TY - JOUR
T1 - A comparison of experimental methods in molecular chronic rhinosinusitis research
AU - Ramanathan, Murugappan
AU - Lane, Andrew P.
PY - 2007/5
Y1 - 2007/5
N2 - 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. Conclusion: 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.
AB - 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. Conclusion: 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.
KW - Epithelial
KW - Immunity
KW - Innate
KW - Methodology
KW - Polymerase chain reaction
KW - Rhinosinusitis
KW - Toll like receptor 9
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U2 - 10.2500/ajr.2007.21.3034
DO - 10.2500/ajr.2007.21.3034
M3 - Article
C2 - 17621826
AN - SCOPUS:34347253610
SN - 1945-8924
VL - 21
SP - 373
EP - 377
JO - American Journal of Rhinology and Allergy
JF - American Journal of Rhinology and Allergy
IS - 3
ER -