The immune response after noise damage in the cochlea is characterized by a heterogeneous mix of adaptive and innate immune cells.

Cells of the immune system are present in the adult cochlea and respond to damage caused by noise exposure. However, the types of immune cells involved and their locations within the cochlea are unclear. We used flow cytometry and immunostaining to reveal the heterogeneity of the immune cells in the cochlea and validated the presence of immune cell gene expression by analyzing existing single-cell RNA-sequencing (scRNAseq) data. We demonstrate that cell types of both the innate and adaptive immune system are present in the cochlea. In response to noise damage, immune cells increase in number. B, T, NK, and myeloid cells (macrophages and neutrophils) are the predominant immune cells present. Interestingly, immune cells appear to respond to noise damage by infiltrating the organ of Corti. Our studies highlight the need to further understand the role of these immune cells within the cochlea after noise exposure.

Protective effect of acetyl-l-carnitine against cisplatin ototoxicity: role of apoptosis-related genes and pro-inflammatory cytokines.

OBJECTIVES: Cisplatin is an anti-neoplastic agent treatment with which causes many side effects including ototoxicity. The aim of this study was to investigate whether acetyl-L-carnitine would have protective effects on cisplatin-induced ototoxicity in vitro, and if present, to reveal roles of apoptotic gene expressions and pro-inflammatory cytokines. MATERIALS AND METHODS: House Ear Institute-Organ of Corti 1 cell line was used for this study. Apoptotic genes were evaluated with an apoptosis PCR array and pro-inflammatory cytokine levels were measured using ELISA. RESULTS: Apoptotic cell death reduced by around 22% with acetyl-L-carnitine-cisplatin treatment compared to cisplatin alone. Genes displaying increase in expression of apoptosis, related to cisplatin treatment, were Casp8, Bcl10, Bcl2, Bcl2l1, Bcl2l2, Bid, Naip1, Bnip3l, Card6, Pak7, Cd40, Trp 53inp1, Cideb and Cd70. The acetyl-L-carnitine-cisplatin combination caused reduced expression of genes Casp8, Fas, Casp1, Tnfrsf11b, Tnfrsf10b induced by cisplatin. Acetyl-L-carnitine-cisplatin also caused reduced levels of IL-6, IL-1ß and TNF-α, pro-inflammatory cytokines, induced by cisplatin. CONCLUSION: Protective mechanisms of aceytl-L-carnitine against cisplatin induced apoptosis, mainly due to activation of anti-apoptotic Bcl family members' genes, and in an Akt-related gene expression dependent manner. This is the first study to indicate that acetyl-L-carnitine can be an effective agent against cisplatin ototoxicity in auditory cells, with induction of anti-apoptotic gene expression and attenuating levels of pro-inflammatory cytokines.

An experimental study of inner ear injury in an animal model of eosinophilic otitis media.

CONCLUSION: As the periods of intratympanic injection of ovalbumin (OVA) to the middle ear became longer, marked eosinophil infiltration in the perilymphatic space was observed. Moreover severe morphological damage of the organ of Corti was observed in the 28-day antigen-stimulation side. These results indicate that eosinophilic inflammation occurred in the inner ear and caused profound hearing loss. OBJECTIVE: The purpose of the present study was to elucidate the inner ear damage in a new animal model of eosinophilic otitis media (EOM) which we recently constructed. METHODS: We constructed the animal model of EOM by intraperitoneal and intratympanic injection of OVA. Infiltrating cells and the inner ear damage were examined by histological study. RESULTS: In the inner ear, a few eosinophils were seen in the scala tympani of the organ of Corti and the dilation of capillaries of the stria vascularis was observed in the 7-day stimulation side. In the 14-day antigen stimulation side, some eosinophils and macrophages were seen in not only the scala tympani but also the scala vestibule. In the 28-day antigen-stimulation side, severe morphological damage of the organ of Corti and many eosinophils, red blood cells, and plasma cells infiltrating the perilymph were observed.

Spiral ligament fibrocytes release chemokines in response to otitis media pathogens.

