Figure 1.

The effect of mCRP on macrophage cytokine production. The differentiated U937 macrophages were pre-treated with acetylcholine (10 μM), nicotine (0.93 μM), tacrine (5 μM), anti-mCRP antibodies 3H12 (1:10) or 8C10 (1:10) for 2 h, followed by stimulated with mCRP (100 μg/mL) for an additional 24 h. The production of TNF-α (A), IL-6 (B), and IL-10 (C) in the supernatant was quantified using ELISA kits (R&D System). The stimulation with LPS (10 ng/mL) for 24 h was used as the positive control for macrophage cytokine production. (D) Shows that treatment with small molecules alone, nicotinic acid receptor antagonist methyllycaconitine (10 μM) or CD16/32/64 to FC-ɤ receptors (1:100) did not affect cytokine (TNF-α) production in U937 cells, although there was a marginal increase in TNF-α in the presence of the mCRP antibodies probably due to residual contamination of low levels of LPS. When cells were pre-treated with CD16/32/64 to FC-ɤ receptors (1:100) for 2 h, complete abrogation of mCRP-induced TNF-α production was shown (E), whilst pre-incubation with the nicotinic acid receptor antagonist methyllycaconitine (10 μM) for 2 h in the presence of either acetylcholine or nicotine did not reverse the anti-inflammatory capacity (F). Results are presented as the mean ± SD from a representative example of three independent experiments. ^*P ≤ 0.05; ^**P < 0.01; ^***P < 0.001 using one-way ANOVA with Bonferroni post-test analysis.