contributing to the down-regulation of innate tissue inflammation at the moment that microorganisms are cleared by the adaptive immune system. The effect of ICs on human myeloid cells, however, is less clearly defined, as several reports indicate that they do not only increase the production of anti-inflammatory mediators such as IL-10 [16–18], but also strongly upregulate pro-inflammatory mediators [19–27]. This discrepancy may be related to differences between species, cell types (monocytes versus macrophages, or dendritic cells), or macrophage polarization conditions. In this study we show that HAGGs in the absence of TLR stimuli do not alter the phenotype and cytokine production of in vitro polarized human macrophage subsets. Costimulation with HAGGs and TLR ligands, however, resulted in markedly increased IL-10 production in most macrophage subsets, while the modulation of TNF and IL-6 production was more discrete and dependent on the macrophage polarization status. As previously shown in mouse macrophages [10,12,13,29], costimulation with ICs and TLR ligands also augmented the IL-10 production of human MWIFN-c, which are prototypical pro-inflammatory cells. These findings raise the critical issue of the exact relationship between polarization, phenotype and function of human macro- phages. The macrophage polarization models originally refer to the production of pro- versus anti-inflammatory mediators to define classically (M1) versus alternatively activated macrophages (M2) [1–3,5–7]. Macrophage polarization is also associated with the expression of specific phenotypic markers [4,8,8,39–41]. Our experiments confirm that polarization affects both the phenotype and the function of macrophages, as exemplified by the distinct cytokine production of MWIFN-c, MWIL-4, and MWIL-10 upon TLR stimulation. Concerning the effect of ICs, we show that HAGGs can modulate the TLR-mediated cytokine production of human polarized macrophages, despite the lack of phenotypic alterations. The capacity of so-called ‘pro-inflammatory’ macrophages to produce significant amounts of IL-10 upon costimulation with HAGGs and TLR ligands illustrate the functional plasticity of these cells. Furthermore, our data indicate that the production of pro- versus anti-inflammatory mediators by macrophages is not only related to their polarization, but also to the type of activation and that, as a consequence, phenotypic polarization markers do not fully correlate to the macrophage function. An important aspect in this context is the expression of FccRs on polarized macrophage subsets. Studies using different IgG subtypes, specific FccR blocking antibodies, or FccR knockout animals demonstrated that the differential expression of the activating versus inhibitory FccRs on immune cells influences their response to ICs [23,28,37,42–45]. We previously confirmed that the high affinity FccRI (CD64) was upregulated on MWIFN-c, while the low affinity FccRII (CD32) and FccRIII (CD16) were specifically expressed by MWIL-10. Furthermore, the activating and inhibitory isoforms of CD32 (CD32a and CD32b, respectively) were differentially expressed on the 3 macrophage subsets, with a lower CD32a/CD32b ratio on MWIL-4 versus MWIL-10 and MWIFN-c [8]. A shifted FccR balance towards FccRIIb on IL-4 primed human monocytes in comparison to IFN-c, TNF, and IL- 10 primed monocytes was previously related to the failure of these cells to upregulate TNF after crosslinking by IgG [37]. Finally, also the expression of TLR2 and TLR4 was differentially modulated by polarization, with lower expression levels on MWIL-4 versus MWIL-10 and MWIFN-c (Figure S1). Although it is beyond the scope of this study to investigate the exact signal transduction in these conditions, the low expression of activating FccRs, TLR2, and TLR4 on MWIL-4 could at least partially explain their poor responsiveness to HAGGs. Effect of Immune Complexes on Macrophage Subsets In an attempt to clarify the discrepancy between our findings and reports indicating increased TNF production by monocytes and macrophages upon stimulation with immobilized IgG [26,27,37], we used immobilized IgG in the same experimental conditions and observed a number of differences with soluble HAGGs. Firstly, immobilized IgG, but not HAGG was able to stimulate cytokine production by polarized macrophages in the absence of TLR co-stimulation. Secondly, whereas MWIL-4 appeared to be poorly responsive to HAGGs, all polarized macrophage subsets reacted strongly upon stimulation with immobilized IgG. Finally and most importantly, HAGGs consistently augmented IL-10 production, whereas immobilized IgG upregulated the production of TNF, IL-6 and IL-10. These data are consistent with previous reports in which prevention of IC phagocytosis by using immobilized IgG was shown to induce persistent ERK signaling and high TNF production by both monocytes and macrophages [26,46,47]. Furthermore, the TLR- induced IL-10 production by human monocytes was higher after immobilized IgG versus HAGG stimulation, and immobilized IgG was able to induce IL-10 production even in the absence of TLR ligands [48]. This data emphasize that, besides many other factors such as IgG isotype, monomeric versus polymeric IgG, antigen- antibody ratio, and natural versus artificial ICs, the capacity to internalize ICs is a key determinant of the functional outcome of IC stimulation of macrophages [16,18–23,42,49]. The differential effects of soluble versus immobilized IgG do not only emphasize the functional plasticity of polarized macrophages, but may also be relevant for our understanding of specific pathological and therapeutic conditions. In autoimmune diseases, ICs are thought to contribute to chronic tissue inflammation when deposited in tissues, with as prototypical examples lupus nephritis and rheumatoid synovitis. In this immobilized form, ICs may promote mainly pro-inflammatory over anti-inflammatory re- sponses by tissue macrophages. Moreover, IC binding to myeloid cells in these conditions was also reported to inhibit their responsiveness to IL-10 [50,51]. In sharp contrast, intravenous administration of soluble immunoglobulins (IVIg) has been used for a long time in the treatment of diverse autoimmune diseases [52–55]. The immunosuppressive effect of macrophage FccR ligation has been attributed to blocking of the activating FccRs, stimulation of FccRIIb, and increased anti- versus pro-inflamma- tory cytokine production [10,12,16,18,45,56]. Our in vitro data thus support the hypothesis that soluble ICs shift the balance of pro- towards anti-inflammatory cytokine production. Interestingly, this effect may be important not only for the therapeutic efficacy of IVIg, but also of targeted biological therapies. It was indeed recently demonstrated that anti-TNF antibodies induce IL-10 producing macrophages in an Fc-dependent manner and that these immunoregulatory macrophages are involved in mucosal healing in inflammatory bowel disease [56,57]. These studies reveal that monoclonal antibody therapy can drive anti-inflam- matory responses by Fc region-dependent and target-independent modulation of macrophage function. In conclusion, we showed here that distinct polarized macro- phage subsets retain an important functional plasticity despite maintenance of their specific phenotype. In particular, we demonstrated that soluble ICs, but not immobilized IgG shifted the balance of human macrophage cytokine production towards IL-10. These findings raise the possibility of therapeutic modula- tion of macrophage function in the context of chronic tissue inflammation. PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35994