CONCLUSION: Spiral ligament fibrocytes (SLFs) may be involved in the innate immune response of the inner ear by producing chemoattractants for recruiting inflammatory cells such as neutrophils and monocytes. OBJECTIVE: The purpose of this study was to investigate the cellular responses of SLFs when challenged by inflammatory stimuli such as components of otitis media pathogens or proinflammatory cytokines. MATERIALS AND METHODS: To detect released inflammatory cytokines and chemokines, cells were treated for 48 h with whole lysates of nontypable Haemophilus influenzae (NTHi), Streptococcus pneumoniae, or with interleukin 1 alpha (IL-1alpha). The culture medium was then collected and applied to protein arrays. To compare mRNA levels of chemokines, total RNA was extracted after 3 h of treatment with the above agents, and quantitative real-time PCR was performed. RESULTS: Protein array analysis showed that in response to NTHi or S. pneumoniae, rat SLFs released monocyte chemotactic protein 1, macrophage inflammatory protein 3 alpha, TNF-alpha, and cytokine-induced neutrophil chemoattractant 2 and 3. Treatment with IL-1alpha, on the other hand, resulted in release of MCP-1 but not the other molecules. Tissue inhibitor of metalloproteinase 1 and vascular endothelial growth factor were released regardless of the inflammatory stimulus used.

Localization of neurotensin immunoreactivity in neurons and organ of Corti of rat cochlea.

The distribution of neurotensin-containing cell bodies and fibers has been observed in the central and peripheral nervous system, including sensory ganglia, but no description has been found in the peripheral auditory system. Here, we investigated the presence of neurotensin immunoreactivity in the cochlea of the adult Wistar rat. Strong neurotensin immunoreactivity was detected in the cytoplasm of the inner hair cells (IHC) and Deiters' cells of the organ of Corti. Outer hair cells (OHC) show weak immunoreaction. Neurotensin immunoreactivity was also found in the neurons and fibers of the spiral ganglia. Quantitative microdensitometric image analysis of the neurotensin immunoreactivity showed a strong immunoreaction in the hair cells of organ of Corti and a moderate to strong labeling in the spiral ganglion neurons. A series of double immunolabeling experiments demonstrated a strong neurotensin immunoreactivity in the parvalbumin immunoreactive IHC and also in the calbindin immunoreactive Deiters' cells. Weak neurotensin immunoreactivity was seen in the calbindin positive OHC. Neurofilament and parvalbumin immunoreactive neurons and fibers in the spiral ganglia showed neurotensin immunoreactivity. Calbindin immunoreactivity was not detected in the spiral ganglion neurons, which are labeled by neurotensin immunoreactivity. The presence of neurotensin in the cochlea may be related to its modulation of neurotransmission in the peripheral auditory pathway.

Evidence for a possible NOS back-up system in the organ of Corti of the guinea pig.

Recently, the two Ca(2+)/calmodulin-regulated nitric oxide synthase isoforms, nNOS and eNOS, and NO itself have been identified in the cochlea of vertebrates using specific antibodies and a new fluorescence indicator. In order to acquire more information about the quantitative and spatial distribution of these two constitutively expressed NOS isoforms (cNOS) in the organ of Corti at the cellular and subcelluar levels, ultrathin sections of London resin (LR) White-embedded cochleae of the guinea pig were incubated with various concentrations of commercially available antibodies to nNOS and eNOS. The immunoreactivity was visualized by a gold-labeled secondary antibody and the amount of the immunoreactions/microm(2) was quantified for different cell types and subcellular regions. Both NOS isoforms were identified to varying degrees in the same cell types and subcellular regions. A prominent eNOS immunoreactivity was identified in nearly every cell type. In all analyzed animals the highest number of gold-coupled anti-eNOS antibodies was always seen in the cells of the reticular lamina, especially in the cuticular structures of outer and inner hair cells, pillar cells and apical Deiters' cells. Also the microtubuli-containing cytoplasmic regions of Deiters' cells were scattered with gold-coupled anti-eNOS antibodies. A clear eNOS immunoreaction was also found in the remaining cytoplasm of inner and outer hair cells and in the apical Deiters' cells. Numerous anti-nNOS antibodies were located in the outer hair cells and in the cuticular structures of the apical Deiters' cells. The amount of the gold-labeled anti-nNOS antibodies in the cuticular plates of the pillar cells and outer hair cells and in the cytoplasm of inner hair cells and apical Deiters' cells were clearly less but still above unspecific background labeling. The spatial co-localization of the two NOS isotypes in the same cell regions was proven in double-labeling experiments. The spatial distribution of the two cNOS isoforms confirmed recent findings of other authors who localized NO distribution and production sites. The cNOS co-expression with similar function in the same cell type and subcellular regions may represent a functional "back-up system" in which one NOS isoform can replace the other in case of pathophysiological malfunction.

