The Human Endolymphatic Sac and Inner Ear Immunity: Macrophage Interaction and Molecular Expression.

Background: The endolymphatic sac (ES) is endowed with a multitude of white blood cells that may trap and process antigens that reach the inner ear from nearby infection-prone areas, it thus serves as an immunologic defense organ. The human ES, and unexpectedly the rest of the inner ear, has been recently shown to contain numerous resident macrophages. In this paper, we describe ES macrophages using super-resolution structured fluorescence microscopy (SR-SIM) and speculate on these macrophages' roles in human inner ear defense. Material and Methods: After ethical permission was obtained, human vestibular aqueducts were collected during trans-labyrinthine surgery for acoustic neuroma removal. Tissues were placed in fixative before being decalcified, rapidly frozen, and cryostat sectioned. Antibodies against IBA1, cytokine fractalkine (CX3CL1), toll-like receptor 4 (TLR4), cluster of differentiation (CD)68, CD11b, CD4, CD8, and the major histocompatibility complex type II (MHCII) were used for immunohistochemistry. Results: A large number of IBA1-positive cells with different morphologies were found to reside in the ES; the cells populated surrounding connective tissue and the epithelium. Macrophages interacted with other cells, showed migrant behavior, and expressed immune cell markers, all of which suggest their active role in the innate and adaptive inner ear defense and tolerance. Discussion: High-resolution immunohistochemistry shows that antigens reaching the ear may be trapped and processed by an immune cell machinery located in the ES. Thereby inflammatory activity may be evaded near the vulnerable inner ear sensory structures. We speculate on the immune defensive link between the ES and the rest of the inner ear.

Innate immune defense in the inner ear - mucines are expressed by the human endolymphatic sac.

The human endolymphatic sac has been shown recently to have immunological capacities and has thus been proposed as the main entity protecting the inner ear from pathogen invasion, equivalent to mucosa-associated lymphoid tissue (MALT). Although the sac expresses molecules of the innate immune system, the potential expression of members of the important mucin family has not been detailed. Thus, this paper explores endolymphatic sac expression of a number of mucins and mucin precursors. Twelve fresh tissue samples from the human endolymphatic sac were obtained during translabyrinthine surgery. The expression of Mucin 1, 2, 5B/AC and 16, as well as the core structure elements (mucin precursors) T-antigen, Tn-antigen and Sialyl-Tn-antigen was investigated by immunohistochemistry. The endolymphatic sac epithelium expressed MUC1 (both apically towards the endolymphatic sac (ES) lumen and basally towards the capillary network), MUC 16 and Tn-antigen. There was no labeling after incubation with antibodies against T-antigen, sialyl-Tn-antigen, MUC2 and MUC5B/AC. We conclude that the human endolymphatic sac epithelium expresses a number of mucin molecules, which supports the hypothesis of the sac as the primary immunological tissue structure of the inner ear, equivalent to MALT in other organs. The mucins may also play a role in the formation and continuous homeostasis of the inner ear fluids, as well as the pathogenesis of Meniere's disease.

Expression of histamine receptors in the human endolymphatic sac: the molecular rationale for betahistine use in Menieres disease.

The human endolymphatic sac (ES) is situated in a duplicature of the dura in the posterior cranial fossa and constitutes a part of the inner ear. The sac possesses immunological capacities and is responsible for a major part of the trans-epithelial ion transport occurring within the inner ear, via molecular mechanisms similar to that of the kidney collecting duct epithelia. Dysfunction of the trans-epithelial ion transport has been hypothesized as the reason for the endolymphatic hydrops occurring in Menieres diseases. Thus, candidate drug selection for medical treatment of Menieres disease has been based on a potential capability of improving trans-epithelial ion transport. However, recent human studies seems to rule out diuretic therapy and The Committee for Medicinal Products for Human Use redrew the recommendation for trimetazidine (Vastarel) treatment in the management of Meniere disease in 2012. This leaves betahistine (Betaserc) as the only drug for potential prevention of the incapacitating attacks of dizziness, tinnitus and hearing loss. However, the histamine receptors targeted by betahistine have never been demonstrated in the human ES. Accordingly, this study aims to investigate the expression of histamine receptors of the human ES epithelium and sub-epithelial stroma. Following sampling of human endolymphatic sac tissue during translabyrinthine surgery, the expression of histamine receptor genes was determined by cDNA microarray analysis. Results were subsequently verified by immuno-histochemistry. The combined results of microarrays and immuno-histochemistry showed expression of the histamine receptor HRH1 in the epithelial lining of the ES, whereas HRH3 was expressed exclusively in the sub-epithelial capillary network. Receptors HRH2 and -4 were not expressed. The present data provide the first direct evidence of a molecular rationale for betahistine treatment in Menieres disease. A potential betahistine effect in Menieres disease may primarily be through the H3-receptor antagonism, leading to inhibition of vestibular neuro-transmission and central vaso-dilation. The H1-receptor localization in the ES epithelium suggests an immuno-regulatory effect.

