Supporting Information Figure S1 Expression of TLR2 and TLR4 on MWIFN-c, MWIL-4, and MWIL-10. Healthy peripheral blood monocytes were cultured for 4 days in medium or in medium supplemented with IFN-c, IL-4, or IL-10. Expression TLR2 and TLR4 was measured by flow cytometry. Bars represent the mean 6 SEM of 4 independent experiments. *p,0.05, **p,0.01 (TIF) References 1. Bogdan C, Vodovotz Y, Nathan C (1991) Macrophage deactivation by interleukin 10. J Exp Med 174: 1549–1555. 2. Goerdt S, Orfanos CE (1999) Other functions, other genes: alternative activation of antigen-presenting cells. Immunity 10: 137–142. S1074-7613(00)80014-X [pii]. 3. Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3: 23–35. 10.1038/nri978 [doi];nri978 [pii]. 4. Stein M, Keshav S, Harris N, Gordon S (1992) Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation. J Exp Med 176: 287–292. 5. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, et al. (2004) The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25: 677–686. S1471-4906(04)00295-9 [pii];10.1016/ j.it.2004.09.015 [doi]. 6. Mosser DM (2003) The many faces of macrophage activation. J Leukoc Biol 73: 209–212. 7. Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8: 958–969. nri2448 [pii];10.1038/nri2448 [doi]. 8. Ambarus CA, Krausz S, van Eijk M, Hamann J, Radstake TR, et al. (2011) Systematic validation of specific phenotypic markers for in vitro polarized human macrophages. J Immunol Methods S0022-1759(11)00294-8 [pii];10.1016/j.jim.2011.10.013 [doi]. 9. Bonney RJ, Naruns P, Davies P, Humes JL (1979) Antigen-antibody complexes stimulate the synthesis and release of prostaglandins by mouse peritoneal macrophages. Prostaglandins 18: 605–616. 10. Gerber JS, Mosser DM (2001) Reversing lipopolysaccharide toxicity by ligating the macrophage Fc gamma receptors. J Immunol 166: 6861–6868. 11. Sutterwala FS, Noel GJ, Clynes R, Mosser DM (1997) Selective suppression of interleukin-12 induction after macrophage receptor ligation. J Exp Med 185: 1977–1985. 12. Sutterwala FS, Noel GJ, Salgame P, Mosser DM (1998) Reversal of proinflammatory responses by ligating the macrophage Fcgamma receptor type I. J Exp Med 188: 217–222. 13. Edwards JP, Zhang X, Frauwirth KA, Mosser DM (2006) Biochemical and functional characterization of three activated macrophage populations. J Leukoc Biol 80: 1298–1307. jlb.0406249 [pii];10.1189/jlb.0406249 [doi]. 14. Anderson CF, Mosser DM (2002) Cutting edge: biasing immune responses by directing antigen to macrophage Fc gamma receptors. J Immunol 168: 3697–3701. 15. Martinez FO, Sica A, Mantovani A, Locati M (2008) Macrophage activation and polarization. Front Biosci 13: 453–461. 2692 [pii]. 16. Berger S, Ballo H, Stutte HJ (1996) Immune complex-induced interleukin-6, interleukin-10 and prostaglandin secretion by human monocytes: a network of pro- and anti-inflammatory cytokines dependent on the antigen:antibody ratio. Eur J Immunol 26: 1297–1301. 10.1002/eji.1830260618 [doi]. 17. Berger S, Chandra R, Ballo H, Hildenbrand R, Stutte HJ (1997) Immune complexes are potent inhibitors of interleukin-12 secretion by human monocytes. Eur J Immunol 27: 2994–3000. 10.1002/eji.1830271136 [doi]. 18. Ronnelid J, Tejde A, Mathsson L, Nilsson-Ekdahl K, Nilsson B (2003) Immune complexes from SLE sera induce IL10 production from normal peripheral blood mononuclear cells by an FcgammaRII dependent mechanism: implications for a possible vicious cycle maintaining B cell hyperactivity in SLE. Ann Rheum Dis 62: 37–42. 19. Abrahams VM, Cambridge G, Lydyard PM, Edwards JC (2000) Induction of tumor necrosis factor alpha production by adhered human monocytes: a key role for Fcgamma receptor type IIIa in rheumatoid arthritis. Arthritis Rheum 43: 608–616. 10.1002/1529-0131(200003)43:3,608::AID-ANR18.3.0.CO;2-G [doi]. 20. Debets JM, Van de Winkel JG, Ceuppens JL, Dieteren IE, Buurman WA (1990) Cross-linking of both Fc gamma RI and Fc gamma RII induces secretion of tumor necrosis factor by human monocytes, requiring high affinity Fc-Fc gamma R interactions. Functional activation of Fc gamma RII by treatment with proteases or neuraminidase. J Immunol 144: 1304–1310. 21. Herrmann F, De Vos S, Brach M, Riedel D, Lindemann A, et al. (1992) Secretion of granulocyte-macrophage colony-stimulating factor by human blood monocytes is stimulated by engagement of Fc gamma receptors type I by solid- phase immunoglobulins requiring high-affinity Fc-Fc gamma receptor type I interactions. Eur J Immunol 22: 1681–1685. 