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.

Neuronal Fibers and Neurotransmitter Receptor Expression in the Human Endolymphatic Sac.

INTRODUCTION: Recent studies suggest that the human endolymphatic sac (ES) may have multiple functions, including an ion-transport capacity comparable to the kidney, an immunological capacity and a possible natriuretic capacity. Further, there have been speculations of a yet undefined role in intracranial pressure homeostasis. The anatomical location towards the sigmoid sinus would suggest a possible endo- and/or paracrine signaling. However, neuronal connections may also apply, but it remains very scarcely explored in the human ES. STUDY DESIGN: DNA micro-arrays and immunohistochemistry were used for analyses of fresh human ES tissue samples. METHODS: A total of 30 tissue samples from the human ES 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 neuronal signaling was determined and results for selected key molecules verified by immunohistochemistry. Transmission electron microscopy was used for ultrastructural analysis. RESULTS: For the transmission electron microscopy analysis, a direct innervation of the ES was observed with unmyelinated fibers imbedded in the ES epithelial lining. The microarrays confirmed, that several molecules involved in neuronal signaling were found expressed significantly in the ES DNA profile, such as the Cholecystokinin peptide and related receptors, Dopamine receptors 2 and 5, vesicular monoamine transporter 2 (VMAT2), plasma monoamine transporter (PMAT), and Serotonin 1D. All peptides were verified by immunohistochemistry. CONCLUSIONS: Based on global gene expression profiling and immuno-histochemical labeling, we conclude that the human ES expresses neuropeptide receptors and monoamine transporters. Combined with the ultrastructural demonstration of unmyelinated axons imbedded within the epithelial lining, the findings suggest that neuro-signaling mechanisms are involved in functions exerted by the ES.

Ephrin-B2 governs morphogenesis of endolymphatic sac and duct epithelia in the mouse inner ear.

Control over ionic composition and volume of the inner ear luminal fluid endolymph is essential for normal hearing and balance. Mice deficient in either the EphB2 receptor tyrosine kinase or the cognate transmembrane ligand ephrin-B2 (Efnb2) exhibit background strain-specific vestibular-behavioral dysfunction and signs of abnormal endolymph homeostasis. Using various loss-of-function mouse models, we found that Efnb2 is required for growth and morphogenesis of the embryonic endolymphatic epithelium, a precursor of the endolymphatic sac (ES) and duct (ED), which mediate endolymph homeostasis. Conditional inactivation of Efnb2 in early-stage embryonic ear tissues disrupted cell proliferation, cell survival, and epithelial folding at the origin of the endolymphatic epithelium. This correlated with apparent absence of an ED, mis-localization of ES ion transport cells relative to inner ear sensory organs, dysplasia of the endolymph fluid space, and abnormally formed otoconia (extracellular calcite-protein composites) at later stages of embryonic development. A comparison of Efnb2 and Notch signaling-deficient mutant phenotypes indicated that these two signaling systems have distinct and non-overlapping roles in ES/ED development. Homozygous deletion of the Efnb2 C-terminus caused abnormalities similar to those found in the conditional Efnb2 null homozygote. Analyses of fetal Efnb2 C-terminus deletion heterozygotes found mis-localized ES ion transport cells only in the genetic background exhibiting vestibular dysfunction. We propose that developmental dysplasias described here are a gene dose-sensitive cause of the vestibular dysfunction observed in EphB-Efnb2 signaling-deficient mice.

Oxygenated fixation demonstrates novel and improved ultrastructural features of the human endolymphatic sac.

OBJECTIVES/HYPOTHESIS: The purpose of the present study is to describe in detail the ultrastructure of the human endolymphatic sac using a new and improved method of fixation as well as a refined surgical approach in obtaining specimens. STUDY DESIGN: Transmission electron microscopy of the human endolymphatic sac, employing an oxygenated fixative. METHODS: Eighteen tissue samples of the human endolymphatic sac were obtained during surgery for vestibular schwannoma using the translabyrinthine approach. The specimens were fixed in 2% glutaraldehyde in an oxygenated fluorocarbon blood substitute vehicle before preparation by routine methods for transmission electron microscopy. We focused on the epithelial cell layer, subepithelial tissue, intraluminal content, and vascular tissue in both the intra- and extraosseous part of the endolymphatic sac. RESULTS: We observed well-defined endolymphatic sac epithelial cell lining in all 18 specimens. In general, we found very well-preserved specimens with well-defined intracellular structures. In contrast to the results in former studies, a minimum of fixation artifacts was observed in the present study. Three different cell types were observed in the intraosseous part of the sac: mitochondria-rich cells, ribosome-rich cells, and nonclassifiable cells. A fourth cell type was found in the extraosseous part. Novel ultrastructural features of the epithelial lining and the subepithelial layer are described and discussed. CONCLUSIONS: The results in the present study indicate an improvement in obtaining human tissue with optimal fixation for ultrastructural analysis and provide several novel morphologic observations. The potential functions of the endolymphatic sac are discussed with reference to former studies.