Fas ligand expression in the organ of Corti.

We have previously demonstrated by FACS analysis and histochemistry that Fas ligand (FasL) increases on cochlear cell surfaces after immune response or stimulation with gamma-interferon (IFN-gamma). To determine whether the appearance of FasL on cochlear cell membranes is related to gene expression or to posttranslational events, cochlear cells were treated with IFN-gamma. They were evaluated for FasL gene expression by real-time PCR and for FasL protein localization by confocal microscopy of permeabilized and immunolabeled cells. Real-time PCR analysis of cDNAs generated from unstimulated or IFN-gamma-stimulated organ of Corti demonstrated no change in the transcription of the gene encoding FasL. In contrast, confocal microscopy revealed dramatic changes in the cellular distribution of FasL, consistent with movement from the endoplasmic reticulum to the cytoplasm and cell membrane. The results suggest that recruitment of preformed FasL from intracellular compartments, rather than its biosynthesis, is responsible for the increase in FasL on the cell surface following IFN-gamma stimulation. This is similar to the response of cytotoxic T lymphocytes in which gene expression is not involved in FasL surface appearance. Presumably, the use of preformed FasL increases the rapidity of this response. FasL localization to the membrane may be involved in protecting the inner ear from autoimmunity or inflammation. Alternatively it may be related to cochlear cell death in response to inflammatory stress.

Immunodetection of surfactant proteins in human organ of Corti, Eustachian tube and kidney.

The presence of surfactant proteins was investigated in the human organ of Corti, Eustachian tube and kidney tissues. It has previously been shown that lamellar bodies are present in hairy cells of organ of Corti, in the cytoplasm of secretory and lumen of tubal glands of Eustachian tube and kidney renal basement membrane. No evidence for the presence of surfactant proteins in the organ of Corti and kidney has been presented until now. The aim of this study was to find out if surfactant proteins were expressed in other epithelia such as organ of Corti, Eustachian tube and kidney. Surfactant proteins were identified using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. On one-dimensional Western blots, bands for surfactant protein A in human Eustachian tube (SP-A, 34 kDa) and in kidney extracts, and for surfactant protein D (SP-D, 43 kDa) in Eustachian tube and in kidney extracts (SP-D, 86 kDa), and for surfactant protein B (SP-B, 8 kDa) in human Eustachian tube and organ of Corti extracts were detected. Bands corresponded to monomeric forms of lung surfactant proteins. These results indicate the presence of SP-A and SP-D in kidney epithelium, SP-A, SP-B and SP-D in Eustachian tube and SP-B in the organ of Corti.

Induction of MHC class II antigens on cells of the inner ear.

Growing evidence supports the concept that immune reactions occur in the cochlea, where they can function either in protection or as a source of inflammation. Since immunity is generally initiated by antigen presentation of foreign substances to T cells, antigen-presenting cells expressing major histocompatibility complex (MHC) class II molecules are required. Under resting conditions, cochlear cells usually express no MHC class II. However, we show that exposure to -interferon in vitro induces an increase in MHC class II expression in neonatal cochlear cells of mice. In addition, MHC class II immunoreactivity was observed in the inner ear of adult mice after induction of sterile labyrinthitis in vivo. It is concluded that the induction of MHC class II molecules by inflammation may render cochlear cells competent to initiate and participate in immune reactions and may therefore contribute to both immunoprotective and immunopathological responses of the inner ear.

Localization of gamma-aminobutyric acid A receptor subunits in the rat spiral ganglion and organ of Corti.

