In vitro effects of acid and pepsin on mouse middle ear epithelial cell viability and MUC5B gene expression.

OBJECTIVE

To examine whether in vitro exposure of mouse middle ear epithelial cells (mMEECs) to conditions that mimic physiologic reflux upregulates Muc5b gene expression and alters cell viability.

DESIGN

In vitro mMEEC model.

SETTING

Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC.

PARTICIPANTS

Cells from the immortalized mMEEC line.

MAIN OUTCOME MEASURES

Cell viability, the quantity of Muc5b messenger RNA abundance, and Muc5b promoter activity.

RESULTS

The 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide assays demonstrated an acidic dose-dependent decrease in cell survival, with pH less than 4 significantly decreasing viability at 1 hour. Pepsin had a mild protective effect up to 8 hours, with greater cell viability, in the pH range of 5.0 to 7.6. Reverse-transcriptase polymerase chain reaction demonstrated induction of Muc5b messenger RNA levels over controls after exposure to acidic pH levels of 5.7, and 4 with and without pepsin. Similarly, a pH of 4.0 significantly increased Muc5b promoter activation 5.4-fold. Pepsin at neutral or acidic pH values did not significantly alter Muc5b expression or promoter activity.

CONCLUSIONS

Despite decreasing cell viability, acidic pH drives middle ear epithelial Muc5b gene expression in vitro, which perhaps explains how laryngopharyngeal reflux can contribute to otitis media. Pepsin at neutral or acidic pH levels had minimal effects on Muc5b gene expression; thus, although pepsin may be a useful marker for detecting the presence of reflux, our results suggest that acid itself is a more likely pathologic component of gastric juice in the middle ear.