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.


METHODS
In vitro mMEEC model.


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


METHODS
Cells from the immortalized mMEEC line.


METHODS
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.
Block
Bradley B
BB
Division of Pediatric Otolaryngology-Head and Neck Surgery, Children's National Medical Center, 111 Michigan Ave NW, Washington, DC 20010, USA.
Kuo
Elaine
E
Zalzal
Habib G
HG
Escobar
Hugo
H
Rose
Mary
M
Preciado
Diego
D
D000143:Acids / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004432:Ear, Middle / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D051379:Mice; D055257:Mucin-5B / Q000235:genetics*; D010434:Pepsin A / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
D000143:Acids; D055257:Mucin-5B; D012333:RNA, Messenger; D010434:Pepsin A