Gene expression demonstrates an immunological capacity of the human endolymphatic sac.

OBJECTIVES/HYPOTHESIS: The purpose of the present study is to explore, demonstrate, and describe the expression of genes related to the innate immune system in the human endolymphatic sac. It is hypothesized that the endolymphatic sac has a significant immunological function in the human inner ear. STUDY DESIGN: DNA microarrays and immunohistochemistry were used for analyses of fresh human endolymphatic-sac tissue samples. METHODS: Twelve tissue samples from the human endolymphatic sac were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression using adjacent dura mater as control. The expression of genes specific for the innate immune system was determined and results for selected key molecules verified by immunohistochemistry. RESULTS: A comprehensive overview of expressed genes of the innate immune system was obtained. Multiple key elements of both the cellular and humoral innate immune system were expressed, including Toll-like receptors 4 and 7, as well as beta-defensin and lactoferrin. CONCLUSIONS: The present data provides the first direct evidence of an immunological capacity of the human endolymphatic sac. At the molecular level, the endolymphatic sac is capable of antigen recognition and processing for initiation of an immune response. In addition, potent molecules directly toxic to invading pathogens are expressed by the sac epithelium. This evidence strongly supports the endolymphatic sac as a significant immunological entity of the inner ear. LEVEL OF EVIDENCE: N/A.

Endolymphatic sac involvement in bacterial meningitis.

The commonest sequelae of bacterial meningitis are related to the inner ear. Little is known about the inner ear immune defense. Evidence suggests that the endolymphatic sac provides some protection against infection. A potential involvement of the endolymphatic sac in bacterial meningitis is largely unaccounted for, and thus the object of the present study. A well-established adult rat model of Streptococcus pneumoniae meningitis was employed. Thirty adult rats were inoculated intrathecally with Streptococcus pneumoniae and received no additional treatment. Six rats were sham-inoculated. The rats were killed when reaching terminal illness or on day 7, followed by light microscopy preparation and PAS-Alcian blue staining. The endolymphatic sac was examined for bacterial invasion and leukocyte infiltration. Neither bacteria nor leukocytes infiltrated the endolymphatic sac during the first days. Bacteria invaded the inner ear through the cochlear aquaduct. On days 5-6, the bacteria invaded the endolymphatic sac through the endolymphatic duct subsequent to invasion of the vestibular endolymphatic compartment. No evidence of direct bacterial invasion of the sac through the meninges was found. Leukocyte infiltration of the sac occurred prior to bacterial invasion. During meningitis, bacteria do not invade the endolymphatic sac through the dura, but solely through the endolymphatic duct, following the invasion of the vestibular system. Leukocyte infiltration of the sac occurs prior to, as well as concurrent with bacterial invasion. The findings support the endolymphatic sac as part of an innate immune defense system protecting the inner ear from infection.

Audiological manifestations of allergic rhinitis.