Gamma-aminobutyric acid (GABA) is thought to be the major inhibitory neurotransmitter in the central nervous system. Although it is distributed in the olivo-cochlear bundles, which constitute the mammalian cochlear efferent system, its function in the cochlea is still obscure. In this study, we investigated the localization of GABAa receptor subunits (alpha1-6, beta1-3, gamma) in the rat cochlea in order to determine the role of GABA in the cochlea. Most spiral ganglion cells were intensely immunolabeled with all the anti-GABAa receptor subunit antibodies. In the organ of Corti, punctate immunoreactivities were observed in inner hair cell regions corresponding to the distribution of GABA. These data suggest that GABAa receptor was present in afferent nerve terminals in inner hair cell regions, and that GABA regulated afferent nerve transmission contacting efferent nerve endings by means of the axo-dendritic synapse function.

Immunological damage to the inner ear: current and future therapeutic strategies.

There is considerable evidence to suggest that hearing and vestibular function can be influenced by immunity in the inner ear. While immunity can protect against infections of the labyrinth, immune response also has the capacity to damage the delicate tissues of the inner ear. Antigenic challenge of the inner ear of sensitized animals leads to rapid accumulation of leukocytes, antibody production, hearing loss and tissue damage. Moreover, a number of systemic autoimmune disorders include hearing loss and vertigo as part of their constellation of symptoms. It also appears that autoimmune damage can exist as an entity confined to the labyrinth. Immune disorders of the inner ear are of special interest since they are among the few forms of hearing loss that are currently amenable to medical treatment. In addition, recent developments in understanding the intracellular pathways that participate in damage to the inner ear provide new opportunities for pharmacotherapy of immune-mediated disorders of hearing and balance.

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. METHODS: An immune response was created in guinea pigs by systemically sensitizing the animals to keyhole limpet hemocyanin and subsequently challenging the inner ear with the protein. Animals were allowed to survive for 1 to 5 weeks, after which the cochlea was evaluated histologically. Hearing was measured by auditory brainstem response before the inner ear challenge, during the survival period, and prior to sacrifice. RESULTS: Inflammatory cells infiltrated the cochlea from the circulation. Surface preparations and plastic sections of the organ of Corti 1 and 2 weeks after the initiation of the inflammation demonstrated degeneration of the sensory and supporting cells in cochlear turns containing inflammatory cells. Good preservation of structures was seen in the more apical cochlear turns with little or no inflammatory cells. In cochleas from animals that survived 5 weeks, most of the infiltrated cells were cleared after undergoing apoptosis and the inflammatory matrix in the scala tympani began to calcify. Hearing loss was moderate to severe depending on the amount of inflammation. CONCLUSION: Although in general the immune response serves to protect an organism from infection, these results demonstrate that bystander injury associated with local immune responses in the cochlea, an organ incapable of regeneration, causes permanent cochlear destruction and hearing loss.

Distribution of endothelin-1-like activity in the cochlea of normal guinea pigs.

Distribution of endothelin-1 (ET-1) in the cochlea of normal guinea pigs was determined by immunohistochemistry. ET-1 activity was identified with the mouse anti-human ET-1 IgG1 monoclonal antibody. ET-1 activity was distributed in the modiolus, spiral ligament, stria vascularis, spiral prominence. Reissner's membrane, supporting cells of the organ of Corti and spiral ganglion cells. These findings suggest that ET-1 may be involved in the regulation of fluid volume and ions of the cochlea.

The binding site on cochlear stereocilia for antisera raised against renal Na+ channels is blocked by amiloride and dihydrostreptomycin.

