Arthritis & RheumatismVolume 33, Issue 2 p. 153-159 ArticleFree to Read A role for immunogenic dna in the pathogenesis of systemic lupus erythematosus David S. Pisetsky MD, PhD, Corresponding Author David S. Pisetsky MD, PhD Chief, Rheumatology, Durham VA Medical Center, and Associate Professor of Medicine, Duke University Medical Center Medical Research Service, Durham Veterans Administration Hospital, and the Division of Rheumatology and Immunology, Department of Medicine, Duke University Medical Center, Durham, North Carolina.Durham VA Hospital, Box 151G, 508 Fulton Street, Durham, NC 27705Search for more papers by this authorJane P. Grudier BS, Jane P. Grudier BS Senior Research Technician, Duke University Medical Center Medical Research Service, Durham Veterans Administration Hospital, and the Division of Rheumatology and Immunology, Department of Medicine, Duke University Medical Center, Durham, North Carolina.Search for more papers by this authorGary S. Gilkeson MD, Gary S. Gilkeson MD Associate in Medicine, Duke University Medical Center Medical Research Service, Durham Veterans Administration Hospital, and the Division of Rheumatology and Immunology, Department of Medicine, Duke University Medical Center, Durham, North Carolina.Search for more papers by this author David S. Pisetsky MD, PhD, Corresponding Author David S. Pisetsky MD, PhD Chief, Rheumatology, Durham VA Medical Center, and Associate Professor of Medicine, Duke University Medical Center Medical Research Service, Durham Veterans Administration Hospital, and the Division of Rheumatology and Immunology, Department of Medicine, Duke University Medical Center, Durham, North Carolina.Durham VA Hospital, Box 151G, 508 Fulton Street, Durham, NC 27705Search for more papers by this authorJane P. Grudier BS, Jane P. Grudier BS Senior Research Technician, Duke University Medical Center Medical Research Service, Durham Veterans Administration Hospital, and the Division of Rheumatology and Immunology, Department of Medicine, Duke University Medical Center, Durham, North Carolina.Search for more papers by this authorGary S. Gilkeson MD, Gary S. Gilkeson MD Associate in Medicine, Duke University Medical Center Medical Research Service, Durham Veterans Administration Hospital, and the Division of Rheumatology and Immunology, Department of Medicine, Duke University Medical Center, Durham, North Carolina.Search for more papers by this author First published: February 1990 https://doi.org/10.1002/art.1780330202Citations: 89AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 Tan EM: Autoantibodies to nuclear antigens: their immunobiology and medicine. 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CRC Crit Rev Biochem 4: 305–340, 1977 23 Cheng S-C, Herman G, Modrich P: Extent of equilibrium perturbation of the DNA helix upon enzymatic methylation of adenine residues. J Biol Chem 260: 191–194, 1985 24 Gilkeson GS, Grudier JP, Pisetsky DS: The antibody response of normal mice to immunization with single-stranded DNA of various species origin. Clin Immunol Immunopathol 51: 362–371, 1989 25 Gilkeson GS, Grudier JP, Pisetsky DS: Induction of anti-dsDNA antibodies by immunization with bacterial DNA. J Immunol 142: 1482–1486, 1989 26 Christian CL, Desimone AR, Abruzzo JL: Anti-DNA antibodies in hyperimmunized rabbits. J Exp Med 121: 309–321, 1964 27 Chused