Peter B. Billings

Evidence Linking the 68 Kilodalton Antigen Identified in Progressive Sensorineural Hearing loss Patient Sera with Heat Shock Protein 70

Immunoblotting against bovine inner ear extracts has previously identified a 68 kd antigen reactive with 22% to 58% of sera of patients with rapidly progressive sensorineural hearing loss (PSNHL) of suspected autoimmune causation. Efforts to purify and characterize this diagnostic antigen suggest that it is ubiquitous rather than inner ear-specific, and may represent the highly inducible heat shock protein (hsp) 70. The antigens identified by PSNHL sera and anti-hsp 70 monoclonal antibodies copurify on ion exchange and adenosine triphosphate affinity chromatography, and comigrate on one- and two-dimensional gel electrophoresis. Additionally, immunoblotting with positive patient sera shows dramatically increased expression of the 68 kd antigen by bovine kidney cells following heat shock in culture. Reactivity with stress proteins of various classes has been reported in a number of autoimmune diseases; however, anti—hsp 70 appears uniquely associated with ulcerative colitis and PSNHL.

Assessment of serum antibodies in patients with rapidly progressive sensorineural hearing loss and Menière's disease.

The immunoreactivity of sera from patients with rapidly progressive sensorineural hearing loss (SNHL) or Menieres disease with bovine inner ear material was determined using the Western blot technique. Patients with other otologic conditions, autoimmune disorders, or arthritic disorders and age‐matched randomly chosen patients with no hearing complaints served as controls. Twenty‐two percent of the patients with bilateral rapidly progressive SNHL and 30% of the patients with Menieres disease had antibodies that reacted with a 68 kd antigen in the inner ear material. In the control groups, the incidence of reactivity was 5.0% (P<.001).

Tumor Necrosis Factor-α, an Initiator, and Etanercept, an Inhibitor of Cochlear Inflammation†‡

Objectives/Hypothesis The inner ear rapidly mounts an immune response that can lead to cochlear degeneration and permanent hearing loss. Identification of proinflammatory cytokine expression during the initiation of the response should lead to rational therapeutic strategies that block the response, reducing damaging sequelae.

Proinflammatory Cytokine Expression in the Endolymphatic Sac During Inner Ear Inflammation

The inner ear is capable of rapidly mounting an immune response that can ultimately lead to cochlear degeneration and permanent hearing loss. The role of the endolymphatic sac in this immune process is not clear. In order to investigate the cytokine expression of cells within the endolymphatic sac, a secondary inner ear immune response to keyhole limpet hemocyanin (KLH) was created in mice. The animals were sacrificed 3–48 h and 7 days following initiation of the immune response. The cochleas and endolymphatic sacs were assayed by immunocytochemistry for IL-1β, TNFα, and IL-6. Three hours after KLH challenge of the scala tympani, the perisaccular tissue of the endolymphatic sac contained more inflammatory cells than the scala tympani or endolymphatic sac lumen. Only a few of these cells, however, expressed the proinflammatory cytokines IL-1β and TNFα between 3 and 12 h after KLH injection. On the other hand, TNFα, which plays an important role in the cochlear secondary immune response, was expressed in cells in the endolymphatic sac lumen. The maximum percentage of cells expressing TNFα was seen later than in the scala tympani. Animals treated with systemic injection of the TNF blocker, etanercept, showed a reduction in the number of cells in the endolymphatic sac lumen. It is concluded that the cells in the endolymphatic sac lumen contribute to the amplification of the adaptive immune response by expressing TNFα, while the infiltration of cells into the perisaccular connective tissue is part of the nonspecific, innate, cochlear immune response.

Immune cell recruitment following acoustic trauma.

