Time course of IRP activity in monocytes and monocyte-derived macrophages from control subjects treated with LPs/IFN-γ. Monocytes from healthy blood donors were cultured in RPMI 1640 medium containing 20% autologous serum for 4, 8, and 24 hours in the presence (A) and absence (B) of cytokines (100 U/mL IFN-γ + 1 μg/mL LPS). Cells were also maintained in culture for 6 days to allow differentiation to macrophages and were then treated with cytokines for the same time periods (C). Cytoplasmic extracts (2 μg protein) were analyzed for IRE-binding activity by RNA-bandshift assay with excess³²P-labeled IRE probe in the absence (upper panels) and presence (lower panels) of 2% 2-mercaptoethanol. TNF-α production was assayed in the culture medium by enzyme-linked immunoassay. After a transient increase, IRP activity returned to that of untreated cells and was eventually downregulated at 24 hours after stimulation with LPS/IFN-γ in both monocytes and monocyte-derived macrophages.