Steven H. Hefeneider

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.

C3H/HeJ mouse model for spontaneous chronic otitis media.

Objectives/Hypothesis: Chronic otitis media is a significant clinical problem. Understanding the mechanisms of chronic otitis media is critical for its control. However, little is known of these processes as a result of lack of animal models of spontaneous otitis media. The C3H/HeJ mouse has a single amino acid substitution in its toll‐like receptor 4 (TLR4), making it insensitive to endotoxin. As a result, these mice cannot clear Gram‐negative bacteria. The chronically inflamed middle ear in this animal provides us the opportunity to study spontaneous chronic otitis media.

Failure of elevated heat shock protein 70 antibodies to alter cochlear function in mice

Heat shock protein 70 (HSP70) has been suggested as the putative cochlear antigen underlying a proposed autoimmune etiology in certain cases of Menieres disease and idiopathic hearing loss. To determine if antibodies to this cellular protein are capable of altering cochlear function, BALB/c (N= 3) and CBA/J (N= 9) mice were inoculated with bovine HSP70 by intraperitoneal injections (10 microg in saline) every 10 days for 7 or 10 months, respectively. An equal number of control mice were injected with PBS according to the same schedule. ABR thresholds at 4, 8, 16, and 32 kHz in the HSP70-inoculated mice did not change over the 10 month period and were similar to saline controls. Furthermore, serum immune complexes and antinuclear antibodies did not increase over the inoculation period. ELISA analysis demonstrated the mice created antibodies to the foreign HSP70, but these apparently caused no abnormalities in the auditory or immune systems. It was concluded that foreign HSP70 is antigenic and inoculation with it will raise antibodies, but these antibodies were neither immunopathogenic nor cochleopathic. Therefore, these findings do not support current theories that elevated anti-HSP70 antibodies are the underlying cause of hearing loss in patients with such antibodies present.

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.

Steroid treatment improves cochlear function in the MRL.MpJ-Faslpr autoimmune mouse

Corticosteroid therapy is used to reverse autoimmune sensorineural hearing loss, although little is known of the mechanism by which this occurs. This has been due to the lack of a suitable animal model with spontaneous hearing loss that is steroid responsive. The present study examined the effects of prednisolone treatment on auditory thresholds in the MRL.MpJ-Fas(lpr) autoimmune mouse to determine its suitability as such a model. Autoimmune mice at 3.5-4. 5 months of age were evaluated by pure-tone auditory brainstem response (ABR) to establish threshold elevations due to the disease. The steroid treatment group was then given prednisolone in their drinking water for 2.5 months, while untreated controls were given tap water. Significantly more steroid treated mice survived to the time of post-treatment ABR evaluation. Half of the steroid treated ears demonstrated either improvement or no change in cochlear function compared to only 25% in the untreated controls. Overall, cochlear thresholds in the untreated controls increased by 14.7 dB, whereas no significant threshold increase was seen in the steroid treated group (4.3 dB) over the treatment period. No qualitative anatomical differences were seen in the ears of those mice surviving to the end of the study. These findings establish the autoimmune mouse as a model for studies of steroid responsive mechanisms within the ear. This could apply to autoimmune sensorineural hearing loss, as well as any hearing disorder for which steroid therapy is recommended.

Inner ear DNA receptors in MRL/lpr autoimmune mice: potential 30 and 70 kDa link between autoimmune disease and hearing loss.

Inner ear function and systemic autoimmune disease were evaluated in the MRL/lpr mouse to determine their relationship with alterations in cell surface DNA receptors of 28-30 and 68-70 kDa size. Auditory brainstem response thresholds in the autoimmune disease mice were significantly elevated as early as 2 months of age when compared to MRL/++ controls. Hearing thresholds continued to rise with progression of the disease, manifested as increasing spleen weights, antinuclear (anti-DNA) antibodies, and serum immune complexes. Cochlear membranous labyrinth cells in the autoimmune mice bound less DNA, suggesting the DNA receptors were abnormally occupied by circulating antibodies. Western blots of a murine T-cell line probed with autoimmune mouse sera demonstrated reactivity to 28-30 and 68-70 kDa proteins after disease onset. It is hypothesized that cell surface DNA binding molecules could be masked or down-regulated by circulating antibodies in autoimmune disease. This interference with DNA receptor activity may be occurring within the inner ear and underlie the cochlear dysfunction seen in autoimmune sensorineural hearing loss.

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.