One mechanism of macrophages regulating the immune response was by releasing proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6. Overproduction of these cytokines, however, was detrimental by prolonging inflammation and aggravating tissue damage. To control the unnecessary inflammation, macrophages also produced negative immunoregulatory cytokines, such as IL-10 and transforming growth factor (TGF)-β, to dampen macrophage activation. To determine whether AME affected the production of proinflammatory and anti-inflammatory cytokines, we used ELISA to measure the concentrations of TNF-α, IL-6, and IL-10 secreted in conditioned media of resting and activated RAW264.7 cells treated with PBS or AME. The amount of TNF-α, IL-6, and IL-10 was further normalized by the total protein present in the each cell lysate extracted by RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS). Control resting cells secreted a detectable amount of TNF-α (1057 ± 27 pg/mg protein), even without stimulation
(Fig. 5A) . The induction of TNF-α production by IFN-γ, particularly by LPS or IFN-γ/LPS, was rapid. After 4 hours of stimulation, TNF-α production was significantly increased by IFN-γ–, LPS-, and IFN-γ/LPS–activated macrophages to 2342 ± 270, 16,397 ± 324, and 23,307 ± 216 pg/mg protein, respectively. In comparison, AME significantly reduced TNF-α levels in resting cells (320 ± 81 pg/mg protein;
P = 0.006) and in IFN-γ, LPS, or IFN-γ/LPS activated cells (1344 ± 135, 11,864 ± 216 and 17,144 ± 351 pg/mg protein;
P = 0.01, 0.0007 and 0.002, respectively). Compared with TNF-α, IL-6 secreted into the culture medium was lower and slower. After 4 hours of stimulation, IFN-γ/LPS induced barely detectable IL-6 secretion in the culture medium, which was not significantly different between the control and AME-treated group (
P = 0.06). After 24 hours of stimulation, IFN-γ produced barely detectable IL-6, a result similar to what has been reported by others.
18 19 In contrast, LPS and IFN-γ/LPS induced significant amounts of IL-6
(Fig. 5B) . Under the latter two conditions, AME significantly reduced IL-6 production (
P = 0.04 and
P = 0.02, respectively). IL-10 levels were barely detectable after 4 hours of stimulation of IFN-γ, LPS, or IFN-γ/LPS but was significantly increased after 24 hours of stimulation by IFN-γ and more so by LPS and IFN-γ/LPS. AME significantly enhanced IL-10 production under each stimulation by IFN-γ, LPS, and IFN-γ/LPS (
P = 0.05,
P = 0.04, and
P = 0.02, respectively). These data consistently showed that AME downregulated the production of proinflammatory cytokines while it upregulated that of anti-inflammatory cytokines, likely leading to a dampening of macrophage activation and a reduction of inflammation.