Expression of anion exchangers in cultured human endolymphatic sac epithelia.

HYPOTHESIS: Pendrin acts as a Cl-/HCO3- exchanger and is responsible for endolymphatic fluid volume and pH homeostasis in human endolymphatic sac epithelial cells. BACKGROUND: The endolymphatic sac (ES) is part of the membranous labyrinth in the inner ear that plays an important role in maintaining homeostasis of the endolymphatic fluid system. However, the exact mechanism of fluid volume and pH regulation is not fully understood yet. We aimed to demonstrate the expression of various anion exchangers (AEs), including pendrin, in cultured human endolymphatic sac epithelial (HESE) cells. METHODS: Endolymphatic sac specimens were harvested during acoustic neuroma surgery (n = 24) using the translabyrinthine approach and then subcultured with high epidermal growth factor (EGF) (25 ng/ml) media and differentiated using low-EGF (0.5 ng/ml) media. The cultured cells were classified according to the morphology on TEM. The Cl-/HCO3- exchanger activity was assessed by pHi measurement using pH sensitive dye 2', 7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF/AM). We performed reverse transcriptase-polymerase chain reaction and immunohistochemical staining for AEs. RESULTS: We determined that 7.3 ± 6.7% of cells differentiated into mitochodria-rich cells and 50.2 ± 15.1 of cells differentiated into ribosome-rich cells. bAE3, AE4, SLC26A4, SLC26A6, and SLC26A11 were also expressed in cultured HESE cells. The cultured cells had Cl-/HCO3- and Cl-/formate exchange activity on the luminal membrane, which is sensitive to anion channel inhibitors (DIDS 500 µM). Furthermore, we showed that pendrin (SLC26A4) was expressed in cultured HESE cell membranes. CONCLUSION: Our results suggest that AEs, including pendrin, are expressed in epithelia of ES and may have role in maintaining ionic homeostasis, and the HESE culture system are useful for uncovering the functional role of ES epithelial cells.

Establishment of cell lines from the human middle and inner ear epithelial cells.

The middle ear infection is the most common childhood infection. In order to elucidate the cell and molecular mechanisms involved in bacterial recognition and innate immune response, we have established a stable human middle ear cell line, which has contributed to the current knowledge concerning the molecular pathogenesis of the middle ear infection. The inner ear, a sensory organ responsible for hearing and balance, is filled with inner ear fluid, and disturbance of the fluid homeostasis results in dizziness and hearing impairment. It has been suggested that the endolymphatic sac (ES) may play a critical role in the fluid homeostasis of the inner ear. We have established a stable human ES cell line and are undertaking cell and molecular characterization of this cell line.

The detailed localization pattern of Na+/K+/2Cl- cotransporter type 2 and its related ion transport system in the rat endolymphatic sac.

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na(+)/K(+)/2Cl(-) cotransporter type 2 (NKCC-2) and Na(+)/K(+)-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na(+)/K(+)-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na(+)/K(+)-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules.

[Ultrastructural localization of aquaporin 1 in endolymphatic sac of the mouse].

OBJECTIVE: To study the ultrastructural localization of aquaporin 1 (AQP1) in the endolymphatic sac (ES) of the mouse inner ear and explore the function of the AQP1 in ES. METHODS: The cellular localization of AQP1 in ES of the mouse inner ear was investigated by immunocytochemistry. The ultrastructural localization of AQP1 in the mouse inner ear was performed by immunogold electron microscopy which is characterized as cryoprotection and high sensitivity. RESULTS: In the ES, strong AQP1 labeling was observed in the sub-epithelial connective tissue. Fibroblasts of sub-epithelial connective tissue of the ES present densely labeling of gold particles. But the epithelial cells of the ES were devoid of labeling. AQP1 was localized on the cell processes of the fibrocytes. CONCLUSIONS: AQP1 in the ES may play an important role in absorbing water and regulate the balance of fluid and ion in the inner ear.

