Inhibition of IFNβ signaling results in partial abrogation of poly (I∶C)-induced ISG upregulation.

<p>(a) ECC-1 cells (<i>n = 2</i>) were cultured on cell inserts until confluent and subsequently stimulated with multiple doses of recombinant human IFNβ (10¹, 10² & 10³ U/ml) for 24 hours (left panel) in the presence or absence of either neutralizing antibody for IFNβ (αIFNβ) (middle panel) or blocking antibody against interferon receptor 2 (αIFNAR2) (right panel). For the neutralization experiments αIFNβ and IFNβ were first mixed together in an eppendorf tube and then added to both compartments for 24 hours. For the receptor blockade experiments cells were pretreated apically and basolaterally with αIFNAR2 or IgG control (not shown) for 1 hour prior to apical stimulation with poly (I∶C) for a subsequent 24 hours during which αIFNAR2 was maintained in the cell culture media. (b) Primary human UEC from two individual patients were cultured on cell inserts until confluent and then stimulated with 10³ U/ml of recombinant IFNβ for 24 hours. (c) Concurrently, the UEC were treated with poly (I∶C) for 24 hours in the presence or absence of neutralizing antibody for IFNβ (αIFNβ). (d) ECC-1 cells (<i>n = 3</i>) or primary human UEC (Patient 8) (representative of 2 separate experiments) were cultured on cell inserts until confluent. Cells were pretreated with αIFNAR2 or IgG control (not shown) for 1 hour prior to apical stimulation with poly (I∶C) for a subsequent 24 hours during which αIFNAR2 was maintained in the cell culture media. Total cellular mRNA was recovered and analyzed for gene expression. mRNA is expressed as a fold change over untreated samples (assigned a value of 1).