Increased TNF-α production by MPN monocytes after stimulation with TLR agonists. (A) MPN (n = 2 PV, 2 ET, and 1 MF) and normal (n = 5) monocytes were stimulated with TLR agonists LPS or R848 at the concentrations shown. After 24 hours of culture, supernatant was harvested and TNF-α was measured by ELISA. (B) MPN (n = 8 PV, 3 ET, and 2MF) and normal (n = 8) monocytes were stimulated with 10 ng/mL LPS or 5 μM R848 and incubated with brefeldin A for 4 hours to prevent protein export. Intracellular staining for TNF-α was performed, and cells were analyzed by flow cytometry. (C) MPN (n = 2 PV and 5 ET) and normal (n = 5) monocytes were stimulated with 10 ng/mL LPS or 5 μM R848 for 15 minutes or 2 hours before fixation and permeabilization. Cells were stained for phospho-p38 and analyzed on a flow cytometer. *P < .05.