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.

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.

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.

Differential expression of transthyretin in papillary tumors of the endolymphatic sac and choroid plexus.

Aggressive papillary tumors of the temporal bone, occurring sporadically or as part of von Hippel-Lindau disease, have been shown to originate within the endolymphatic sac or duct. Also implicated as a potential precursor from which some of these tumors may arise is ectopic choroid plexus epithelium. To aid in the differentiation between papillary tumors of endolymphatic sac and duct origin and those arising from choroid plexus, an immunohistochemical study using stains for transthyretin (TTR), cytokeratins, S-100 protein, epithelial membrane antigen (EMA), and glial fibrillary acidic protein (GFAP) was carried out on archival specimens of normal and neoplastic endolymphatic sac and duct and choroid plexus epithelium. Transthyretin, a marker for choroid plexus epithelium, was found to show differential expression between choroid plexus papillomas and aggressive papillary tumors of the endolymphatic sac or duct. Therefore the use of TTR in concert with other immunohistochemical stains appear to aid in the differentiation between intracranial and intratemporal papillary tumors arising from choroid plexus and endolymphatic sac or duct epithelium.

Preliminary study of the role of endothelin-1 in the homeostasis of the inner ear.

Endothelin (ET), originally characterized as a 21-residue vasoconstrictor peptide from endothelial cells, has been reported to act as a local hormonal regulator of pressure, fluid, ions, and neurotransduction. Our previous studies suggested an important role of ET-1 in the inner ear. The present study investigated the time kinetics of ET-1 in the epithelium of the endolymphatic sac (ES) of guinea pigs and its relation to the development of endolymphatic hydrops (EH) following locally mounted secondary immune reaction. In the duration between 12 h and day 1, ET-1-like activity completely disappeared from the epithelium of the ES and was associated with the accumulation of inflammatory cells in the ES and a rapid development of EH. On day 7, ET-1-like activity recovered as a consequence of the decrease of inflammatory cells and reduction of EH. These findings suggest that ET-1 may play an important role as one of the regulators maintaining the fluid balance.

[A study on monoclonal antibodies against epithelial cell of endolymphatic sac in guinea pigs].

Using a microdissected endolymphatic sac of the guinea pig (n = 35) as an initial antigen preparation, serials of monoclonal antibodies were established which were used to label the epithelial cells of endolymphatic sac. The antibodies showed strong immunoreactivity with kidney, but not with other organs. It also showed isotopes IgG1, IgG2b and IgM. The results of Western blotting and SDS-PAGE indicated that the epitopes of monoclonal antibodies were proteins or glyoprotein with a molecular weight of approximately 7,400. The locations of the epitopes in epithelial cells suggest that it may play some roles in construction and function of the endolymphatic sac.

Allergic and immunologic aspects of Meniere's disease.

Meniere's disease, although idiopathic by definition, has been ascribed to a variety of causes, which more recently include autoimmune factors. Interest in the role of allergy in Meniere's disease has also increased. Studies from this institution and elsewhere provide evidence that allergy and immunologic factors play a role in Meniere's disease in at least some patients. The symptoms of Meniere's disease are thought to be produced by a sudden influx of fluid into the endolymphatic sac, producing a rupture of Reissner's membrane in the cochlea. The endolymphatic sac is capable of trapping antigen and generating its own immune response. It has a highly vascular subepithelial space containing numerous fenestrated blood vessels that are peripheral and "leaky." At least three mechanisms by which allergy may play a role in the production of fluid in the endolymphatic sac are described: the endolymphatic sac itself might be a "target organ" of mediator released from systemic inhalant or food reactions; deposition of circulating immune complex may produce inflammation and interfere with the sac's filtering capability; and a predisposing viral infection in childhood that produces a mild impairment of endolymphatic sac function may interact with allergies in adulthood and cause the endolymphatic sac to decompensate, resulting in endolymphatic hydrops. The endolymphatic sac is the seat of immune reactivity in the inner ear. Repeated inflammatory reactions can produce sac dysfunction and eventual production of Meniere's disease.

