroitin sulfate, dermatan sulfate, and heparin. ________________________________________ Induction of anti DNA antibodies in Balb/c mice Immunization of Balb/c mice with DNA-methylated BSA (DNA mBSA) complex in alum resulted in induction of anti DNA antibodies of both IgG and IgE isotypes. More than 90% of induced IgG anti DNA antibodies were of the IgG1 subclass-distinct from IgG2a/IgG2b predominance seen in SLE prone mice. Competitive ELISA and SPRIA inhibition demonstrated specificity for nDNA, dnDNA, and dsDNA, but not RNA. These antibodies cross reacted with cibacron blue and chondroitin sulfate, but not with other proteoglycans, nucleosides, or nucleotides. Passive cutaneous anaphylaxis in rats confirmed that these antibodies can induce mast cell degranulation in vivo, supporting the concept that IgE anti DNA antibodies may contribute to immediate type hypersensitivity phenomena (e.g., urticaria) and localization of IgE-nucleic acid complexes in SLE. ________________________________________ Induction of anti DNA antibodies by immunization with serum albumins Immunization of rabbits and multiple mouse strains with bovine serum albumin (BSA) resulted in induction of anti DNA antibodies demonstrable by AB microELISA. Binding was abolished by DNase I treatment of DNA coated wells. Cross inhibition using affinity purified anti BSA IgG antibodies revealed that a subset of anti BSA antibodies recognizes DNA, with higher affinity for BSA than for DNA. Immunization with serum albumins from different species showed that only BSA and guinea pig serum albumin (GSA) induced anti DNA antibodies. Binding inhibition studies showed that RNA, tRNA, poly I·poly C, and cardiolipin-but not mononucleotides-could inhibit binding. Four of 30 SLE patients had demonstrable BSA specific antibodies. Immunization of Balb/c mice with mouse anti BSA idiotypic antibodies led to production of anti idiotypic antibodies capable of competing with both BSA and DNA for binding to the idiotypic antibody. These anti idiotypic antibodies recognized autoantibodies in the sera of 16 out of 30 SLE patients. The correlation between idiotypes recognizing DNA and BSA specific anti idiotypic antibodies was strong. ________________________________________ Induction of anti DNA antibodies using BSA conjugates of nucleosides Immunization of Balb/c mice with BSA conjugates of guanosine, adenosine, and ATP resulted in two distinct populations of antibodies (carrier specific and hapten specific), separable by immunoaffinity chromatography. Antibodies in both groups recognized DNA, though only one population bound the hapten. Inhibition studies showed: • BSA specific antibodies cross reacted with both nDNA and dnDNA • Hapten specific antibodies bound only to dnDNA ________________________________________ Conclusion These studies support the theory of molecular mimicry in the production of autoantibodies and highlight the role of idiotype-anti idiotype interactions in the induction of anti DNA antibodies. They reinforce the notion that DNA itself may not be the primary immunogen responsible for anti DNA autoantibody production in SLE; instead, structurally mimicking antigens such as proteins may play a central role.