Sharon L. McCoy

Nucleosomes and DNA bind to specific cell-surface molecules on murine cells and induce cytokine production

The molecular basis for the cellular interaction of DNA and nucleosomes and the physiological consequences of this binding were examined. Both DNA and nucleosomes were demonstrated to bind specifically to the surface of human peripheral blood mononuclear cells and the murine T cell line S49. Western blots of S49 cell membranes, using probes of biotin-labeled DNA and nucleosomes, showed reactivity at 29 and 69 kDa. Functionally, the interaction of DNA and nucleosomes with murine spleen cells stimulated the release of significant amounts of IL-6 activity. There is evidence that nucleosomes, a product of apoptosis, are the major component of circulating DNA found in the plasma of patients with systemic lupus erythematosus (SLE). The interaction of nucleosomes with cell-surface DNA binding molecules may have physiological relevance to some of the immune aberrations observed in patients with SLE.

Identification of a Peptide Derived from Vaccinia Virus A52R Protein That Inhibits Cytokine Secretion in Response to TLR-Dependent Signaling and Reduces In Vivo Bacterial-Induced Inflammation

TLRs recognize and respond to conserved motifs termed pathogen-associated molecular patterns. TLRs are characterized by an extracellular leucine-rich repeat motif and an intracellular Toll/IL-1R domain. Triggering of TLRs by pathogen-associated molecular patterns initiates a series of intracellular signaling events resulting in an inflammatory immune response designed to contain and eliminate the pathogen. Vaccinia virus encodes immunoregulatory proteins, such as A52R, that can effectively inhibit intracellular Toll/IL-1R signaling, resulting in a diminished host immune response and enhancing viral survival. In this study, we report the identification and characterization of a peptide derived from the A52R protein (sequence DIVKLTVYDCI) that, when linked to the nine-arginine cell transduction sequence, effectively inhibits cytokine secretion in response to TLR activation. The peptide had no effect on cytokine secretion resulting from cell activation that was initiated independent of TLR stimulation. Using a mouse model of otitis media with effusion, administration of heat-inactivated Streptococcus pneumoniae into the middle ears of BALB/c mice resulted in a significant inflammatory response that was dramatically reduced with peptide treatment. The identification of this peptide that selectively targets TLR-dependent signaling may have application in the treatment of chronic inflammation initiated by bacterial or viral infections.

A human gene coding for a membrane-associated nucleic acid-binding protein.

Studies to clone a cell-surface DNA-binding protein involved in the binding and internalization of extracellular DNA have led to the isolation of a gene for a membrane-associated nucleic acid-binding protein (MNAB). The full-length cDNA is 4.3 kilobases with an open reading frame of 3576 base pairs encoding a protein of ∼130 kDa (GenBank accession numbers AF255303 andAF255304). The MNAB gene is on human chromosome 9 with wide expression in normal tissues and tumor cells. A C3HC4 RING finger and a CCCH zinc finger have been identified in the amino-terminal half of the protein. MNAB bound DNA (K D ∼4 nm) and mutagenesis of a single conserved amino acid in the zinc finger reduced DNA binding by 50%. A potential transmembrane domain exists near the carboxyl terminus. Antibodies against the amino-terminal half of the protein immunoprecipitated a protein of molecular mass ∼150 kDa and reacted with cell surfaces. The MNAB protein is membrane-associated and primarily localized to the perinuclear space, probably to the endoplasmic reticulum or trans-Golgi network. Characterization of the MNAB protein as a cell-surface DNA-binding protein, critical in binding and internalization of extracellular DNA, awaits confirmation of its localization to cell surfaces.

Evaluation of the mouse model for acute otitis media

Various animal models have been employed for otitis media research. The mouse has been studied less, in spite of its many advantages. To better understand the suitability of the mouse for studies of otitis media, an evaluation was made of its middle ear inflammatory processes following inoculation with heat-killed Streptococcus pneumoniae (strain 6A), one of the three most common bacteria to cause otitis media in the human. A total of 94 BALB/c mice were injected transtympanically with three concentrations of heat-killed bacteria (10(4), 10(6), and 10(9) organisms per ml) and inflammation evaluated with both histologic examination and auditory brainstem response audiometry. Dose-related measures of the time course of inflammation showed it was maximal at 3 days. PBS-injected control mice also demonstrated some degree of middle ear inflammation. Therefore, inflammation measures from PBS injected mice were used as the threshold above which histologic inflammatory changes would be considered a response to bacteria. These quantitative comparisons of bacterial and PBS inoculations revealed the most significant middle ear measures of inflammation were amount of fluid in the middle ear, tympanic membrane thickness, and number of inflammatory cells. The induction of middle ear inflammation in the mouse demonstrated the applicability of this model for investigations of otitis media.

A novel inhibitory peptide of Toll-like receptor signaling limits lipopolysaccharide-induced production of inflammatory mediators and enhances survival in mice.

Sepsis resulting from gram-negative bacterial infections is characterized by an excessive inflammatory immune response initiated by exposure of the host innate immune system to either bacteria or bacterial products, primarily lipopolysaccharide (LPS). Engagement of the Toll-like receptor (TLR) 4 on immune cells by LPS induces production of inflammatory mediators, leading to tissue damage. We recently identified a peptide, termed P13, which was previously shown to be a potent inhibitor of in vitro TLR signaling. In this study, we demonstrate that the use of this novel peptide significantly reduces the in vitro production of inflammatory mediators seen after exposure of hepatocytes/nonparenchymal cell cocultures and endothelial cells to LPS. In addition, in vivo treatment of mice with this peptide was effective at inhibiting LPS-induced production of inflammatory mediators and significantly limited liver damage. Peptide treatment significantly increased survival of LPS-/D-galactosamine-treated mice and mice treated with high-dose LPS. These results demonstrated the therapeutic potential of peptide P13 to limit an LPS-induced inflammatory response and enhance survival in murine models of inflammation.