BACKGROUND: Allergic rhinitis is associated with excess specific immunoglobulin E. Inner ear involvement (via both cellular and humoral immunity) is poorly understood, but appears to arise from the endolymphatic sac and duct. AIMS: To assess the otological and audiological status of patients with allergic rhinitis. METHODOLOGY: Thirty allergic rhinitis patients (14 men, 16 women; age 17-45 years, mean 31 years) and 20 controls (12 men, eight women; age 21-42 years, mean 27 years) underwent audiological investigation. RESULTS: All study group patients had sensorineural (rather than conductive) hearing loss, worse at high frequencies. All had abnormal transient evoked otoacoustic emissions and 27 had abnormal distortion product otoacoustic emissions. All had a statistically significantly prolonged wave I latency, and shortened absolute wave I-III and I-V interpeak latencies, compared with controls. CONCLUSION: Allergic rhinitis patients had a higher prevalence of hearing loss and otoacoustic emission abnormalities than controls. The endolymphatic sac can process antigens and produce its own local antibody response; the resulting inflammatory mediators and toxic products may interfere with hair cell function. Additional research is needed to determine the clinical value of audiometry and otoacoustic emission testing in allergic rhinitis.

Controlled study of the frequency of anti-HSP 70 with the ELISA and the Western Blot methods in patients with Ménière Disease.

OBJECTIVES: To establish the frequency of auto-antibodies anti-HSP 70 using ELISA and Western Blot (WB) methods and to compare the results of each method among patients with the Ménière's Disease (MD) and internal ear diseases (IED) who do not fulfill criteria for MD. Sensibility, specificity and predictive values of anti-HSP70 test in diagnosis of MD were calculated. STUDY: Prospective, case-control. METHODS: Blood samples were collected from 31 patients with MD and 78 patients with non Ménière IED. Data regarding cochlear and vestibular symptoms were obtained and blood sample was tested. RESULTS: ELISA tests results were positive in 4(13%) patients and results of WB were positive in 8(26%) patients. Among patients with positive ELISA results, 1 patient presented active disease and in the remaining 3 patients the disease was inactive. Among the 8 WB positive patients, only 2 patients presented active disease. Statistical analyses did not establish any association between serologic findings and clinical factors of MD. CONCLUSION: The presence of anti-HSP70 using the ELISA and the WB methods did not demonstrate clinical value for the diagnosis of MD. We did not find association between idiopathic MD nor unspecific etiology MD and the presence of anti-HSP70 auto-antibodies.

Distribution of bone marrow-derived cells in the vestibular end organs and the endolymphatic sac.

CONCLUSION: Bone marrow-derived cells (BMDCs) are constitutively present in the vestibular end organs and in the endolymphatic sac, and may play a role in the maintenance of inner ear homeostasis. OBJECTIVES: The aim was to examine the distribution and characteristics of BMDCs in the vestibular end organs and in the endolymphatic sac. METHODS: Bone marrow-chimeric mice were generated by bone marrow transplantation from mice genetically labeled with enhanced green fluorescent protein to C57 Bl/6 mice to visualize BMDCs. Three months after bone marrow transplantation, inner ear specimens were processed for histochemical analyses. RESULTS: BMDCs were widely distributed in the vestibular end organs and the endolymphatic sac, whereas there were differences in the phenotype and the distribution between the vestibular end organs and the endolymphatic sac. A subpopulation of BMDCs in the vestibular end organs expressed antigen-presenting protein MHC class II. Moreover, the density of BMDCs in the vestibular end organs increased in response to local mechanical stress.

Expressions of aquaporin-2, vasopressin type 2 receptor, transient receptor potential channel vanilloid (TRPV)1, and TRPV4 in the human endolymphatic sac.

OBJECTIVE: To localize aquaporin (AQP)2, vasopressin type 2 receptor (V2-R), and transient receptor potential channel vanilloid subfamily 1, 4 (TRPV1, TRPV4) in the human endolymphatic sac (ES). METHODS: Three samples of human ES were sampled during the removal of vestibular schwannoma by way of the translabyrinthine approach. The samples were immediately fixed in 4% paraformaldehyde and embedded in OCT compound; immunohistochemistry was performed with AQP2, V2-R, TRPV1, and TRPV4 polyclonal antibodies. RESULTS: AQP2, V2-R, TRPV1, and TRPV4 proteins were detected in the epithelial layer of the ES but were not observed in connective tissue around the ES. TRPV1 was also expressed in blood vascular endothelial cells of the connective tissue of ES. CONCLUSIONS: AQP2, V2-R, and TRPV4 were expressed in the luminal epithelium of human ES. The same characteristic distribution of water and ion channels is seen in the kidney, where a significant amount of fluid is filtrated and resorbed. ES probably plays an active role in the homeostasis of the endolymph.

