Antigenic triggers and molecular targets for anti-double-stranded DNA antibodies.

While anti-double-stranded (ds)DNA antibodies are a characteristic serologic hallmark for SLE, the triggering antigen is unknown. Using phage display libraries, we identified DWEYSVWLSN as a peptide mimic of DNA for a pathogenic anti-dsDNA antibody. Peptide immunization of non-autoimmune mice induced anti-dsDNA as well as other lupus-associated antibodies. Molecular analysis of the induced anti-dsDNA antibodies revealed several similarities with anti-dsDNA antibodies that appear spontaneously in lupus mice. Furthermore, lupus-prone mice immunized with this peptide DNA mimic had higher autoantibody titers as well as more severe nephritis. Anti-DNA antibodies may contribute to lupus nephritis via cross-reactivity with renal antigen. Using western blotting of lysates of mesangial cells from a lupus mouse, we found that a pathogenic anti-DNA antibody binds to alpha-actinin. High titers of anti-alpha-actinin antibodies were present in the sera and kidney eluates of lupus mice with active disease. Binding to alpha-actinin was diminished in mesangial cells derived from BALB/c mice, suggesting that target antigen expression may play a role in determining autoantibody binding to the kidney. We conclude that a pathogenic, lupus-like autoantibody response can be induced by a peptide antigen, and that alpha-actinin is a cross-reactive renal target for the pathogenic anti-dsDNA autoantibody response in lupus mice.

Molecular analysis of the autoantibody response in peptide-induced autoimmunity.

Immunization of nonautoimmune BALB/c mice with multimeric DWEYSVWLSN, a peptide mimotope of DNA, induces anti-DNA and other lupus-associated Abs. To further investigate the pathogenesis of the autoantibody response induced by peptide immunization, we generated hybridomas from peptide-immunized mice that bound peptide, dsDNA, cardiolipin, Sm/ribonucleoprotein (RNP), or some combination of these Ags. Analysis of 24 IgM Abs led to the identification of three groups of Abs: 1) Abs reactive with peptide alone, 2) anti-peptide Abs cross-reactive with one or more autoantigens, and 3) autoantibodies that do not bind to peptide. The gene families and particular VH-VL combinations used in those hybridomas binding DNA were similar to those used in the anti-DNA response in spontaneous murine lupus. Another similarity to the spontaneous anti-DNA response was the generation of arginines in the complementarity-determining region-3 of DNA-binding hybridomas. Interestingly, one Ab had the VH-VL combination present in the original R4A anti-DNA Ab used to select the DWEYSVWLSN peptide from a phage display library. Many of the heavy and light chains displayed evidence of somatic mutation, suggesting that they were made by Ag-activated B cells. Analysis of the Ab repertoire in peptide-induced autoimmunity may provide insights into the generation of anti-DNA Abs following exposure to foreign Ag. Furthermore, the recovery of an Ab with the heavy and light chain combination of the Ab originally used to isolate the immunizing peptide confirms the utility of phage display peptide libraries in generating true molecular mimics.

Splice-site mutations: a novel genetic mechanism of Crigler-Najjar syndrome type 1.

Crigler-Najjar syndrome type 1 (CN-1) is a recessively inherited, potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from deficiency of the hepatic enzyme bilirubin-UDP-glucuronosyltransferase. In all CN-1 patients studied, structural mutations in one of the five exons of the gene (UGT1A1) encoding the uridinediphosphoglucuronate glucuronosyltransferase (UGT) isoform bilirubin-UGT1 were implicated in the absence or inactivation of the enzyme. We report two patients in whom CN-1 is caused, instead, by mutations in the noncoding intronic region of the UGT1A1 gene. One patient (A) was homozygous for a G-->C mutation at the splice-donor site in the intron, between exon 1 and exon 2. The other patient (B) was heterozygous for an A-->G shift at the splice-acceptor site in intron 3, and in the second allele a premature translation-termination codon in exon 1 was identified. Bilirubin-UGT1 mRNA is difficult to obtain, since it is expressed in the liver only. To determine the effects of these splice-junction mutations, we amplified genomic DNA of the relevant splice junctions. The amplicons were expressed in COS-7 cells, and the expressed mRNAs were analyzed. In both cases, splice-site mutations led to the use of cryptic splice sites, with consequent deletions in the processed mRNA. This is the first report of intronic mutations causing CN-1 and of the determination of the consequences of these mutations on mRNA structure, by ex vivo expression.