TM, Steinberg AD, Talal N: The clearance and localization of nucleic acids by New Zealand and normal mice. Clin Exp Immunol 12: 465–476, 1972 28 Emlen W, Mannik M: Kinetics and mechanisms for removal of single-stranded DNA in mice. J Exp Med 147: 684–699, 1978 29 Bennett RM, Gabor GT, Merritt MS: DNA binding to human leukocytes: evidence for receptor-mediated association, internalization, and degradation of DNA. J Clin Invest 76: 2182–2188, 1985 30 Robey FA, Jones KD, Steinberg AD: C-reactive protein mediates the solubilization of nuclear DNA by complement in vitro. J Exp Med 161: 1344–1356, 1985 31 Rozenberg-Arska M, van Strijp JAG, Hoekstra WPM, Verhoef J: Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. J Clin Invest 73: 1254–1262, 1984 32 Grabar P: Autoantibodies and immunologic theories: an analytic review. Clin Immunol Immunopathol 4: 453–466, 1975 33 Cameron FH, Russell PS, Sullivan J, Geczy AF: Is a Klebsiella plasmid involved in the aetiology of ankylosing spondylitis in HLA B-27 positive individuals? Hum Immunol 20: 563–567, 1983 34 Kraus W, Ohayama K, Snyder DS, Beachy EH: Autoimmune sequence of streptococcal M protein shared with the intermediate filament protein vimentin. J Exp Med 169: 481–492, 1989 35 Chin JH, Kono DH, Yong Z, Park MS, Oldstone MBA, Yu DTY: A Yersinia pseudotuberculosis protein which crossreacts with HLA B-27. J Immunol 139: 3003–3011, 1987 36 Schwimmbeck PL, Yu DTY, Oldstone MBA: Autoantibodies to HLA B-27 in the sera of HLA B-27 patients with ankylosing spondylitis and Reiter's syndrome. J Exp Med 166: 173–181, 1987 37 Query CC, Keene JD: A human autoimmune protein associated with U1 RNA contains a region of homology that is crossreactive with retroviral p30gag antigen. Cell 51: 211–220, 1987 38 Hochberg MC: The incidence of systemic lupus erythematosus in Baltimore, Maryland, 1970–1977. Arthritis Rheum 28: 80–86, 1985 39 Day NK, Geiger H, McLean R, Michael A, Good RA. C2 deficiency: development of lupus erythematosus. 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The administration of certain monoclonal anti-Sm antibodies (2G7, 7.13) induced most MRL/lpr mice to become anti-Sm positive by 5 mo of age, although other anti-Sm monoclonals (Y2, Y12) suppressed the spontaneous response. Positive anti-Sm antibody enhancement occurred efficiently only in MRL/lpr mice and not in other systemic lupus erythematosus mice that have little spontaneous anti-Sm production. The enhancement by anti-Sm antibodies was specific for the anti-Sm response. The mechanism of the passive antibody enhancement was apparently not isotype- or idiotype-related. The fine specificity of the anti-Sm monoclonal antibody may be essential to its enhancing or suppressing effects, since both enhancing monoclonals recognized only the D Sm polypeptide, whereas both suppressing monoclonals saw the D and the B polypeptides. Furthermore, analysis of serial bleeds from unmanipulated MRL mice that developed anti-Sm positivity showed that the D specificity almost always appeared first. We hypothesize, therefore, that those animals in which an anti-Sm response is initiated by D-specific B-cell clones can become serologically positive with the aid of a positive feedback loop. In contrast, animals in which the initial specificity is for both B and D peptides would be prevented from developing a full anti-Sm response.