Interferon-gamma expression in the inner ear of rats following secondary immune reaction in the endolymphatic sac.

Recently, we demonstrated increased intercellular adhesion molecule-1 (ICAM-1) expression in the inner ear of systemically pre-sensitized rats after antigen [keyhole limpet hemocyanin (KLH)] challenge into the endolymphatic sac (ES), in good correlation with the cellular infiltration. Interferon-gamma (IFN-gamma) is an important cytokine that upregulates the expression of ICAM-1. Here, we report upregulation of IFN-gamma expression in the inner ear of systemically pre-sensitized rats after antigen (KLH) challenge into the ES. Immunoreactivity for IFN-gamma was detected in the spiral ligament, suprastrial region, spiral modiolar veins, spiral collecting venules, surface membrane of the perilymphatic compartment and perilymphatic space of immunized, but not control, rats. IFN-gamma expression was detected at 1.5 h post-challenge, peaked at 6 h and gradually returned to baseline levels after 7 days. Interestingly, the time kinetics of IFN-gamma expression were in good correlation with those of ICAM-1. These observations demonstrate that antigen challenge into the ES induces IFN-gamma expression, which can then upregulate ICAM-1 expression and induce cell infiltration, suggesting that IFN-gamma may play a crucial role in immune-mediated inner ear diseases.

[The endolymphatic sac: its roles in the inner ear]. / Le sac endolymphatique: ses fonctions au sein de l'oreille interne.

The endolymphatic sac is a non-sensory organ of the inner ear. It is connected to the endolymphatic compartment that is filled with endolymph, a potassium-rich fluid that bathes the apical side of inner ear sensory cells. The main functions ascribed to the endolymphatic sac are the regulation of the volume and pressure of endolymph, the immune response of the inner ear, and the elimination of endolymphatic waste products by phagocytosis. Functional alteration of these functions, leading to deficient endolymph homeostasis and/or altered inner ear immune response, may participate to the pathophysiology of Ménière's disease, an inner ear pathology that causes episodes of vertigo, sensorineural hearing loss and tinnitus, and is characterized by an increase in volume of the cochleo-vestibular endolymph (endolymphatic hydrops).

Cell proliferation in the endolymphatic sac in situ after the rat Waldeyer ring equivalent immunostimulation.

OBJECTIVES/HYPOTHESIS: It has been recognized that immunological mechanisms could be involved in the pathogenesis of different inner ear disorders, such as progressive sensorineural hearing loss, Meniere's disease, and even sudden deafness. The endolymphatic sac acts as an immune control organ for the inner ear and has been considered as an effector site of the mucosa-associated lymphoid tissue. The purpose of the study was to determine the potentially immunological relationship between endolymphatic sac and Waldeyer ring equivalent, one of the most important affector organs in mucosa-associated lymphoid tissue. STUDY DESIGN: Animal model. METHODS: Thirty-six rats were employed. Two animals were killed for histological observation of Waldeyer ring equivalent, and another 34 animals were randomly divided into experimental and control groups and received bilateral intranasal immunizations with keyhole limpet hemocyanin or Freund adjuvant, respectively. The ears of immunized animals and control animals were examined for keyhole limpet hemocyanin-positive memory cells and immunoglobulin G-positive plasma cells with the technique of immunohistochemical analysis. The histopathological changes and cell proliferation in those ears were also assessed. RESULTS: There were paired and organized lymphoid tissues in the lateral wall of the first choana in the rat. Keyhole limpet hemocyanin-positive lymphocytes appeared within the endolymphatic sac at 3 days after the first anti-genetic stimulus of the Waldeyer ring equivalent. Endolymphatic hydrops in the cochlea, elevated amounts of immunocompetent cells, and increased activity of cell proliferation within the endolymphatic sac were also considered after four intranasal injections of keyhole limpet hemocyanin. CONCLUSION: Presumably, endolymphatic sac is supplied with immunocompetent cells from Waldeyer ring equivalent and has an ability of co-reaction with Waldeyer ring equivalent.

