Dan Eilat

Lupus anti-DNA autoantibodies cross-react with a glomerular structural protein: a case for tissue injury by molecular mimicry

Anti‐DNA autoantibodies are the hallmark of human and murine systemic lupus erythematosus (SLE), an autoimmune rheumatic disease of unknown etiology. Some of these antibodies are believed to be pathogenic for kidney tissue and to initiate immune glomerulonephritis. However, the mechanisms by which anti‐DNA antibodies participate in tissue injury remain controversial. We have studied the in vivo pathogenicity of anti‐DNA monoclonal antibodies in immune deficient mice, using a panel of murine B cell hybridomas. No consistent genetic or immunochemical differences were found between pathogenic and non‐pathogenic anti‐DNA antibodies. However, the two antibody populations differed in their cross‐reaction with the acidic actin‐binding protein, α‐actinin, that is known to play a major role in the structural integrity of glomerular filtration components. These results suggest that kidney dysfunction in SLE may be facilitated by protein‐nucleic acid antigenic mimicry.

Murine B Cell Response to TLR7 Ligands Depends on an IFN-β Feedback Loop

Type I IFNs play an important, yet poorly characterized, role in systemic lupus erythematosus. To better understand the interplay between type I IFNs and the activation of autoreactive B cells, we evaluated the effect of type I IFN receptor (IFNAR) deficiency in murine B cell responses to common TLR ligands. In comparison to wild-type B cells, TLR7-stimulated IFNAR−/− B cells proliferated significantly less well and did not up-regulate costimulatory molecules. By contrast, IFNAR1−/− B cells did not produce cytokines, but did proliferate and up-regulate activation markers in response to other TLR ligands. These defects were not due to a difference in the distribution of B cell populations or a failure to produce a soluble factor other than a type I IFN. Instead, the compromised response pattern reflected the disruption of an IFN-β feedback loop and constitutively low expression of TLR7 in the IFNAR1−/− B cells. These results highlight subtle differences in the IFN dependence of TLR7 responses compared with other TLR-mediated B cell responses.

Treatment with a Laminin-Derived Peptide Suppresses Lupus Nephritis

The role of DNA as the target for pathogenic lupus autoantibodies in systemic lupus erythematosus is equivocal and renal damage may be due to cross-reactivity of lupus Abs with glomerular components. We have previously shown that lupus autoantibodies bind to the laminin component of the extracellular matrix. In the present work, we have analyzed the fine specificity of the interaction of pathogenic murine lupus autoantibodies with this molecule and the effect of inhibiting their binding to laminin during the course of the disease. We have found that pathogenic murine lupus autoantibodies react with a 21-mer peptide located in the globular part of the α-chain of laminin. Immunization of young lupus-prone mice with this peptide accelerated renal disease. Analysis of transgenic, congenic, and RAG-1−/− mice confirmed the importance of this epitope in the pathogenesis of lupus renal disease. We have synthesized a panel of peptides that cross-react with the anti-laminin Abs and have found that the binding of lupus autoantibodies to the extracellular matrix could be inhibited in vitro by some of these competitive peptides. Treatment of MRL/lpr/lpr mice with these peptides prevented Ab deposition in the kidneys, ameliorated renal disease, and prolonged survival of the peptide-treated mice. We suggest that laminin components can serve as the target for lupus Abs. The interaction with these Ags can explain both the tissue distribution and the immunopathological findings in lupus. Moreover, inhibition of autoantibody binding to the extracellular matrix can lead to suppression of disease.

An autoimmune disease prevented by anti- retroviral drugs

BackgroundBoth Aicardi-Goutières syndrome, a Mendelian mimic of congenital infection, and the autoimmune disease systemic lupus erythematosus can result from mutations in the gene encoding the enzyme Trex1. In mice, the absence of Trex1 causes severe myocarditis. The enzyme is thought to degrade endogenous retroelements, thus linking them to autoimmune disease. However, inhibition of reverse transcription by the inhibitor zidovudine (AZT) did not ameliorate the disease, weakening the link to retroelements.FindingsHere, we show that two other FDA-approved drugs that inhibit reverse transcriptase can ameliorate the myocarditis in Trex1-null mouse.ConclusionsThe result suggests that retroelements contribute to this hereditary form of autoimmunity, and that treatment with retroelement inhibitors might ameliorate Aicardi-Goutières syndrome in humans.

STRUCTURE-FUNCTION CORRELATES OF AUTOANTIBODIES TO NUCLEIC ACIDS. LESSONS FROM IMMUNOCHEMICAL, GENETIC AND STRUCTURAL STUDIES

Nucleic acid binding autoantibodies are the hallmark of the human autoimmune disease, systemic lupus erythematosus (SLE) and are also prevalent in mouse models of this disease. The immunologic stimuli for the production of these antibodies as well as their pathogenic mechanisms are not well understood. However, extensive immunochemical and genetic studies, together with initial crystallographic analysis and computer modeling, have suggested several structure-function correlates which will form the basis for future research. The anti-DNA and anti-RNA autoantibodies comprise a continuous spectrum of specificities in which a delicate balance exists between the binding to the sugar-phosphate backbone and the interactions with the heterocyclic bases of the nucleic acid. Prominent in these interactions are the products of specific V-region immunoglobulin genes, some of which appear to be uniquely suitable for nucleic acid binding. Other structural elements encoded by D minigenes, N sequences and somatic mutations, help to increase the affinity of the binding interaction, and may also increase the repertoire of nucleic acid binding antibodies by combining with a relatively large number of additional V-gene products. Initial crystallographic analyses of anti-DNA antibodies indicate some fundamental differences in the structure and shape of ssDNA and dsDNA antibody combining sites. However, they also suggest a considerable degree of flexibility of both antibody and antigen, which is induced by their binding interaction.