DNA Binding to Mouse Cells is Mediated by Cell-Surface Molecules: The Role of These DNA-Binding Molecules as Target Antigens in Murine Lupus

Autoimmunity to a 28-29-kDa cell-surface DNA-binding molecule has previously been described in patients with systemic lupus erythematosus and related autoimmune diseases. This report describes experiments that implicate a similar antigen-antibody system in the evolution of autoimmunity in lupus-prone mice. DNA binding to murine spleen cells was found to be a saturable phenomenon that was inhibited by excess cold DNA and trypsinization. The role of autoimmunity to murine cell-surface DNA-binding molecules in lupus-prone mice (MRL lpr/lpr, MRL + / +, BXSB) was compared to normal mice (BALB/c, C3H.SW) by means of an assay that measured the inhibition of cell-surface DNA binding. Only sera from lupus strains had inhibitory activity and this component was shown to be an IgM autoantibody. Furthermore, we isolated a spontaneously occurring IgM monoclonal antibody from the spleen of an MRL/lpr mouse, which inhibited DNA binding to mouse cells. Time-course studies indicated that young female MRL/lpr mice lacked detectable activity against cell- surface DNA-binding molecules; however, by 8-10 weeks maximal inhibitory activity was observed. This response occurred prior to the development of significant antinuclear antibody activity. With the appearance of overt disease and anti-DNA antibodies, inhibition of DNA- binding activity became undetectable. These findings mirror previous studies on autoimmunity to a cell-surface DNA-binding molecule on human leucocytes, but have the added advantage of permitting the study of the temporal evolution of this inhibitory activity in relation to disease expression.

Autoimmune mouse antibodies recognize multiple antigens proposed in human immune-mediated hearing loss.

Hypothesis: Autoimmune diseased mice with hearing loss will have autoantibodies against the various cochlear antigens proposed in clinical autoimmune inner ear disease. Background: Serum antibodies of patients with hearing loss recognize several proteins that are proposed as possible antigenic targets in the ear. This often leads to a clinical diagnosis of autoimmune inner ear disease, although it is not clear how these antibodies cause inner ear disease. Therefore, to better understand the relationship of autoantibodies and ear disease, an examination was made of serum autoantibodies in the MRL/MpJ-Faslpr autoimmune mouse with hearing loss. Similar antibody patterns in the mouse would provide an animal model in which to investigate potential autoimmune mechanisms of this clinical ear disorder. Methods: Sera from MRL/MpJ-Faslpr autoimmune mice and normal C3H mice were tested by the enzyme-linked immunosorbent assay technique for reactivity against various reported cochlear antigens: heat shock protein 70 (bovine, human, bacterial), laminin, heparan sulfate proteoglycan, cardiolipin, and collagen types II and IV. Results: The autoimmune mouse sera showed significantly greater antibody reactivity against all of the antigens when compared with normal mouse sera. Conclusions: Serum antibodies from autoimmune mice recognized several putative autoantigens reported for patients with hearing loss, suggesting that comparable antigen–antibody mechanisms might be operating. However, the recognition of multiple antigens did not identify any one as being the specific target in autoimmune hearing loss. The correlation of antibodies in the MRL/MpJ-Faslpr autoimmune mouse and human studies indicates this animal model should aid further investigations into potential cochlear antigens in autoimmune hearing loss.

Immunization of balb/c mice with a monoclonal anti-DNA antibody induces an anti-idiotypic antibody reactive with a cell-surface DNA binding protein

DNA binds to cell-surface proteins on human and murine leukocytes and induces secretion of the cytokine interleukin 6 (IL-6). Cell-surface DNA binding molecules have been shown to serve as target antigens for the production of autoantibodies in patients with systemic lupus erythematosus (SLE), and in lupus-prone mice. Recent studies have demonstrated that a subset of anti-anti-DNA antibodies, isolated from patients with SLE, are idiotypically related to antibodies reactive with a cell-surface DNA binding molecule. We now report that immunization of normal mice with a murine monoclonal anti-DNA antibody induces an anti-idiotypic response which has reactivity with a cell-surface DNA binding molecule. An anti-idiotypic anti-DNA monoclonal antibody (LB17) was isolated from the spleen of an immunized mouse. This monoclonal antibody blocked the binding of DNA to murine splenocytes and mimicked the functional effect of DNA by stimulating the secretion of IL-6. These experiments provide further evidence for an idiotypic connectivity between antibodies to cell-surface DNA binding proteins and anti-DNA antibodies. It is hypothesized that this idiotypic system is part of the network of natural autoantibodies and that its perturbation may give rise to pathogenic antibodies.

Quantification of DNA binding to cell-surfaces by flow cytometry.

DNA binding to cell-surfaces has been documented in several studies. The interaction of DNA with cells has been shown to have therapeutic potential as a non-viral form of gene delivery and DNA vaccination. Recently, bacterial DNA binding and internalization has been demonstrated in some cells to trigger secretion of cytokines and cell activation. Previous studies to quantify DNA binding to cells have used radiolabeled DNA. Here we report a non-radioactive assay for quantification of cell-surface DNA binding based on the isoparametric analysis of flow cytometric data as described by Chatelier et al., Embo J., 5 (1986) 1181. This assay has the advantage over previously used procedures in not employing radioactive material and being able to discriminate viable from non-viable cells that bind DNA. With the importance of understanding the interaction of DNA with cells, this assay may have application for the identification and characterization of reagents designed to either enhance or inhibit DNA binding to cells.