Detection of viral antigen in the endolymphatic sac.

A study was devised to determine whether or not any immune defense mechanism is present when a virus invades the human endolymphatic sac (ES). The ES was removed from 14 fresh autopsy cases having no known pre-mortem diseases in the middle and inner ears. Specimens were then examined for viral antigens including herpes simplex (HSV) type 1 and 2, mumps and cytomegalovirus using immunohistochemical methods. DNA examination by in situ hybridization was also performed for HSV. HSV antigen and DNA were observed in 9 of the 14 cases studied. These findings suggest that the virus invades the ES but is impeded by an immune defense mechanism under normal conditions. Since disease may alter host defenses, further studies are warranted to study the relationship between HSV and patients with Meniere's disease.

Antigen diffusion from the perilymphatic space of the cochlea.

The diffusion pattern of horseradish peroxidase (HRP) injected into the scala tympani of the cochlear basal turn of guinea pigs was studied to test whether antigen presented in this manner can gain access to the endolymphatic sac. By two hours, HRP reaction product was found throughout the cochlea, with the greatest amounts in the spiral ligament, spiral limbus, basilar membrane, and organ of Corti. In several cochleas, very weak labeling was seen in the stria vascularis. HRP reaction product was maximal in the basal turn. By two hours, HRP reaction product was also observed in the endolymphatic sac lumen, epithelial cells, subepithelial tissue, and perisaccular connective tissue. It was more common in the proximal portion. At this time, macrophages within the lumen already appeared to have phagocytosed the HRP. By 72 hours after injection, the inner ear was cleared of HRP. The results of this study support the hypothesis that antigen in the scala tympani gains access to the endolymphatic sac lumen, where it may be presented by macrophages to the systemic immune system. Antigen most likely does not gain access to the endolymphatic space in the cochlea, but it gets to the endolymphatic sac through the perilymph and the perisaccular tissue.

Immunological pathogenesis of endolymphatic hydrops and its relation to Menière's disease.

This study was designed to investigate an immunologically induced endolymphatic hydrops (ELH) and to focus on the issue of its pathogenesis in relation to Menière's disease. The time course of ELH was evaluated by light microscopy in a 2-hour to 7-month period following direct antigen challenge to the endolymphatic sac (ELS) in systemically pre-sensitized guinea pigs. ELH began to appear in the vestibule and the basal turn 5-7 hours after inner ear challenge and developed gradually. During the interval from the second day to the first week, ELH rapidly developed in all the cochlear turns and reached a maximum size. During the period from the second week to the eighth week, ELH gradually reduced. After 9 weeks, ELH of the saccule and the cochlea gradually recurred. During the interval from the first week to the eighth week, the time course of ELH correlated well with the grade of cellular infiltration of the perisaccular tissue. These results suggest that recurrent immunological reaction in the ELS may result in disorders of the ELS which finally lead to the onset of Menière's disease.

Evidence of direct communication of bone marrow cells with the endolymphatic sac in experimental autoimmune labyrinthitis.

In experimental autoimmune labyrinthitis, we found that the guinea pigs sensitized with bovine inner ear antigen (IEAg) developed cellular infiltration within the endolymphatic sac (E. sac). In this study, we investigated the distribution of immune mediated cells within bony vascular channels. The channels, which are normally found around the E. sac, are tiny and contain few immune-mediated cells. In contrast, in guinea pigs sensitized with IEAg bony vascular channels were large and contained immune-mediated cells. We hypothesize that immune-mediated cells in these channels are the result of the immune response directed against inner ear antigen, and that some immune-mediated cells seen in the E. sac during inner ear inflammatory events have migrated from the adjacent bone marrow.

Functional morphology of the human endolymphatic sac. A review.

Modern immunohistochemical methods allow a functional characterization of the human endolymphatic sac (ES) and its associated cell populations. The currently available immunohistochemical data on the extraosseous part of the human ES support the assumption that the epithelium is metabolically active and capable of both secretion and absorption. The reactivity of some epithelial cells with antibodies against neuroendocrine antigens implies a paracrine activity of the human ES. Further results provide evidence for a possible role of the human ES in inner ear immune defense and indicate a putative functional relationship of the human ES to the common mucosa-associated immune system.