The mechanoelectrical transduction channels on hair cells have been suggested to be operated by tip links that are stretched when the hair bundle is deflected in the direction of the tallest row of stereocilia. Localising these channels is therefore an important test of this hypothesis. The transduction channels are known to be amiloride-sensitive and immunogold labelling with antibodies raised against the amiloride-sensitive epithelial Na+ channel from kidney (alpha NaCh), has suggested that sites with similar characteristics are located in the region where the tips of the shorter stereocilia appear to come into contact with the sides of the adjacent taller stereocilia rather than being associated directly with the tip links. Now, further immunocytochemical experiments have been performed to determine if amiloride and dihydrostreptomycin, both of which can block transduction, can affect this labelling. Immunofluorescent labelling of the stereocilia is obtained when surface preparations of the organ of Corti are fixed and incubated with alpha NaCh followed by an appropriate secondary antibody. This labelling is abolished by trypsinization prior to fixation but retained if the tissue is pretreated with amiloride and then trypsinized in its presence. Because amiloride is known to protect amiloride-binding sites from degradation by trypsin, these results suggest that alpha NaCh is revealing amiloride-binding sites on the stereocilia. Similarly, immunofluorescent labelling of the stereocilia is abolished if cochlear tissue is pretreated with dihydrostreptomycin (DHS) and fixed in its presence prior to incubation with alpha NaCh. Quantitative analysis of colloidal gold labelling using transmission electron microscopy shows that DHS treatment produces a significant reduction in the number of gold particles on stereocilia, especially in the region of contact between them. These results suggest that anti-Na+ recognises a site with characteristics similar to the mechanoelectrical transduction channels.

Natural killer cell response in the inner ear.

Heterologous tumor cells (human chronic myelogenous leukemia K-562 cells) were injected into the perilymph and skin in guinea pigs in order to study the induction of NK cell activity in the inner ear. An in vitro transmission electron microscopic and immunohistochemical study showed that K-562 cells were attacked by guinea pig large granular lymphocytes. K-562 cells injected through the round window membrane were found to be targeted by NK cells emerging from surrounding venules after 7 to 9 days. During this time morhological changes occurred in the organ of Corti and stria vacularis. These findings suggest that the inner ear response to foreign cells induces activation and invasion of NK cells which occur relatively late compared with those in other organs such as skin.

Monoclonal antibody induced hearing loss.

Monoclonal antibodies KHRI-3 and KHRI-5 identify antigens expressed on inner ear supporting cells and auditory hair cells respectively. To determine if these antibodies affect inner ear function groups of syngeneic Balb/c mice were inoculated with hybridomas KHRI-3, KHRI-5 and other Ig-secreting hybridomas. Hybridomas UM-A9, UM-7F11, the non-secreting SP2/0 myeloma and mice with no hybridoma were used as controls. Animals were tested for auditory brainstem responses (ABR) for frequencies of 4, 8, 16 and 24 kHz, before the inoculation of the hybridomas and at intervals of 6 to 10 days thereafter or daily once tumors became palpable. In normal mice there were no changes in ABR thresholds over the course of the experiment. Other control animals showed little change in ABR even when the growth of the hybridoma or myeloma tumors were far advanced. Of the KHRI-5 hybridoma bearing animals only one of seven animals exhibited threshold shifts greater than 15 dB. In contrast, most mice bearing the KHRI-3 hybridoma exhibited high frequency threshold shifts of 40-50 dB that coincided temporally with the growth of the hybridoma, the presence of circulating KHRI-3 antibody, and greatly increased immunoglobulin titers. Ears from KHRI-3-bearing mice that developed high frequency hearing loss also had a novel type of lesion in the basal turn of the cochlea that was characterized by loss of outer hair cells and absence of typical supporting cell scars. Such changes were not found in control hybridoma-bearing mice. These findings suggest that KHRI-3 antibody has an effect on hearing that is secondary to damage to the organ of Corti and loss of outer hair cells. Our results have important implications for antibody-mediated mechanisms of hearing loss and provide an animal model in which to study this phenomenon.

The localization and specificity of guinea pig inner ear antigenic epitopes.

In this study, we investigated the relative localization of some antigenic epitopes in the inner ear. The inner ear protein antigens were extracted from various parts of the guinea pig inner ear. Brain, kidney, lung, heart and liver extracts were also obtained. We found by SDS-polyacrylamide gel electrophoresis that total inner ear extracts separated into three high concentration polypeptide bands with molecular weights of approximately 30, 42, 58 kd and three low density bands of 20, 25 and 35 kd. The 30 kd band was found mainly in the extract of the spiral ganglion and the acoustic nerve in the modiolus. The 42 and 58 kd bands were detected in the extract of the spiral ligament and the stria vascularis. The Organ of Corti and the basilar membrane extract gave rise to three bands of 30, 42 and 58 kd. Twenty-eight of the 75 sera from patients with inner ear disease reacted with the 30 and 58 kd bands of the inner ear protein extracts by immunoblotting. Sixteen of these 28 positive sera were then used to probe immunoblots of the brain, kidney, lung, heart and liver extracts. The 58 kd band was also found in protein extracts of the brain, the lung and the liver. This study suggests that the 30 kd antigenic epitope may be mainly related to the acoustic nerve and that the 58 kd antigenic epitope is not cochlear specific.