To characterize further polyspecific interactions of antibodies to DNA, the binding of sera from autoimmune MRL-lpr/lpr mice to Escherichia coli beta-galactosidase (beta-gal) was analyzed. This protein was selected for study because of preliminary observations that sera from autoimmune mice bound unexpectedly to cloned fusion protein constructions containing beta-gal. Using ELISA assays, sera from MRL-lpr/lpr mice demonstrated high levels of antibodies to both DNA and beta-gal, in titers significantly greater than those of BALB/c controls. Affinity chromatography using beta-gal-Sepharose demonstrated that antibodies enriched for anti-beta-gal activity bound both DNA as well as beta-gal, indicating the presence of a population of cross-reactive anti-DNA antibodies. Furthermore, anti-DNA mAb of MRL-lpr/lpr strain origin also bound beta-gal by ELISA, although these levels were lower than those to DNA. Together, these results extend the range of polyspecific binding of murine anti-DNA antibodies to bacterial proteins. They further suggest caution in the interpretation of immunoassays using fusion protein constructions containing beta-gal, especially with sera from autoimmune mice.

To determine the genetic requirements for the development of inflammatory arthritis in MRL-lpr/lpr mice, clinical, serologic, and pathologic features of lpr/lpr and +/+ mice of MRL, B6, C3H, and AKR strains were studied. Arthritis was evaluated by histopathologic examination of the knee joint, while sera were tested for the presence of rheumatoid factor (RF) and anti-DNA activity by ELISA. Of the strains tested to age 7 months, only the MRL-lpr/lpr mice developed histologic evidence of arthritis. All lpr mice, however, produced both IgM RF and IgG RF, although amounts varied among strains. These results indicate that the lpr gene as well as another gene(s) in the MRL background are necessary for the development of inflammatory arthritis and that this lesion may be independent of RF production.

To further assess the mechanism for the induction of anti-DNA antibodies, the response of BALBc mice to immunization with single-stranded DNA of various species origin was determined. Anti-DNA levels of mice immunized with Escherichia coli DNA as complexes with methylated BSA in adjuvant were significantly greater by ELISA than those from mice immunized similarly with calf thymus DNA. Furthermore, comparison of the responses of mice immunized with complexes of DNA from calf thymus, chicken blood, Clostridium perfringens, E. coli, human placenta, or salmon testes indicated that the bacterial DNAs induced the highest antibody levels. The antibody response to E. coli DNA was shown by inhibition ELISA to have two populations, one binding unique determinants in E. coli DNA and the other cross-reactive with determinants expressed on all DNAs tested. These results indicate that DNA molecules, when complexed to a protein carrier, differ in their immunogenic potential, likely because of the presence of unique sequences or structures rarely presented by mammalian host DNA.

To investigate mechanisms for the induction of anti-DNA antibodies in systemic lupus erythematosus (SLE), the specificity of anti-DNA antibodies was determined in sera from SLE patients and normal control subjects. As a marker of these responses, the reactivity to single-stranded DNA of various mammalian and bacterial species origin was tested by enzyme-linked immunosorbent assay. Patients with SLE demonstrated serum antibodies to all six types of DNA tested, whereas normal control subjects showed appreciable antibody responses only to DNA obtained from Micrococcus lysodeikticus (MC) and Staphylococcus epidermidus (SE). Anti-DNA antibodies in normal sera appeared to recognize unique sites on the DNA because MC DNA failed to inhibit antibody binding to SE DNA, and vice versa; in contrast, SLE antibody binding to MC DNA could be inhibited by SE as well as other DNA, suggesting recognition of a more widely shared epitope. The expression in normal sera of antibodies specific for certain bacterial DNA is consistent with their induction by structural determinants on these DNA molecules that are immunogenic. DNA may therefore represent another bacterial macromolecule capable of inducing cross-reactive antibodies in human autoimmune disease.

To investigate whether IgG antinuclear antibodies have cross-reactive rheumatoid factor activity, monoclonal IgG antibodies to DNA and Sm from autoimmune MRL-lpr/lpr mice were assayed by ELISA for binding to IgG antigens. Of the nine anti-DNA and anti-Sm monoclonals tested, six showed significant binding to affinity-purified rabbit IgG (RIgG) and human IgG (HIgG). To confirm that cross-reactivities were due to a single antibody, immunoabsorption of a representative polyspecific monoclonal termed C11 (anti-DNA, anti-Sm) on either Sepharose-DNA or Sepharose-RIgG resulted in marked loss of activity to the three antigens DNA, Sm and RIgG compared with immunoabsorption on Sepharose-bovine serum albumin. The monomolecular nature of the cross-reacting antibody was also suggested by inhibition analysis of C11; DNA inhibited C11 binding to RIgG 64%, whereas Sm inhibited binding to RIgG 33%. Aggregated RIgG and HIgG, however, did not inhibit binding of C11 to DNA, Sm, or solid-phase RIgG, probably reflecting the low affinity of this antibody for fluid phase Ig. Together, these findings suggest that antinuclear autoantibodies of the IgG, as well as the IgM, class have polyspecific IgG binding activity and suggest that IgG antinuclear antibodies may emerge from rheumatoid factor responses.