Soluble megalin is accumulated in the lumen of the rat endolymphatic sac.

The endolymphatic sac (ES) is believed to play an important role in maintaining homeostasis in the inner ear by the absorption and endocytosis of endolymph. Megalin is a 600-kDa multiligand endocytic receptor expressed in certain types of absorptive epithelia including kidney proximal tubules. We analyzed the immunoreactivity for megalin in rat ES by immunofluorescence, immunogold electron microscopy, and immunoblotting. With immunostaining, the luminal substances of the ES were strongly stained for megalin. Megalin was also localized in luminal macrophage-like cells and both types of epithelial cell (mitochondria-rich cells and ribosome-rich cells). In these cells, the megalin was localized in the lumen of endosomes, but was not membrane associated. This localization pattern indicates that the megalin in these cells is not a membrane receptor, but merely one of the constituents that are endocytosed from the lumen of the ES. Immunoblotting indicated that the megalin in the ES is a 210-kDa molecule lacking a cytoplasmic domain. This suggests that the megalin in the ES may be a soluble form, different from the 600-kDa membrane-bound receptor expressed in kidneys. Taken together, it is likely that the megalin in the ES lumen is a soluble component and may be endocytosed by the ES epithelial cells. Furthermore, we found that the tectorial membrane, an acellular structure in the cochlea, gave a strong megalin immunoreaction. Since the cochlea is connected to the ES, the megalin may be transported alone or with the components of the tectorial membrane from the cochlea to the ES lumen through longitudinal flow.

Differences in endolymphatic sac mitochondria-rich cells indicate specific functions.

OBJECTIVE/HYPOTHESIS: The purpose of the study was to examine the specific involvement of endolymphatic sac mitochondria-rich cells in endolymph homeostasis. STUDY DESIGN: Transmission electron microscopy and immunohistochemistry were performed on the endolymphatic sac of young adult rats, and two important developmental stages were also investigated. METHODS: Ultrastructural characteristics of endolymphatic sac mitochondria-rich cells were studied more concisely and compared with renal mitochondria-rich cells (i.e., the intercalated cells). In addition, expression of cytokeratins 7 and 19 was determined. RESULTS: Until birth, only one type of mitochondria-rich cell is observed in the rat endolymphatic sac. In young adult animals, distinct differences in mitochondria-rich cell ultrastructure in the endolymphatic sac enables classification into subtypes or configurations. Comparison of endolymphatic sac mitochondria-rich cells with renal intercalated cells reveals striking similarities and provides additional information on their specific function in endolymph homeostasis. Furthermore, differences in cytokeratin expression are determined in endolymphatic sac mitochondria-rich cells. CONCLUSIONS: Differences in morphology of endolymphatic sac mitochondria-rich cells develop after birth and may reflect a distinct functional or physiological state of the cell. In analogy to renal intercalated cells, the distribution patterns of H+-adenosine triphosphatase and Cl-/HCO3- exchanger may differ between subtypes. We propose that subtype A mitochondria-rich cells, from which protruding A mitochondria-rich cells are the activated state, are involved in proton secretion (apical H+-adenosine triphosphatase) and thus are potential candidates for hearing loss accompanying renal tubular acidosis. Subtype B mitochondria-rich cells are the most likely candidates to be affected in Pendred syndrome because of the assumed function of pendrin as apical Cl-/HCO3- exchanger.

Vascular occlusion in the endolymphatic sac in Meniere's disease.

In 2 patients with severe Meniere's disease (MD), there was histologic evidence of occlusion of the vein of the vestibular aqueduct (VVA). This finding coincided with total or partial occlusion of numerous small vessels around the endolymphatic sac (ES), flattening of epithelium, extensive perisaccular fibrosis, and signs of new bone formation. Ultrastructural analysis of the occluding material showed foci with dense connective tissue, calcification, lipid deposits, and layers of basement membrane, sometimes concentrically arranged. The exact nature of the occluding material was unknown. In another 2 MD patients, the VVA was not visualized, and the ES vessels showed no signs of occlusion. Seven controls with acoustic schwannoma or meningioma had normal vasculature. The presence of vascular impairment in the ES in MD patients indicated that altered hemodynamics may contribute to the pathogenesis of endolymphatic hydrops and MD.

