Effect of Immune Complexes on Macrophage Subsets Figure 4. IL-10 production by MWIFN-c, MWIL-4, and MWIL-10 after stimulation with HAGGs and LPS, or R848. Healthy peripheral blood monocytes were cultured for 4 days in medium or in medium supplemented with IFN-c, IL-4, or IL-10. Polarized macrophages were not stimulated, or stimulated for 20 hours with LPS, or R848, in the presence or absence of HAGGs. Bars represent the mean 6 SEM of 6 independent experiments. *p,0.05. doi:10.1371/journal.pone.0035994.g004 stimulation with iHAGGs or immobilized IgG [26,27,37], we next investigated the effect of immobilized IgG on the cytokine production of differentially polarized macrophages. In contrast with HAGGs, even in the absence of P3C stimulation, immobilized IgG increased the production of TNF (p,0.05 in unpolarized macrophages, p,0.01 in MWIL-10, and p,0.001 in MWIFN-c), IL-6 (p,0.05 in MWIL-10) and IL-10 (p,0.05 in MWIL-10) (Figure 7). Similarly, immobilized IgG strongly augmented the TLR-induced production of TNF (p,0.05 in MWIL-4, p,0.01 in unpolarized macrophages, and p,0.001 in MWIL-10), IL-6 (p,0.05 in MWIL-4 and MWIL-10) and IL-10 (p,0.05 in MWIL-10, and p,0.01 in MWIL-4). Comparable results were obtained when using LPS or R848 as TLR stimuli (data not shown). These data indicate that immobilized IgG augments the spontaneous, as well as the TLR-induced cytokine production of polarized macrophages, without shifting the balance from pro-inflammatory cytokines to IL-10. Figure 5. IL-10 production by MWGM-CSF and MWM-CSF after stimulation with HAGGs and TLR ligands. Healthy peripheral blood monocytes were cultured for 4 days in medium supplemented with GM-CSF or M-CSF. MWGM-CSF and MWM-CSF were not stimulated, or stimulated for 20 hours with P3C, LPS, or R848, in the presence or absence of HAGGs. Bars represent the mean 6 SEM of 6 independent experiments. *p,0.05, **p,0.01. doi:10.1371/journal.pone.0035994.g005 PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35994