Analysis of Cultured Spiral Ligament Fibrocyles for revealing immune mechanisms of the inner ear

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1. An immunocytochemical strategy to classify the three types of fibrocytes found in the murine spiral ligament (SL) was established. A combination of staining for caldesmon, S-100 protein, and Na-K-ATPase was useful for this classification.2. Murine cochlear lateral wall was dissected for secondary culture. Cultures were positive for caldesmon and S-100 protein, and negative for Na-K-ATPase, suggesting that the cells were type I fibrocytes.3. Cultures were stimulated with IL-1β or TNF-α, and secretion of various mediators was measured. After stimulation, TNF-α, IL-6, MCP-1, KC, MIP-2, slCAM-1, and VEGF levels were elevated. Secretion of these chemokines and other mediators could induce inflammatory cell movement, which would prolong the inflammatory response.4. Cultures were stimulated with IL-1β or TNF-α, and expression of various adhesion molecules was investigated. Cultures without stimulation did not show staining for VCAM-1, ICAM-1, or MAdCAM-1. Staining was also absent after stimulation with IL-1β; however, VCAM-1 and ICAM-1 staining was observed after cells were stimulated with TNF-α. RT-PCR analysis showed that both VCAM-1 and ICAM-1 mRNAs were present after fibrocytes were stimulated with TNF-α. The findings suggest that activated fibrocytes may cause inflammatory cells to persist in the SL.5. Immunostaining for glucocorticoid receptors (GRs) was performed, and GRs were found throughout in the murine inner ear. The SL was among the regions where GRs were strongly expressed.6. Secretion of various mediators was measured after cultures were stimulated with TNF-α and dexamethasone, and the results were compared with those of cells stimulated with TNF-α alone. Dexamethasone suppressed secretion of IL-6, MCP-1, and KC by fibrocytes, suggesting that glucocorticoids have an anti-inflammatory effect on SL fibrocytes.