In vitro growth of human endolymphatic sac cells: a transmission electron microscopic and immunohistochemical study in patients with vestibular schwannoma and Ménière's disease.

HYPOTHESIS: Human endolymphatic sac cells have been notoriously difficult to maintain in culture. It was hypothesized that an in vitro environment intended for growth of keratinocytes would also be suitable for human endolymph sac cells. BACKGROUND: Studies on cell physiology of human endolymphatic sac cells have been hampered by difficulties in maintaining them in culture. METHODS: Human endolymphatic sac cells were taken from 10 patients during translabyrinthine skull base surgery for vestibular schwannoma, one of whom also had Ménière's disease. Cell lines of proliferating epithelial cells were obtained after trypsinization and growth in a 3:1 mixture of Dulbecco's modified Eagle medium and Ham's F12 medium supplemented with 10% fetal calf serum. Fibroblast overgrowth was counteracted by the use of so-called cloning rings. During various stages, cells were investigated with transmission electron microscopy and/or immunohistochemistry. RESULTS: Proliferation took place after 2 to 3 days of primary cell culture. The cells were cytokeratin-positive and pleomorphic, and they had abundant polarized microvillus-like projections, numerous coated cytoplasmic pits and vesicles, and a well-developed rough endoplasmic reticulum. CONCLUSION: Cell lines of proliferating human endolymphatic sac cells can be produced with the technique described here and may be a valid tool in studies of human endolymph sac physiology.

Ultrastructural analysis of 20 intraosseous endolymphatic sacs from patients with cerebello-pontine angle tumours. A surgically obtained control material for histopathological studies.

OBJECTIVE: The purpose of this work was to collect a surgically obtained, freshly fixed material of the human intraosseous endolymphatic sac. This biopsy material was used to describe the normal ultrastructure as well as to serve as a control material for histopathological studies on Ménière's disease in particular. METHOD: The specimens, obtained during surgery for cerebello-pontine angle tumours, were fixed by immersion and then prepared by routine methods for transmission electron microscopy. The ultrastructural analysis was focused on intraluminal content, epithelial cell layer, subepithelial space, and morphological signs of immunological activity. The ultrastructure was analysed in relation to inner ear sensory function, tumour diagnosis, and patient's age and sex. RESULTS: As it was possible to obtain numerous specimens with an intact bony shell, the intraluminal substance could be analysed. Two separate epithelial cell types are described: one less abundant, often lighter and mitochondria-rich cell type; the other, often darker, epithelial cell with fever mitochondrias. Some of the latter cell types showed signs of active secretion. The subepithelial space was characterized by loose connective tissue adjacent to the epithelial lining, being more dense toward the bone. Elastic fibres were seen surrounding the entire endolymphatic sac. Macrophages in the intraluminal space and lymphocytes in the epithelial and subepithelial layers are described. No distinct morphology correlating to inner ear sensory function, tumour diagnosis, or patient's age and sex was revealed. CONCLUSIONS: This study confirms previously described, extensive variations in form and structure of the human endolymphatic sac. Various factors, such as surgical trauma, previous treatment, and processing method, can affect the ultrastructure and must be taken into consideration. The specimens described in this work appear to constitute a good control material for histopathological study of the human endolymphatic sac. It is still necessary to obtain large control materials such as this, as surgical specimens from patients with Ménière's disease are uncommon.

Ultracytochemical localization of Ca2+-ATPase activity in the endolymphatic sac of the chick.

The localization of Ca2+-ATPase activity was examined ultracytochemically in the endolymphatic sac (ES) of the 1-day-old chick. The reaction products showing Ca2+-ATPase activity were localized along the microvilli and the apical cell membranes of the epithelial cells of the ES. The lateral cell membranes also showed mild activity. These reactions were completely abolished when either Ca2+ or ATP was omitted from the incubation medium. The results suggest that Ca2+-ATPase plays a significant role as a Ca2+ pump for regulation of the Ca2+ concentration in the endolymph of the ES.

Ultrastructure of the endolymphatic sac in two-phase endolymphatic hydrops in the guinea pig.