Autoimmune inner ear disease: diagnostic and therapeutic approaches in a multidisciplinary setting.

Autoimmune Inner Ear Disease (AIED) is a clinical syndrome of uncertain pathogenesis. It is associated with bilateral rapidly progressive hearing loss. The hearing loss may be associated with vestibular symptoms. Autoimmunity has been proposed as the pathogenesis of this sort of hearing loss, although the mechanism of the disease is poorly understood. It is well accepted that the endolymphatic sac is an immunocompetent organ and circulating autoantibodies against inner ear antigens have been reported, as have viral antigens in the endolymph, although the sensitivity, specificity, and roles of those antibodies in a disease process are poorly defined. We will describe the clinical aspects of the disease, the histopathology, the immunologic indicators, the types of presentation, both from the audiologic and vestibular point of view, clinical trials for treatment and the follow-up. One of our conclusions is that many of these patients respond favorably to the treatment Methotrexate.

[Immuno-pathogenesis in Meniere's disease].

Since the report of Duke in which an allergic etiology was considered to be the cause of Meniere's disease, the hypothesis that a certain type of Meniere's disease is generated through immuno-pathological mechanisms has been advocated for 70 years. During this period, another entity of immune-mediated inner ear disorders, i. e., autoimmune inner ear disease was introduced. Fundamental immunological phenomena of the inner ear have been rapidly elucidated since 1980. The endolymphatic sac is the only site which contains immuno-competent cells within the inner ear. The inner ear is capable of mounting active immune responses when appropriately stimulated and the endolymphatic sac plays an integral function for inner ear immune response. Actually, many reports have been published that link immunity and Meniere's disease with a variety of proposed immune-related etiologies from autoimmunity to non-autoimmunity. It is suggested that immune injury to the endolymphatic sac plays an important role in the pathogenesis of Meniere's disease. These functional and morphological circumstances strongly suggest that an immunological etiology of Meniere's disease is not theoretically unfounded.

[Clearance function of endolymphatic sac to extraneous TD-antigen].

OBJECTIVE: To study the clearance function of the rat endolymphatic sac to TD antigen. METHOD: 30 healthy adult S-D rats were employed in the experiment. At two weeks after being sensitized systemically with keyhole limpet hemocyanin (KLH) (Thymus-dependent antigen), the animals were inoculated into the labyrinth through cochlea base tune with the same antigen. At 1, 3 h, 1, 3,7 and 14 day after labyrinth vaccination, the rats were sacrificed and the temporal bones were removed, then the frozen-sections of the decalcified samples were examined respectively. Using Anti-KLH McAb, the SABC immunohistochemical methods were utilized to investigate the changes of KLH in the endolymphatic sac. RESULT: At 3 h after labyrinth vaccination the antigen appeared in the endolymphatic sac. While the KLH were trapped and phagocytized in the endolymphatic sac at 1-7 day after labyrinth vaccination, the antigen in the cochleae gradually reduced. At the 14th day only feeble KLH positive stains can be found in the endolymphatic sac. CONCLUSION: The rat endolymphatic sac can phagocytize, dispose and removal extraneous antigen, which verifies the immune defense function of endolymphatic sac in inner ear immune response.

Th1: mediator lymphocytes in experimental autoimmune labyrinthitis.

A previous study on experimental autoimmune labyrinthitis (EAL) consistently demonstrated transient infiltration of lymphocytes only into the inner ear of mice. To clarify the profile of lymphocytes in the initiation of EAL, the present study investigated cell surface antigens, as well as cytokines, from Day 4 to Day 35, using immunohistochemical techniques. Many CD4+ cells mainly infiltrated the endolymphatic sac as early as Day 4 and gradually spread to the rest of the inner ear. Infiltration peaked on Day 12 and persisted in most animals until Day 35, although the number of cells gradually decreased. In contrast, very few CD8+ cells were found to have appeared in the inner ear of all animals on Day 10, and the number of cells rapidly decreased. Many cells positive for IFN-gamma and IL-2 were identified in the endolymphatic sac on Day 4. These results suggest that helper T1 lymphocytes, rather than cytotoxic T lymphocytes, may play a central role in the initiation of EAL.

