A comparison of experimental methods in molecular chronic rhinosinusitis research.
BACKGROUND
Research into the molecular pathogenesis of chronic rhinosinusitis (CRS) requires the collection and analysis of sinonasal tissue. Recent gene expression studies have used either surgical tissue specimens or isolated epithelial cell preparations. Here, we compare cultures of nasal epithelial cells, nasal brush biopsy, and whole ethmoid mucosa with respect to expression of innate immune genes.


METHODS
Ethmoid mucosa was collected intraoperatively from 12 CRS and control patients. This tissue either was processed whole for mRNA extraction or was used to generate primary nasal epithelial cell cultures. After 6 weeks, epithelial cells in culture were assessed for multiple innate immune proteins by flow cytometry. In parallel, middle meatal brush biopsy specimens were obtained from the same patients and studied acutely in a similar fashion by flow cytometry. Expression of innate immune genes was determined in whole tissue samples by real-time polymerase chain reaction.


RESULTS
Flow cytometry revealed that brush biopsy specimens contain 75% epithelial cells, whereas primary nasal epithelial cell cultures were pure. Epithelial cells derived from individual subjects expressed very similar levels of innate immune markers whether studied acutely or after 6 weeks in culture. Whole tissue mRNA levels were variable and not correlated to epithelial expression.


CONCLUSIONS
The choice of experimental methodology can greatly influence the results and interpretation of CRS research. Primary nasal epithelial cells maintain their innate immune receptor expression profile when grown in prolonged culture in vitro. These findings imply that alterations in innate immune gene expression in CRS may be intrinsic to the epithelial cells, even outside of their in vivo microenvironment.
Ramanathan
Murugappan
M
Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins School of Medicine, Baltimore, Maryland 21287-0910, USA.
Lane
Andrew P
AP
D001706:Biopsy; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured / Q000166:cytology; D002908:Chronic Disease; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D009297:Nasal Mucosa / Q000166:cytology / Q000502:physiology / Q000503:physiopathology; D010256:Paranasal Sinuses / Q000166:cytology / Q000473:pathology; D012016:Reference Values; D012107:Research Design; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012220:Rhinitis / Q000473:pathology / Q000503:physiopathology*; D012852:Sinusitis / Q000473:pathology / Q000503:physiopathology*
D017931:DNA Primers; D018076:DNA, Complementary