Lymphocyte-macrophage activity in the human endolymphatic sac.

The human endolymphatic sac was analysed electron microscopically in patients undergoing acoustic Schwannoma surgery or vestibular nerve section. In addition, endolymphatic sacs from cadavers were analysed light microscopically. The results show that the human sac is endowed with a variable number of leucocytes and that there is a continuous recirculation of immuno-competent cells in this area of the inner ear that may be of importance for clearance of the inner ear from foreign substances and microorganisms derived from nearby located infection-prone areas. The possibility in Meniere's disease of a disturbed immunological activity in the sac is discussed.

Scanning electron microscopy and immunoglobulins of the endolymphatic sac in normal human subjects and sensorineural deafness. With special reference to Menière's disease.

The pathophysiology of the endolymphatic sac (ES) in Meniere's disease was studied by scanning electron microscopy and staining of immunoglobulins in the intradural portion of the ES. The peroxidase-antiperoxidase method by means of paraffin sections was used for staining of immunoglobulins. First, subjects without hearing impairment and malformation of the temporal bone, ranging from a 7-month-old fetus to an 80-year-old adult, were investigated. All subjects, including fetuses, showed well-arranged epithelial cells by scanning electron microscopy. The epithelial cells in the proximal portion of the intradural ES were oval, showing a tendency of transitional change to be flat as they drew near the distal portion of the ES. The epithelial cells consisted mostly of light cells, but sporadic dark cells were seen. Regarding the immunoglobulins. IgG was slightly positive in the epithelial and subepithelial layers. All 15 patients with Meniere's disease showed various types of degeneration of the epithelial cells though to varying degrees. However, these findings were also seen in cases of cochlear deafness. On the other hand, the ES of acoustic tumors, with retrocochlear or neural deafness revealed a normal finding, as found in healthy subjects. Inner ear deafness experimentally produced in animals by Kanamycin sulfate (KM) injection showed degeneration of the epithelial cells of the ES similar to that found in human cochlear deafness. IgG of the ES in Meniere's disease showed moderately evident deposits compared to normal subjects. However, this was also found not only in inner ear deafness other than Meniere's disease, but also in animal deafness experimentally produced by KM injection. It is very interesting to note that moderate endolymphatic hydrops was found in animals one year after Preyer's reflex had disappeared. It is postulated that endolymphatic hydrops develop because of impairment of endolymphatic fluid resorption at the rugose portion and stenosis of the lumen in the same portion, due to degeneration of the epithelial cells. From the above results, it is argued that degenerated epithelial cells and immunoglobulins of the ES in Meniere's disease may arise from the sequelae of cochlear deafness. It is also hypothesized that endolymphatic hydrops--at least in the terminal stage of Meniere's disease--may be consistent with the same pathophysiological conditions as in animal experiments.

Human endolymphatic sac: evidence for a role in inner ear immune defence.

In an immunohistochemical study, monoclonal and polyclonal antibodies have been used to identify cells and structures in the extraosseous part of endolymphatic sacs (ES) which were removed at autopsy from 30 persons. Intraluminal, intraepithelial, intravascular and perisaccular cells expressed the leukocyte common antigen. Immunostaining with antibodies against the CD4 and CD8 antigens revealed the predominance of CD4-positive T lymphocytes in the ES. A few lymphoid cells were found to express the major histocompatibility antigen class II. Interdigitating cells of the Langerhans type were rarely found in the epithelial layer. B lymphocytes were present in the lumen and the stroma of the ES and IgA- or IgG-containing cells in the stroma only. IgA, secretory component and the J chain were detected within epithelial cells and in the lumen of the ES. Macrophages were observed in the lumen and the stroma. Our findings are in accordance with previously published data in animals and man and give further evidence of an important role of the ES in inner ear immune defence.