levels were not affected [10–12]. Polarization of mouse bone- marrow derived macrophages (BMDMs) with IFN-c, followed by stimulation with ICs and LPS resulted also in an increased IL-10 production, which led to the conclusion that ICs modulate the macrophage cytokine production profile towards alternative activation, in a similar fashion as IL-10, TGF-b, or glucocorticoids [5,7,13–15]. Although this model has been confirmed by several studies, two important aspects remain incompletely understood. Firstly, it is unclear whether ICs induce macrophage polarization to a distinct subset or rather modulate the function of polarized macrophages. The previously mentioned experiments using IFN-c polarized BMDMs could suggest namely either that M1 polarization can be reversed by ICs, or that ICs modulate the function of macrophages irrespective of their polarization status. Secondly, most of these experiments were performed in mice and only few studies analyzed the effects of ICs on human myeloid cells. In human monocytes, cross-linking of FccRs decreased IL-12 and increased IL-1ra, IL-10, and PGE2 production, which is in agreement with the M2 profile in mice [16,17]. The increased IL-10 production was not only observed after monocyte stimulation with artificial ICs, but also with ICs from SLE sera [18]. At the same time, however, the production of pro-inflammatory factors such as TNF, GM-CSF, IL-6, IL-8, and IL-1b by monocytes was also increased by FccR cross-linking [19–24]. This was not only observed in human monocytes, since we demonstrated previously that costimulation of human monocyte-derived DCs with ICs and TLR ligands leads to increased production of TNF and IL-6 [25]. Similarly, stimulation of M-CSF polarized human macrophages (MWM-CSF) with immobilized HAGGs (iHAGGs) or ACPA- containing ICs induced higher TNF production [26,27]. In order to clarify the effect of ICs on human macrophages and to assess whether the existing discrepancies in the literature are due to interspecies differences or to specific polarization conditions, we systematically studied the effect of HAGGs in the presence or absence of TLR stimuli on the phenotype and cytokine production of human polarized macrophages. Materials and Methods Ethics statement This study was conducted with the approval of the Medical Ethical Committee of the Academic Medical Center/University of Amsterdam and all blood donors gave their written informed consent. Monocyte isolation and in vitro polarization Monocyte isolation and in vitro polarization were performed as previously described [8]. Briefly, monocytes from peripheral blood of healthy volunteers were isolated by gradient centrifugation with Lymphoprep (Axis-Shield PoPAS, Oslo, Norway) and, subse- quently, Percoll gradient separation (GE Healthcare, Uppsala, Sweden). Monocytes were cultured at a concentration of 0.56106/ ml in Iscove’s Modified Dulbecco’s Medium (Invitrogen, Breda, The Netherlands) supplemented with 10% fetal calf serum (FCS) (PAA Laboratories, Co¨ lbe, Germany) in 6 well culture plates (Corning Incorporated, New York, NY, USA). Cells were cultured in medium alone or polarized with human recombinant IFN-c (50 ng/ml; R&D Systems, Abingdon, UK), IL-4 (40 ng/ml; Miltenyi Biotec, Bergisch Gladbach, Germany), IL-10 (50 ng/ ml; R&D Systems), GM-CSF (50 ng/ml, R&D Systems), or M- CSF (50 ng/ml, R&D Systems) for 4 days. Polarization with LPS (100 ng/ml, E. coli 0111:B4; Sigma Aldrich, Zwijndrecht, The Netherlands) was additionally used for the phenotypic experi- Effect of Immune Complexes on Macrophage Subsets ments. Human HAGGs (50 mg/ml), prepared as previously described [28], were added to each polarizing condition. Flow cytometric analysis Cultured macrophages were recovered by scraping of the plate. Surface marker expression was analyzed by flow cytometry (BD FACS Calibur Flow Cytometer, Erembodegem, Belgium) using fluorochrome-labeled monoclonal antibodies against CD14 (clone 61D3, eBioscience, San Diego, CA), CD16 (clone DJ130c, AbD Serotec, Du¨ sseldorf, Germany), CD32 (clone AT10, abcam, Cambridge, UK), CD64 (clone 10.1, BioLegend, Uithoorn, The Netherlands), CD80 (clone L307.4, BD Pharmingen, Breda, Nederland), CD163 (clone GHI/61, BD Pharmingen), CD200R (clone OX108, AbD Serotec), TLR2 (clone T2.5, abcam) and TLR4 (clone 76B357.1, abcam). Equivalent concentrations of matched isotype controls were included in all experiments. Before staining, Fc receptors were blocked with 10% human serum (Lonza, Cologne, Germany). Data were analyzed with Flow Jo Flow Cytometry Analysis software (Tree Star, Ashland, OR) after gating on the myeloid population in the FSC/SSC window. Values were expressed as the ratio of the geometric mean fluorescence intensity (gMFI) of the marker of interest over the gMFI of the isotype control. Cytokine production To assess the cytokine production of polarized macrophages, the in vitro polarized cells were harvested and extensively washed at day 4. Macrophages were subsequently activated with HAGGs (50 mg/ml) and/or the following TLR ligands: LPS (100 ng/ml; Sigma Aldrich), Pam3CSK4 (5 mg/ml; EMC microcollections, T u¨bingen, Germany), or R848 (2 mg/ml; InvivoGen, San Diego, CA). In order to study the effect of immobilized IgG, macrophages were also cultured on IgG coated plates (96-well plate, Corning). The supernatant of these cultures was recovered after 20 hours of stimulation and analyzed using commercially available Luminex kits (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. Cytokine levels of TNF, IL-6, and IL-10 were measured and analyzed with the Bio-Plex system (Bio- Rad). The sensitivity of the cytokine assay was ,5 pg/ml for all measured cytokines. Quantitative real-time PCR Total RNA was isolated from in vitro polarized macrophages using GenEluteTM Mammalian Total RNA Miniprep Kit (Sigma Aldrich) and reverse transcribed using RevertAidTM H Minus First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany). RNA concentration was determined with the Nanodrop (Nano- drop Technologies, Wilmington, DE). Quantitative real-time PCR was performed using StepOnePlusTM Real-Time PCR System (Applied Biosystems, Foster City, CA). Each 12 ml reaction was performed in a 96-well format with 5 ng of cDNA, 10 ml of SYBR green PCR Master Mix (Applied Biosystems) and a concentration of 50 nmol of each primer. The primers comprised IL-10 (forward 59-GATGCCTTCAGCAGAGTGAA, reverse 59-CCCAGGT- AACCCTTAAAGTCC), IL-23p19 (forward 59-TTCTCTGCT- CCCTGATAGCC, reverse 59-CCTCAGGCTGCAGGAGTT), and IL-12p35 (forward 59- ACCAGGTGGAGTTCAAGACC, reverse 59-TGGCACAGTCTCACTGTTGA), respectively. All reactions were performed in duplicate. The mRNA expression levels were normalized to those of the human housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Oligonu- cleotide primers were designed using the online tool for Real-time PCR (TaqMan) Primer Design (Genscript) and obtained from Invitrogen. PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35994