Expression of IL-6 and TNFα in macrophages. (A) RAW264.7 cells were stimulated with 100 ng/ml LPS and 500 pg/ml IFNγ, and samples of the medium were removed every 2 h over a 6-h time course. The graph shows the levels of IL-6 as determined by cytokine-specific ELISA secreted by macrophages over the time course. (B) Macrophages were activated with LPS and IFN for 6 h before fixation, permeabilization, and staining for intracellular IL-6 (red) and TNFα (green). Epifluorescence staining shows the coexpression of endogenous IL-6 and TNFα and their coaccumulation in the perinuclear Golgi complex. (C) Immuno-EM of newly synthesized cytokines in Golgi complexes (G), demonstrating IL-6–GFP (arrows) alone or together with TNFα (arrowheads), labeled with 5 or 10 nm gold particles as indicated. (D) Cells were activated with combinations of LPS and IFN for 6 h in the presence or absence of 5 μg/ml brefeldin A, and IL-6 in cell extracts was determined by Western blotting. Consistent with the ELISA data, increased levels of intracellular IL-6 are stimulated in the presence of IFN and LPS together, and disruption of the Golgi complex leads to the intracellular accumulation of IL-6. Bars (B), 5 μm; (C) 100 nm.