IFN-α up-regulates IL-27 and IL-27 receptor mRNA expression. (A) Monocyte-derived macrophages (n = 3) were stimulated with IFN-α (10 ng/mL, 4 hours) in parallel with mock-treated cultures, and RNA was extracted and processed for microarray analysis using the Affymetrix system. Expression of IL-27 p28 and EBI3 was up-regulated by IFN-α. (B) 6 × 10⁶ monocyte-derived macrophages were treated with IFN-α (10 ng/mL) for 4 hours in DMEM with 10% FCS. Cells were lysed and processed for RNA following Qiagen's RNeasy protocol for conventional RT-PCR (primers were: 5′-TTCCCTTGCTCCTGGTTCAAG-3′ [forward], 5′-TGGAGATGAAGCAGAGACGCTC-3′ [reverse]; representative data) or real-time PCR (n = 5). (C) Inhibition of IFN-α–induced IL-27 transcription by JAK inhibitor (JAK Inh 1 and 10 nM) added 1 hour before IFN-α(10 ng/mL) and macrophage RNA processed for RT-PCR 4 hours after addition of IFN-α (mean ± SEM; *P < .01; representative experiment, n = 2). (D) Control or IFN-α treated macrophage cultures (n = 3) were processed for real-time PCR for IL-27R-α after 24 hours, and GAPDH was used for normalization.