Figure 1.

5-LO is activated and translocates to the nucleus in human IELs, a process that is critical for NKG2D-mediated cytotoxicity. (A) Schematic of the various eicosanoid biosynthetic pathways. Upon liberation from membrane phospholipids by cPLA2, AA can be used to synthesize the various eicosanoids. Our previous work established a role for cPLA2 and AA in the NKG2D cytolytic pathway and CD pathogenesis (Tang et al., 2009). This work focuses on the pathways downstream of cPLA2 and, in particular, on the role of eicosanoids in NKG2D-mediated cytolysis and CD. (B) Three human IE-CTL lines were pretreated with vehicle control or 5-LO inhibitor MK886 for 30 min before stimulation with anti-NKG2D or anti-CD3 mAbs for the indicated time points. Translocation was determined by immunoblot analysis of nuclear extracts using an anti–5-LO antibody, with equal loading being assessed using an antibody directed against the nuclear marker TBP. Scanning densitometry was used to determine fold change with respect to unstimulated controls. A representative blot is shown at the top, with fold change ± standard deviation from three independent experiments compiled below. (C) Effector cells were pretreated for 30 min with vehicle control, 10 µM CF3, or 10 µM MK886 before the assay, which was performed against ⁵¹Cr-labeled EL4-MICA cells at an effector/target ratio of 18:1. Data are means ± standard deviation of four independent experiments using three different cell lines. (D) TALL cells were transfected with siRNAs against 5-LO, COX2, or a scrambled control siRNA by electroporation and allowed to recover for 24 h. Efficacy of knockdown was determined by Western blot (representative blots shown on the right). NKG2D-mediated cytotoxic capacity was assessed using ⁵¹Cr-labeled MICA-EL4 target cells at an effector/target ratio of 5:1. Data are means ± standard deviation of four independent experiments. (E) TALL-104 cells were pretreated with 10 µM MK886 or vehicle control for 30 min before incubation with ⁵¹Cr-labeled FcγR⁺ P815 target cells at the indicated effector/target ratios in the presence of antibodies against NKG2D (left) or CD3 (right). Data are means ± standard deviation of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.