Two-phase endolymphatic hydrops is a subtle experimental model for Meniere's disease. Chronic dysfunction of the endolymphatic sac, induced by dissection of the most distal part without causing damage to the intermediate part, is combined with increased endolymph production induced by administration of aldosterone which stimulates the N/K-ATPase in the stria vascularis. A transmission electron microscopic study was performed on the endolymphatic sacs of four groups of guinea pig cochleas: controls: non-operated aldosterone-treated cochleas; operated (dissection of the endolymphatic sac) cochleas; operated and aldosterone-treated cochleas. Light and electron microscopy showed a normal morphology in the controls. Aldosterone treatment had no visible effect. Dissected ears revealed severe deviations. The epithelium of the intermediate sac was low, showed dilated lateral intercellular spaces indicating elevated fluid transport and displayed serious degenerative processes. Distally, the endolymphatic sac was completely blocked by newly formed bone. Additional aldosterone treatment had no cumulative effect on the dissected ears.

Stereocilia-like structures in the endolymphatic sac in Ménière's disease and acoustic neuroma.

The vestibular aqueduct was surgically removed in 3 patients undergoing labyrinthectomy due to severe Ménière's disease (MD). Stereocilia-like structures were found in the luminal contents of the endolymphatic sac (ES) in all of these patients. The ES from 18 patients with acoustic neuroma were used as controls. In 1 of these, numerous stereocilia-like structures were found in the ES and in 3 additional patients, a few isolated cilia-like structures were disclosed. The findings may suggest an ongoing hair cell degeneration in the inner ear that is more advanced in patients with MD. The data also suggest that the endolymphatic duct is patent and that a longitudinal flow of endolymph also occurs in patients with MD.

"Endolymphatic sacitis" in a case of active Menière's disease. An ultrastructural histopathologic investigation.

An ultrastructural analysis of an entire intraosseous endolymphatic sac (ES) from a patient with active, well-documented Menière's disease was performed for the first time. The results were compared with those obtained from ES biopsy material from patients with acoustic neuromas. The ES was small in size and showed signs of focal inflammation with intraepithelial invasion by mononuclear cells. At these places the normal fine structure, including the vascular anatomy, was altered. The possible relationship between these changes and Meniere's disease is discussed.

Endocytosis and transepithelial transport of endolymph in the endolymphatic sac.

The fate of cationized ferritin (CF) injected into the endolymphatic space of the endolymphatic sac was observed by transmission electron microscopy. At 10 min after the injection, CF particles bound to the apical plasma membrane of epithelial cells of the sac and were then endocytosed with coated pits. However, they never passed through the junctional complexes between the epithelial cells. At 30 min after the injection, the CF particles were transferred to endosomes and lysosomes by small vesicles of 100-150 nm in diameter. CF particles were also found in small vesicles close to Golgi cisternae and in multivesicular bodies. Acid phosphatase positive lysosomes were found close to endosomes containing CF particles. In addition, a small fraction of the small vesicles containing CF particles became inserted into the basolateral plasma membrane. At 60 min after the injection, many CF particles were found in acid phosphatase positive secondary lysosomes. These observations suggest that endocytosis of endolymph is actively performed by the epithelial cells of the sac, and transepithelial vesicular transport across the epithelial cells occurs.

[Ultracytochemical localization on Na(+)-K(+)-ATPase activity in the guinea pig endolymphatic sac].

It is well-known that Na(+)-K(+)-ATPase plays an important role in active transportion of sodium (Na) and potassium (K) ions across the epithelium. This paper reports the ultrastructural localization of the K(+)-NPPase reflecting Na(+)-K(+)-ATPase activity in the guinea pig endolymphatic sac (ES) with the lead citrate one step method. The ultrathin sections viewed in the transmission electron microscope revealed that the reaction product was restricted to the cytoplasmatic side of the basolaterl plasma membranes of the ES epithelial cells. And the reactivity could be traced all the way up to the level of tight junctions close to the luminal aspect of the cells. On the other hand, the luminal plasma membrane of the epithelial cells showed no enzyme activity. Our findings suggest that the rich Na(+)-K(+)-ATPase enzyme activity is preferentially localized on the basolateral plasma of membrane of the ES epithelial cells. Possible physiological mechanism of Na(+)-K(+)-ATPase in the Na(+)-K+ ion transport of ES epithelial cells is discussed.