Acoustic trauma induces cochlear inflammation. We hypothesized that chemokines are involved in the recruitment of leukocytes as part of a wound healing response. The cochleas of NIH-Swiss mice, exposed to octave-band noise (8-16 kHz, at 118 dB) for 2h, were examined after the termination of exposure. Leukocytes were identified immunohistochemically with antibodies to CD45 and F4/80. Gene array analysis followed by RT-PCR was performed on cochlear tissue to identify up-regulation of chemokine and adhesion molecule mRNA. The expression of the adhesion molecule ICAM-1 was also investigated immunohistochemically. Few CD45- or F4/80-positive leukocytes were observed in the non-exposed cochlea. Following acoustic trauma however, the number of CD45-positive cells was dramatically increased especially after 2 and 4 days, after which time the numbers decreased. F4/80-positive cells also increased in number over the course of a week. Gene array analysis indicated increased expression of monocyte chemoattractant protein 5 (MCP-5), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein-1beta (MIP-1beta) and ICAM-1. RT-PCR, performed using primers for the individual mRNA sequences, confirmed the increased expression of MCP-1, MCP-5, MIP-1beta, and ICAM-1 relative to non-exposed mice. In the normal cochlea, ICAM-1 immunohistochemical expression was observed in venules, spiral ligament fibrocytes and in endosteal cells of the scala tympani. Expression increased to include more of the spiral ligament and endosteal cells after acoustic trauma. A cochlear inflammatory response is initiated in response to acoustic trauma and involves the recruitment of circulating leukocytes to the inner ear.

Blockage of immune-mediated inner ear damage by etanercept.

Hypothesis Etanercept will be able to reduce the inflammation and hearing loss associated with experimentally induced labyrinthitis. Background Inner ear immune responses cause hearing loss that may be reversible with pharmacologic treatment. Etanercept, tumor necrosis factor receptor blocker, was investigated in a guinea pig model of immune-mediated hearing loss. Sterile labyrinthitis was created by injection of keyhole limpet hemocyanin into the inner ear after systemic sensitization to keyhole limpet hemocyanin with adjuvant. Labyrinthitis involves infiltration of inflammatory cells and hearing loss detectable 3 to 5 days after challenge with keyhole limpet hemocyanin. Methods Etanercept was administered either systemically (2.5 mg) 30 minutes before intracochlear challenge with keyhole limpet hemocyanin, with a second intraperitoneal dose (2.5 mg) 3 days later or locally by long-term infusion into the scala tympani with an osmotic pump (5.0 &mgr;g/h for 7 days). Auditory evoked brainstem response thresholds were measured before and after treatment to determine hearing loss. Cochleas were evaluated for the amount of inflammation. Results Hearing loss in the untreated systemic group averaged 71 ± 21 dB versus 37 ± 32 dB in the etanercept-treated animals (t test, P < 0.001). There was also less inflammation in the cochleas from etanercept-treated animals (t test, P < 0.01). Hearing loss with local administration of etanercept was 59 ± 31 dB in the nontreated ears and 18 ± 8 dB in the treated ears (t test P < 0.02). Inflammation was also less (t test, P < 0.01). Etanercept was not ototoxic. Conclusion Prompt intervention with the anti-inflammatory drug etanercept significantly reduces inflammation sufficient for substantive hearing preservation.

Characterization of an experimentally induced inner ear immune response.

Objective To determine the effects of a sterile immune response on the structure and function of the cochlea.

Comparison of Anti-Heat Shock Protein 70 (Anti-hsp70) and Anti-68-kDa Inner Ear Protein in the Sera of Patients with Meniere's Disease†

The 68‐kDa antigen detected in the sera of patients with autoimmune inner ear disease is known to represent the highly inducible heat shock protein 70 (hsp70). To evaluate the existence of anti‐hsp70 in the sera of patients with Menieres disease and to develop a more reliable method to detect this antibody, the sera of patients and controls were examined.

Innate Immunity Contributes to Cochlear Adaptive Immune Responses

Inner ear immune responses mediated by antigen-specific processes are thought to contribute to hearing loss in humans. Systemic activation of innate immunity contributes to immune responses in the central nervous system. We hypothesized that activation of innate immunity can prime the inner ear for adaptive immune responses and exacerbate disease. Mice were systemically immunized with antigen. Three weeks after initial antigen exposure, the antigen was injected intrathecally coincident with systemic injection of lipopolysaccharide (LPS), an activator of innate immunity. Responses were measured by quantifying the leukocyte infiltrate and cochlear IL-1β expression. LPS stimulation markedly amplified the adaptive immune response to exogenous antigen in the inner ear. These data indicate that the cochlea is activated by systemic events that stimulate innate immunity and when antigen is present in the inner ear, a robust cochlear adaptive response is generated. If true in humans, this implies that priming may be an important component in the development of immune-mediated hearing loss.

Connection between the inner ear and the lymphatic system.

Objective The aim of this study was to identify the lymphatic drainage of the inner ear in guinea pigs.