Expression of inducible nitric oxide synthase (iNOS) in middle ear epithelial cells by IL-1β and TNF-α

Objective: The importance of nitric oxide (NO) in the development of mucoid middle ear effusion (MMEE) has been reported, but the mechanism regulating NO release is unclear. We hypothesized that middle ear epithelial cells (MEEC) are an important source of NO and that cytokines may be responsible for inducible nitric oxide synthase (iNOS) mRNA expression in middle ear epithelial cells. This study aims to identify and localize iNOS in middle ear epithelium, and to characterize the effects of cytokines IL-1β and TNF-α on the expression and regulation of iNOS in rat middle ear epithelial cells. Methods: In vitro study: 40 Healthy adult Sprague-Dawley rats weighing 200-250 g were used as donors of MEEC. Cultured MEEC were exposed to IL-1β (10 ng/ml), TNF-α (5 ng/ml) or PBS (negative control) stimulation for 16 h. In vivo study: A total of 45 healthy adult Sprague- Dawley rats weighing 200-250 grams were used for this study. The tympanic bullae were exposed bilaterally by a submandibular approach. Animals were equally divided into three groups and inoculated with either 250 ng of IL- 1β, 250 ng of TNF-α or PBS. A PBS group served as control. Expression of iNOS mRNA in MEEC from both in vivo and in vitro studies was determined by RT-PCR using specific primers. Expression of iNOS protein in MEEC was determined by immunocytochemistry and Western blot using specific anti-iNOS antibody. Results: Primary culture of rat MEEC was positively stained by cytokeratin antibody, but not by vimentin, indicating the epithelial origin of the cultured cells. RT-PCR revealed that cultured MEEC without treatment of IL-1 β or TNF-α did not express iNOS mRNA whereas cultured MEEC treated with IL-1β or TNF-α for 16 h expressed iNOS mRNA. Both immunocytochemistry and Western blot demonstrated the expression of iNOS protein in the majority of cultured MEEC treated with IL-1β or TNF-α for 16 h, whereas the expression of iNOS protein was not detectable in MEEC without treatment. Expression of iNOS protein in vivo was observed in middle ear mucosa treated with IL-1β and TNF-α by immunohistochemistry. Conclusion: Expression of iNOS mRNA and iNOS protein is induced in MEEC following the treatment of cytokines IL-1β or TNF-α both in vivo and in vitro. The results of the present study demonstrate that rat MEEC possess the capacity to express iNOS after IL-1β and TNF-α stimulation. (C) 2000 Elsevier Science Ireland Ltd.