Detection of single-stranded DNA in the hydropic vestibule after the direct injection of antigen into the endolymphatic sac of guinea pigs.

Immunohistochemical study for single-stranded DNA (ssDNA) with vestibule of guinea pigs was performed after the injection of keyhole limpet hemocyanin (KLH) into the right endolymphatic sac. Endolymphatic hydrops became evident by expansion of the Reissner's membrane in the cochlea of all animals 1 day after the injection of KLH. Increased ssDNA expression was detected in the sensory epithelium and transitional area, while temporal bones in the control group did not show any ssDNA immunoreactivities. ssDNA is accompanied with the apoptotic change in the vestibule. Our results suggest that apoptotic changes could be involved in the hydropic vestibule and these phenomena lead inner ear disturbance as seen in endolymphatic hydrops.

Immune response and immunopathology of the inner ear: an update.

Immune-mediated inner-ear disease includes clinical conditions associated with unilateral or bilateral rapidly progressive forms of sensorineural hearing loss. A systemic autoimmune disorder can be present in less than one-third of cases. Because of the lack of well defined detection methods to identify immune-mediated processes within the inner ear, and the fact that the human inner ear is not amenable to diagnostic biopsy, there has been great interest in developing animal models. Experimental models of sterile and virus-induced labyrinthitis support the participation of the immune system in the aetiopathogenesis of inner-ear disorders: interleukin-2 emanates from the endolymphatic sac and assists in changing the spiral modiolar vein, as in the expression of intercellular adhesion molecule 1, which allows the egrees of immune cells from the circulation. The formation of a fibro-osseous matrix ultimately results in degeneration of the inner ear. These investigations have allowed us to alter the immune response for the purpose of regulating its intensity and the subsequent damage to patients.

[Cell proliferation in endolymphatic sac during the immune response of inner ear].

OBJECTIVE: To study the cell proliferation in endolymphatic sac(ES) during the secondary immune response in inner ear. METHODS: Fifteen healthy, female SD rats were employed in the experiment. Sensitized systematically with keyhole limpet hemocyanin (KLH), the animals were inoculated with the same antigen through cochlea basal turn into the labyrinth. Administrated 5-bromo-2'-deoxyuridine (BrdUrd) intraperitoneally, the rats were sacrificed and the temporal bones were harvested at 3, 7, 14 day after labyrinth vaccination respectively. The frozen sections of the decalcified samples were dealt with H-E staining and immunohistochemical methods to investigate the cellular infiltration, BrdUrd and IgG positive cells in the ES. RESULTS: While the ES lumen and perisaccular region were infiltrated with mononuclear-phagocyte at the 3rd day post-vaccination, plasmocyte and lymphatic cells become the predominant infiltrating cells in the ES at the 7th day. The KLH in the ES lumen were phagocytized at the 3-7th day post-vaccination. S-phase cells and IgG positive cells in ES increased markedly in the 3rd day and 7-14 days post-vaccination respectively with similar localization. CONCLUSION: The activity of cell proliferation in the ES enhance and local proliferated cells may differentiate in situ into immunocompetent cells during the secondary immune response against TD antigen in the inner ear. This indicates that ES plays an important role in immune response of inner ear.

T cells infiltrating from the systemic circulation proliferate in the endolymphatic sac.

It has been reported that autoimmune mechanisms are involved in the development of inner ear disorders such as Meniere's disease and steroid-responsive sensorineural hearing loss. In the present study, using an animal model for graft-versus-host disease, we investigated the immune regulatory mechanism in the endolymphatic sac and demonstrated that donor T cells injected into the systemic circulation of recipients infiltrate and proliferate in the perisaccular region. These findings suggest that immunocompetent cells are supplied from the systemic circulation through blood-labyrinth and blood-endolymph barriers into the endolymphatic sac, and that the endolymphatic sac allows these cells to proliferate locally as a local immune defense. It therefore seems likely that the endolymphatic sac plays a crucial role in not only graft-versus-host disease but also autoimmune inner ear disorders.