IFNγ priming for signaling and responsiveness to LPS.

Human fresh monocytes were preincubated overnight with IFNγ or IFNα at 500 U/mL or with medium alone and then exposed to different concentrations of LPS as indicated in each lane. (A) After a treatment with LPS (10 ng/mL) for 45 minutes, cells were lysed and IRAK activation was determined. The figure shows autophosphorylation of IRAK (p-IRAK). (B) Cells were lysed after a 2-hour LPS treatment and nuclear extracts analyzed for NF-kB DNA binding activity by EMSA. CP: the 100 ng/mL LPS-treated extract in lane 6 was also incubated with³²P-labeled NF-kB probe plus a 100-fold excess unlabeled NF-kB probe. (C) After exposure to LPS (100 ng/mL) for 6 hours, supernatants were analyzed for secreted TNFα (gray columns, i) and IL-12 (filled columns, ii). Error bars represent SDs of triplicate samples. Similar results were obtained in monocyte-derived macrophages.