TLR2 is required for the recognition of HSV-1 and the release of IL-15 by monocytic cells. (A) Primary human monocytes were treated separately with 1 μg/mL of anti-TLR2 (neutralizing) mAb, anti-TLR4 (neutralizing) mAb, or isotype-matched control (IgG1) for 30 minutes. Nontreated cells as well as antibody-treated cells were exposed to mock or HSV-1. Cells were harvested 6 hours after treatment for isolation of total RNA to assess IL-15 gene expression by RT-PCR (top panel) and real-time RT-PCR (bottom panel). (B) THP1 cells were treated similarly to primary monocytes, and IL-15 gene expression was assessed with RT-PCR (top panel) and real-time RT-PCR (bottom panel). In both cases, RT-PCR values show that IL-15 mRNA and 18S rRNA are representative of 3 independent experiments; real-time RT-PCR values are expressed as the ratio of IL-15 mRNA in treated samples to the untreated sample (after normalization with reference dye and housekeeping gene). (C) THP1 cells pretreated as indicated were incubated with mock or HSV-1 for 1 hour. Nuclear extracts were subjected to EMSA analysis with radiolabeled NF-κB consensus oligonucleotide in the presence of unlabeled competitor (a lanes) or nonspecific competitor (b lanes). NF-κB binding was visible by autoradiography.