Inner ear-specific autoantibodies.

The sera of patients with idiopathic sensorineural hearing loss (SNHL) were examined, using qualitative immunoblotting (Western blotting), for the presence of inner ear (IE)-specific autoantibodies. The water-soluble extracts and the sodium dodecyl sulfate-soluble extracts were prepared as the antigens from bovine IE tissues and several other organs in order to determine the specificity of autoantibodies in their sera to the IE. Some patients (n = 46) tested had autoantibodies that reacted with 68-, 62-, 55-, 50-, or 47-kd antigenic determinants found in all tissues, while others (n = 13) showed IE-specific autoantibodies which reacted with 220-, 60-, 58-, 33-35-, or 32-kd determinants. The presence of these autoantibodies from patients with progressive SNHL may have important implications with regard to their etiology and possibly their sensitivity to therapeutic intervention. It is now necessary to purify these IE-specific antigens and determine their clinical usefulness.

Gentamicin uptake by cochlear hair cells precedes hearing impairment during chronic treatment.

Immunodetection of gentamicin (GM) was carried out on surface preparations of the whole organ of Corti from cochleas of guinea pigs treated daily with GM at a dose of 60 mg/kg/day and sacrificed at the end of different treatment periods. Cochlear function was determined just before sacrifice, 24 h after the last injection. Threshold elevations, mainly at high frequencies, were noted only after 10-14 days of treatment. However, the presence of GM was observed much earlier, as early as after the second injection, and specifically in the sensory hair cells. GM labelling was essentially observed in the outer hair cells (OHC) and increased from the apex to the base of the cochlea and from the third to the first row of OHC. GM labelling of inner hair cells was less pronounced and was observed only after the 8th day of treatment. These observations demonstrate that GM specifically enters and accumulates in the sensory hair cells and that the uptake precedes the development of functional and cellular damage which may result from a long-term intracellular cytotoxic action of the molecule.

Monoclonal antibodies to inner ear antigens: I. Antigens expressed by supporting cells of the guinea pig cochlea.

Murine monoclonal antibodies against guinea pig cochlear epithelium were generated with the goal of identifying cochlea-specific antigens and elucidating their function. To compensate for the limited amount of cochlear tissue, intrasplenic immunization was used. Hybridoma supernatants were screened by ELISA for antibody production and for binding to homogenates from cochlea, liver, lung, kidney and brain. Hybrids producing antibody to cochlea were subcloned and tested immunocytochemically against frozen sections and surface preparations of paraformaldehyde-fixed cochlear tissue. KHRI-1, a low titer IgM antibody stained only Hensen cells. KHRI-2, also an IgM antibody, stained tectorial membrane, cells of the spiral limbus, cells bordering the space of Nuel, Hensen cells and the root cells of the spiral prominence. KHRI-3, an IgG1 antibody, stained the phalangeal processes of outer pillar cells and the apical portion of phalangeal processes of Deiters' cells in a distinctive wine goblet pattern on surface preparations. KHRI-3 antibody also reacted with peripheral nerves and pia mater of brain in unfixed frozen sections but the antigenic site was not stable to fixation in contrast to the epitope detected in the cochlea. In Western blots of detergent extracts from cochlea KHRI-3 stained a broad tissue-specific band of Mr 70-75 kDa; a narrower band of Mr 68-70 kDa was identified by KHRI-3 in extracts of tongue and brain. KHRI-1 and KHRI-2 did not detect any proteins in Western blots. The monoclonal antibodies KHRI-1, -2, and -3 which define epitopes expressed by discrete populations of supporting cells in the inner ear should be useful in characterizing the nature and function of cellular structures in the cochlea.