Immunization of rabbits with purified RNA polymerase I induces a distinct population of antibodies against nucleic acids as well as anti-RNA polymerase I antibodies, both characteristic of systemic lupus erythematosus.

Abstract

Rabbits were immunized with either RNA polymerase I or poly(A) polymerase that had been purified to apparent homogeneity and was devoid of nucleic acids. Sera from rabbits thus immunized were screened for antibodies against nucleic acids. All seven rabbits injected with RNA polymerase I but none of the four rabbits immunized with poly(A) polymerase produced anti-nucleic acid antibodies. Anti-RNA polymerase I antibodies were induced after a single injection of the enzyme. Anti-polynucleotide antibodies were not detectable until after the second immunization. Anti-RNA polymerase I antibodies could be detected with as little as 100 pg of purified RNA polymerase I in the radioimmunoassay. At least 50 ng of poly(A) or 200 ng of DNA was required to detect anti-nucleic acid antibodies. The immunoreactivity of anti-RNA polymerase I antisera was greater with synthetic polynucleotides than with DNA, particularly early in the immunization schedule. Alkaline phosphatase treatment of poly(A) to remove 5' phosphates nearly abolished its antigenicity with respect to the early sera and decreased antibody binding of later sera by 60%. These results indicate that the anti-nucleic acid antibodies produced early were primarily directed against determinants including the 5'-terminal phosphates while antibodies produced later were directed against other sites. The antinucleic acid antibodies and anti-RNA polymerase I antibodies formed two distinct populations that were not immunologically crossreactive. We suggest that after injection, RNA polymerase I becomes associated with the nucleic acids present in blood plasma which renders them immunogenic; thus, association of nucleic acids with autoimmunogenic RNA polymerase I may be one of the mechanisms by which anti-DNA antibodies are induced in systemic lupus erythematosus.Sera from patients with systemic lupus erythematosus (SLE) and other rheumatic autoimmune diseases contain antibodies against a variety of proteins and nucleic acids in the cell nucleus (reviewed in ref. 1). It has been postulated that phosphodiester bonds of nucleic acids are autoantigenic determinants in both human SLE (2) and the murine lupuslike syndrome (3). Recently, we identified RNA polymerase I (4) and protein kinase NII (5) as target nuclear antigens in SLE, rheumatoid arthritis, and mixed connective tissue disease and demonstrated (6) that phosphorylation of the polymerase can dramatically increase its antigenicity with respect to SLE patient autoantibodies. The importance of phosphate groups in the autoantigenicity of both RNA polymerase I and DNA and the recent reports that anti-DNA autoantibodies were capable of reacting with phospholipids (2, 3) and proteins (7) suggested that anti-RNA polymerase I antibodies might crossreact with DNA. In addition, since RNA polymerase I can interact with DNA as part of its biological function, it is plausible that association of the immunogenic enzyme with DNA, which is present in blood plasma (8), might induce production of anti-DNA antibodies. These possibili...

Immunization of nonautoimmune mice with DNA binding domains of the largest subunit of RNA polymerase I results in production of anti-dsDNA and anti-Sm/RNP antibodies

“…Anti-RNAPI(S1)-NT and -BD antibody production by rabbits immunized with lupus anti-DNA We reported previously that rabbits immunized with purified rat hepatoma RNAPI holoenzyme produce anti-DNA antibodies [34]. Subsequently, we reported that affinity purified anti-DNA (IgG or IgG Fab) from SLE patients or lupus mice produced anti-RNAPI antibodies directed primarily against the S1 of the polymerase [35,36].…”

“…Previously, we reported that immunization of rabbits with RNA polymerase I (RNAPI) holoenzyme resulted in anti-DNA antibody production [34]. Conversely, immunization of rabbits with anti-dsDNA purified from the sera of SLE patients or lupus mice induced production of anti-RNAPI antibodies directed primarily against the largest subunit (194 kDa; S1) of RNAPI [35,36].…”

Immunochemical and molecular characterization of anti-RNA polymerase I autoantibodies produced by tight skin mouse.

“…In previous reports it has been shown that the autoantibodies against rat hepatoma RNA pol I were highly specific and did not cross-react with poly(A) polymerase (25). Therefore, we have examined whether the TSK mAbs also exhibit a similar property by competitive inhibition assays.…”

“…The presence of anti-RNA pol I antibodies in the sera of patients with systemic sclerosis (7) or other rheumatic diseases (6) and in MRL mice (8) was studied using rat hepatocyte RNA pol I (25). In this study, besides the rat enzyme, we have used bacterial (E. coli) and viral (T7 phage) enzymes, since it is well known that self-epitopes on housekeeping enzymes (15), structural proteins (26), and cell receptors (27) Fig.…”

Immunization of Rabbits with Spermine Induces Antibodies to Self Antigens

“…In some cases, it was found that the sera raised in this way were able to cross-react with antigens other than the ones used for im munization. For instance, in New Zealand rabbits immu nized with RNA-polymerase, antibodies reacting with nu cleic acids were obtained [7], while in animals of the same breed immunized with a mixture of total histones complexed with RNA, antibodies were produced that reacted with dsDNA, Ub, a peptide of ubiquitinated H2A, pep tides of Sm-D antigen and poly-(ADP)-ribose [8],…”