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W. Zhang, H.J. Gukasyan, M.D. Trousdale, N. Neamati, K.J. Kim, R. Kannan, V.H. Lee; cDNA Microarray Analysis of Cytokine Expression in an Adenovirus Type 5 Infection Model of Primary Cultured Rabbit Conjunctival Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):709.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Accumulation of subepithelial immune factors demonstrated in a rabbit ocular model of adenovirus type 5 (Ad5) infection closely mimics the clinical features of conjunctivitis (IOVS 33:574, 1992). In this study, we determined whether viral infection due to Ad5 treatment alters the expression of 96 common cytokine genes in primary cultured pigmented rabbit conjunctival epithelial cell layers (RCEC). Methods: RCEC were grown on tissue culture-treated polystyrene dishes for 7 days. On day 4 after cell seeding, RCEC were inoculated with 1×106 plaque forming units of Ad5 McEwen. The efficiency of in vitro viral transduction was determined by immunocytochemical analysis using Ad5 antiserum. On day 7 (3 days post-Ad5 inoculation), total RNA was isolated using the TRIzol® reagent and 3-5 µg was used with ThermoScriptTM RT for labeled probe synthesis (Invitrogen Corp., Carlsbad, CA). Non-radioactive GEArray Q series kit for detection of functionally grouped 96 common cytokines was utilized (Superarray Inc., Bethesda, MD). Results: The response was similar to other inflammatory conditions, including 10 genes significantly diverging in expression level from uninfected controls (p ≤ 0.05). Of these, only 6 genes were upregulated, while the other 4 were downregulated, to an extent ≥ 4 fold. The expression of remaining genes did not change significantly from the starting values. Upregulated genes fell into five functional groups, the individual members of which are listed in decreasing extent of variation from uninfected controls: thrombopoietin > vascular endothelial growth factor B > interleukin (Il)-18 > Il-11 > tumor necrosis factor superfamily member 5 (Tnfsf5), CD40 ligand > hepatocyte growth factor. Downregulated genes fell into three functional groups, where the tumor necrosis factors (Tnfsf12 and Tnfsf13b) and Il-13 were common. The only other significantly downregulated gene (p ≤ 0.05) belonged to the pleiotrophin family. Conclusion: cDNA microarray analysis can offer a rapid and simple method for determining cytokine expression profile under viral or various other inflammatory conditions.
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