Structure and function of anti-DNA autoantibodies derived from a single autoimmune mouse.

Abstract

Four monoclonal anti-DNA antibodies derived from a single autoimmune MRL/lpr mouse were studied. Three of these antibodies showed similarities in DNA binding; the fourth had a much higher specific activity for singlestranded DNA and, in addition, was unique in binding doublestranded DNA and cardiolipin. Complete nucleotide sequences of heavy-and light-chain variable regions demonstrated that all four antibodies are clonally related. The sequences also showed numerous somatic mutations, the distribution of which suggests that positive selection by antigen operated on these clonally related autoantibodies.Antibodies to DNA (anti-DNA) are prominent autoantibodies in the sera of patients with systemic lupus erythematosus (SLE) and of MRL/Mp-lpr/lpr (MRL/lpr) mice, a murine model of SLE. The levels of these autoantibodies have diagnostic and prognostic significance. Moreover, a direct role of anti-DNA in disease pathogenesis has been established by correlation of antibody levels with disease activity and identification of anti-DNA at sites of tissue injury (1-5). Two competing models to explain anti-DNA production have emerged. The first suggests that anti-DNA result from polyclonal B-lymphocyte activation and are the consequence of antigen-nonspecific disturbances of B and/or T cells (6-8).The extensive diversity of anti-DNA has been interpreted as evidence for the polyclonal activation model. In addition, reports of idiotypes expressed on a large fraction of anti-DNA suggest that anti-DNA are encoded by germ-line variable (V)-region genes (9-13). The second model proposes that antigen stimulates anti-DNA production. Although nonspecific immune disturbances may exist in SLE patients, in this model their role is to allow or promote induction of autoantibodies by antigen (14-17). It is reasonable to postulate DNA as the inciting antigen, although a non-DNA self or foreign antigen could fulfill this role (8,(17)(18)(19). To provide further insight into the etiology and structure of anti-DNA, we investigated the specificity and primary structure of monoclonal anti-DNA obtained from spleen cells of an autoimmune MRL/lpr mouse.t The goal was to determine the genetic basis of anti-DNA diversity and thereby to distinguish these models of anti-DNA production.MATERIALS AND METHODS Antibodies. Antibodies used in this study were derived from the fusion of spleen cells from a 6-month-old MRL/lpr mouse (obtained from The Jackson Laboratory, Bar Harbor, ME) with the myeloma cell line NS1 as described (20). Antibodies initially were identified by ELISA using as an antigen source rabbit thymus extract (Pel-Freeze, Rogers, AR) as previously described (21) and subsequently were shown to be anti-DNA (see below). The heavy-and lightchain isotype of all four antibodies, y3 K, was determined using subclass-specific reagents (Litton Bionetics, Charleston, SC).Specificity Analyses. The specificity of antibodies for DNA antigens was tested as described (22). Antibodies to cardiolipin were assayed by ELISA using bovine heart cardiolip...

Molecular Evolution of Catalytic Antibodies in Autoimmune Mice

“…Could the catAbs be derived from an abnormal repertoire that specifically exists in or is enriched in MRL/lpr mice? Autoreactive Abs, such as anti-nuclear Abs, Abs to ribonucleoproteins, ssDNAs, or dsDNAs, which are normally eliminated, but have a high incidence in autoimmune strains such as MLR/lpr mice (33,34), seem like the most likely source of the catAbs. As a consequence, such mouse strains develop multisystem autoimmunity, including a lupus-like glomerulonephritis and arthritis (35,36).…”

Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display

“…The Ig V region sequences of the H and L genes were submitted to the DNAPlot program for IMGT (40) and to FASTA, BLAST-n, and WU-BLASTXϩBEAUTY programs for GenBank/EMBL. The probability that the excess of replacement (R) mutations in the complementarity-determining region (CDR) or framework region (FR) (with respect to the closest germline genes) arose by chance was calculated according to the binomial distribution model (41,42)…”

T Cell Studies in a Peptide-Induced Model of Systemic Lupus Erythematosus

“…In murine lupus models, disruption of T cell activation (2)(3)(4)(5)(6)(7)(8) or an absence of T cells (9,10) abrogates autoantibody production and glomerulonephritis. In addition, nephritogenic anti-dsDNA Abs are high affinity, somatically mutated IgG Abs, all of which are features of a T cell-dependent, Ag-driven immune response (11,12). Sequence analyses of anti-dsDNA Abs, showing a greater than random ratio of replacement to silent mutations in complementarity-determining regions, suggests that DNA could be the selecting Ag for these Abs (13)(14)(15).…”

“…The autospecificities that are correlated with disease manifestations of SLE, specifically anti-dsDNA Abs, are high affinity, IgG, somatically mutated Abs produced by B cells in a T cell-dependent fashion (11,12). Peptide-induced SLE is characterized by antipeptide, anti-DNA cross-reactive Abs that also display somatic mutation (18,21).…”