Interaction of the NK Cell Inhibitory Receptor Ly49A with H-2Dd:Identification of a Site Distinct from the TCR Site

Natural killer cell function is controlled by interaction of NK receptors with MHC I molecules expressed on target cells. We describe the binding of bacterially expressed Ly49A, the prototype murine NK inhibitory receptor, to similarly engineered H-2Dd. Despite its homology to C-type lectins, Ly49A binds independently of carbohydrate and Ca2+ and shows specificity for MHC I but not bound peptide. The affinity of the Ly49A/H-2Dd interaction as determined by surface plasmon resonance is from 6 to 26 microM at 25 degrees C and is greater by ultracentrifugation at 4 degrees C. Biotinylated Ly49A stains H-2Dd-expressing cells. Competition experiments indicate that the Ly49A and T cell receptor (TCR) binding sites on MHC I are distinct, suggesting complex regulation of cells that bear both TCR and NK cell receptors.

Monoclonal autoantibodies: An approach to studying autoimmune disease

Hybridoma technology has paved the way for new insights into mechanisms of autoimmunity and autoimmune disease. Autoantibodies and autoantigens can now be studied at the level of the individual molecule. The specificity of the autoimmune reaction, as well as its pathogenetic role in disease, may be more accurately assessed. Monoclonal autoantibodies which bind to red blood cells, IgG, DNA, RNA and ribonucleoprotein complexes have been prepared by several groups of investigators. The initial molecular and functional characterization of these immunoglobulins and their corresponding antigens is described.

The efficiency of B cell receptor (BCR) editing is dependent on BCR light chain rearrangement status

Anti‐DNA knock‐in mice serve as models for studying B cell tolerance mechanisms to a ubiquitous antigen. We have constructed six strains of double transgenic (C57BL/6×BALB/c)F1 mice, eachexpressing an unmutated or somatically mutated anti‐DNA heavy (H) chain, combined with one of three different light (L) chains, namely Vκ1‐Jκ1, Vκ4‐Jκ4 and Vκ8‐Jκ5. In vitro analysis of the various Ig H/L chain combinations showed that all had a similar specificity for single‐stranded DNA and double‐stranded DNA, but that antibodies encoded by the mutated H chain had higher affinities for the autoantigen. None of the targeted mouse strains exhibited significant levels of serum anti‐DNA activity. However, while B cells from mice carrying the Vκ1‐Jκ1 transgenic L chains were tolerized almost exclusively by L chain receptor editing in an affinity‐independent manner, the mice expressing Vκ8‐Jκ5 L chains have utilized affinity‐dependent clonal anergy as their sole mechanism of B cell tolerance. Vκ4‐Jκ4 targeted mice exhibited an intermediate phenotype with respect to these two mechanisms of B cell tolerance. Our results suggest that receptor editing is the preferred mechanism of B cell tolerance and that the efficiency of L chain editing is directly related to the number of available Jκ segments on the expressed Vκ allele.

Autoimmune NZB/NZW F1 mice utilize B cell receptor editing for generating high‐affinity anti‐dsDNA autoantibodies from low‐affinity precursors

We have previously constructed knock‐in (C57BL/6×BALB/c) F1 mice, each expressing an anti‐DNA heavy (H) chain (D42), combined with one of three different light (L) chains, namely Vκ1‐Jκ1, Vκ4‐Jκ4 or Vκ8‐Jκ5. All of these H/L chain combinations bind DNA with similar affinity and fine specificity. However, while mice carrying Vκ1‐Jκ1‐transgenic L chain were tolerized almost exclusively by L chain receptor editing, the mice expressing Vκ8‐Jκ5 L chains utilized clonal anergy as their principal mechanism of B cell tolerance. Vκ4‐Jκ4 targeted mice exhibited an intermediate phenotype. In the present study, these three H/L chain combinations were backcrossed onto the autoimmune NZB/NZW F1 mice. We find that the mechanism of clonal anergy is abrogated in these mice, but that receptor editing is maintained. Moreover, diseased NZB/NZW mice utilize L chain secondary rearrangements for the generation of high‐affinity, anti‐dsDNA‐producing B cells from low‐affinity precursors. The edited B cell clones are not deleted or anergized in the autoimmune animal; rather they are selected for activation, class‐switching and affinity maturation by somatic mutation. These results suggest that B cell receptor editing plays an important role not only in tolerance induction, but also in generating high‐affinity autoreactive B cells in autoimmune diseases.

Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir

Raltegravir is a recently, Food and Drug Administration-approved, small-molecule drug that inhibits retroviral integrase, thereby preventing HIV DNA from inserting itself into the human genome. We report here that the activity profile of raltegravir on the replication of murine leukemia virus is similar to that for HIV, and that the drug specifically affects autoimmune disease in mice, in which endogenous retroelements are suspected to play a role. While NZW and BALB/c mice, which do not succumb to autoimmune disease, are not affected by raltegravir, lupus-prone (NZBxNZW) F1 mice die of glomerulonephritis more than a month earlier than untreated mice. Raltegravir-treated NZB mice, which share the H-2 haplotype with BALB/c mice, but which are predisposed to autoimmune hemolytic anemia, develop auto-antibodies to their red blood cells >3 months earlier than untreated mice of the same strain. Because nonautoimmune mice are not affected by raltegravir, we consider off-target effects unlikely and attribute the exacerbation of autoimmunity to the inhibition of retroviral integrase.