Up-Regulated Expression of Interferon-Gamma, Interleukin-6 and Tumor Necrosis Factor-Alpha in the Endolymphatic Sac of Meniere's Disease Suggesting the Local Inflammatory Response Underlies the Mechanism of This Disease

Abstract

Background: Immune mediated inflammatory changes affecting the endolymphatic sac (ES) may underlie the pathology of Meniere's disease (MD). The aim of the present study was to explore the differentially expressed cytokines in ES luminal fluid (ELF) of patients with MD, and the correlation between the expression of cytokines in the ELF with that in the serum was determined by quantitatively analyzing the cytokines in human ELF and serum.

Methods: Human ELF, serum and ES tissues were collected from patients with unilateral MD and patients with acoustic neuroma (AN) during surgery. The Simoa Cytokine 6-Plex Panel kit was used to analyze the levels of cytokines in the ELF and blood samples of the patients. Immunohistochemistry and immunofluorescence were subsequently used to validate the relative expression levels of the cytokines in MD.

Results: Significant differences were identified in the expression levels of interferon-γ (IFN-γ) (P < 0.001), interleukin (IL)-6 (P = 0.008) and tumor necrosis factor-α (TNF-α) (P = 0.036) in the luminal fluid of the ES comparing between the MD and AN groups. By contrast, the levels of IFN-γ, IL-10, IL-12p70, IL-17A, IL-6 and TNF-α in the serum of the MD group were not significantly different from those of either the AN group or healthy control subjects. In addition, no significant correlations in the expression levels of cytokines compared between the ELF and serum were found for the patients in either the MD or the AN group. Finally, the detection of positive expression of TNF-α, IL-6 and IFN-γ in the epithelial cells of the majority of ES specimens from patients with MD confirmed the up-regulated expression of these cytokines in the ES of patients with MD.

Conclusions: The identification of up-regulated expression levels of TNF-α, IL-6 and IFN-γ in the ELF in the present study has provided direct evidence for an increased immunologic activity in the microenvironment of the ES in patients with unilateral MD, may suggest the local inflammatory response underlies the mechanism of this disease.

Keywords: ES luminal fluid; Meniere's disease; cytokines; endolymphatic sac; inflammatory.

Figure 1

Schematic illustration of endolymphatic sac (ES; blue) and duct (ED; blue) and its relationship to the cochlea (pink) and the vestibule (light green). The sac can be divided into extraosseous (eES), intermediate (mES) and intraosseous (iES) portions.

Figure 2

Sampling ELF and collecting ES specimen from patient no. 8 with left MD. (A) After blockage of the distal intraosseous portion of the ES using two titanium clips (green arrow) and exposure of the lumen of the ES (yellow arrow), a small amount of fluid (endolymph) could be found (white arrow). Then, 2 μl sterile water (*) was infused into the lumen of the ES. (B) 2 μl diluted luminal fluid (**) was aspirated from ES. (C) A solid and dotted line was used to mark the border of the ES specimen that would be resected. (D) An ES specimen including the distal intraosseous and proximal extraosseous (lateral side) portions (blue arrow) of the ES was resected. PSCC, posterior semicircular canal; ES,endolymphatic sac; ELF, ES luminal fluid; MD, Meniere's disease.

Figure 3

(A) Cytokine levels of ELF in the MD group compared with those in the AN group. (B) Cytokine levels of the serum in the MD group compared with those in the AN group and the healthy control group. *P < 0.05; **P < 0.01; ***P < 0.001; ES, endolymphatic sac; ELF, ES luminal fluid; MD, Meniere's disease; AN, acoustic neuroma.

Figure 4

Hematoxylin-Eosin staining revealed a lumen was patent or slit-like in the intraosseous and extraosseous portions of the ES from patient no. 7 with acoustic neuroma (A), whereas the framed area of a1 in (A) magnified to show an intact folded epithelial (arrow) lining in the lumen of intraosseous part of ES (B) and the framed area of a2 in (A) magnified to show a simple cuboidal epithelium (arrow) lining in the lumen of extraosseous portion of the ES (C). ES, endolymphatic sac. Scale bars: (A) 200 μm; (B,C) 100 μm.

Figure 5

Immunostaining for TNF α showed strong staining of epithelial cells (red arrowhead) and weak staining of the subepithelial fibroblasts (yellow arrow) in patient no. 5 with right MD (A), and weak staining of epithelial cells in the magnified framed area (yellow arrow) in patient no. 3 with left AN (B). Immunostaining for IL 6 revealed strong signal in the ES epithelium in patient no. 6 with left MD (C) and no positive expression of epithelial cells in patient no. 4 with right AN (D). Immunostaining from ES specimen in patient no. 8 with left MD showed strong labeling along the epithelial lining of ES for IFN γ (E) and a negative expression in the epithelial cells of ES for IL-10 (G). Whereas a negative expression of the ES epithelium for IFN γ in patient no. 6 with right AN (F) and IL-10 in patient no. 7 with right AN (H) were detected. MD, Meniere's diseae; AN, acoustic neuroma; ES, endolymphatic sac. Scale bars: (A–H) 200 μm.

Figure 6

Immunofluorescent staining showed a strong cytoplasmic immunofluorescence signal for IL-6 (green) (A), IFN-γ (red) (B) and TNF-α (green) (E), and a minimal cytoplasmic immunofluorescence signal for IL-10 (red) (F) along with the marker of nuclei, DAPI (blue) (C,G) in the proximal extraosseous part of the ES in patient no. 8 with MD. (D) The co-localization regions of IL-6 and IFN-γ showed color in yellow in ES epithelia cells (arrow) in the merged image. The framed area is magnified in (D′), showing that IFN γ and IL-6 were mainly expressed in the cytoplasm and membrane of the ES epithelium. (H) Merged image of immunofluorescent labeling for TNF-α and IL-10 showed color in green in ES epithelia cells (white arrow) and the sub-epithelial tissue and Sub-epithelial fibrocytes (yellow arrow). The framed area is magnified in (H′), showing the expression of TNF-α was localized in the cytoplasm and membrane of the ES epithelium. MD, Meniere's disease; ES, endolymphatic sac. Scale bars: (A–H) 50 μm.