Increasing concentrations of D609 progressively inhibit nitrite accumulation in supernatants of activated human and mouse cells and are effective for at least 72 hr after stimulation
Mouse RAW 264.7 cells were incubated with increasing concentrations of D609 applied 30 min before the addition of 1 µg/ml LPS (A). Human cells were likewise exposed to D609, then activated with IL-1β, IFN-γ, and TNF-α (B). After an 8-hr (dotted line), 24-hr (dashed line), or 48-hr (solid line) incubation period, the culture supernatants were tested for the amount of nitrite present using the Griess reaction. The data are presented as a percentage of the nitrite determined to be present in stimulated cells without D609 treatment. The ability of D609 to suppress nitrite accumulation over a period of several days was shown by adding the inhibitor (50 µg/ml) to stimulated mouse (C) or human (D) cells. Cells were activated as described for A and B, respectively. Unstimulated cells are represented by dotted lines, cytokine or LPS-activated cell responses, dashed lines, and activated, D609-treated cell responses, solid lines. Error bars represent the SEM calculated from three independent experiments.