10.1002/eji.1830220703 [doi]. Effect of Immune Complexes on Macrophage Subsets Author Contributions Conceived and designed the experiments: CAA TRDJR DLPB. Performed the experiments: CAA KCMS LvB MHW. Analyzed the data: CAA KCMS LvB MHW. Contributed reagents/materials/analysis tools: CAA KCMS LvB MHW PPT TRDJR DLPB. Wrote the paper: CAA TRDJR DLPB. 22. Krutmann J, Kirnbauer R, Kock A, Schwarz T, Schopf E, et al. (1990) Cross- linking Fc receptors on monocytes triggers IL-6 production. Role in anti-CD3- induced T cell activation. J Immunol 145: 1337–1342. 23. Mullazehi M, Mathsson L, Lampa J, Ronnelid J (2006) Surface-bound anti-type II collagen-containing immune complexes induce production of tumor necrosis factor alpha, interleukin-1beta, and interleukin-8 from peripheral blood monocytes via Fc gamma receptor IIA: a potential pathophysiologic mechanism for humoral anti-type II collagen immunity in arthritis. Arthritis Rheum 54: 1759–1771. 10.1002/art.21892 [doi]. 24. Polat GL, Laufer J, Fabian I, Passwell JH (1993) Cross-linking of monocyte plasma membrane Fc alpha, Fc gamma or mannose receptors induces TNF production. Immunology 80: 287–292. 25. Radstake TR, van Lent PL, Pesman GJ, Blom AB, Sweep FG, et al. (2004) High production of proinflammatory and Th1 cytokines by dendritic cells from patients with rheumatoid arthritis, and down regulation upon FcgammaR triggering. Ann Rheum Dis 63: 696–702. 10.1136/ard.2003.010033 [doi];63/ 6/696 [pii]. 26. Clavel C, Nogueira L, Laurent L, Iobagiu C, Vincent C, et al. (2008) Induction of macrophage secretion of tumor necrosis factor alpha through Fcgamma receptor IIa engagement by rheumatoid arthritis-specific autoantibodies to citrullinated proteins complexed with fibrinogen. Arthritis Rheum 58: 678–688. 10.1002/art.23284 [doi]. 27. Laurent L, Clavel C, Lemaire O, Anquetil F, Cornillet M, et al. (2011) Fcgamma receptor profile of monocytes and macrophages from rheumatoid arthritis patients and their response to immune complexes formed with autoantibodies to citrullinated proteins. Ann Rheum Dis 70: 1052–1059. ard.2010.142091 [pii];10.1136/ard.2010.142091 [doi]. 28. Radstake TR, Blom AB, Sloetjes AW, van Gorselen EO, Pesman GJ, et al. (2004) Increased FcgammaRII expression and aberrant tumour necrosis factor alpha production by mature dendritic cells from patients with active rheumatoid arthritis. Ann Rheum Dis 63: 1556–1563. 63/12/1556 [pii];10.1136/ ard.2003.016550 [doi]. 29. Anderson CF, Mosser DM (2002) A novel phenotype for an activated macrophage: the type 2 activated macrophage. J Leukoc Biol 72: 101–106. 30. Landmann R, Knopf HP, Link S, Sansano S, Schumann R, et al. (1996) Human monocyte CD14 is upregulated by lipopolysaccharide. Infect Immun 64: 1762–1769. 31. Gratchev A, Kzhyshkowska J, Kothe K, Muller-Molinet I, Kannookadan S, et al. (2006) Mphi1 and Mphi2 can be re-polarized by Th2 or Th1 cytokines, respectively, and respond to exogenous danger signals. Immunobiology 211: 473–486. S0171-2985(06)00076-3 [pii];10.1016/j.imbio.2006.05.017 [doi]. 32. Verreck FA, de Boer T, Langenberg DM, Hoeve MA, Kramer M, et al. (2004) Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco)bacteria. Proc Natl Acad Sci U S A 101: 4560–4565. 10.1073/pnas.0400983101 [doi];0400983101 [pii]. 33. Verreck FA, de Boer T, Langenberg DM, van der Zanden L, Ottenhoff TH (2006) Phenotypic and functional profiling of human proinflammatory type-1 and anti-inflammatory type-2 macrophages in response to microbial antigens and IFN-gamma- and CD40L-mediated costimulation. J Leukoc Biol 79: 285–293. jlb.0105015 [pii];10.1189/jlb.0105015 [doi]. 34. Shanley TP, Schmal H, Friedl HP, Jones ML, Ward PA (1995) Regulatory effects of intrinsic IL-10 in IgG immune complex-induced lung injury. J Immunol 154: 3454–3460. 35. Virgin HW, Unanue ER (1984) Immune complexes suppress cellular immunity. Ann N Y Acad Sci 437: 16–27. 36. Fleetwood AJ, Lawrence T, Hamilton JA, Cook AD (2007) Granulocyte- macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation. J Immunol 178: 5245–5252. 178/8/5245 [pii]. 37. Wijngaarden S, Van de Winkel JG, Jacobs KM, Bijlsma JW, Lafeber FP, et al. (2004) A shift in the balance of inhibitory and activating Fcgamma receptors on monocytes toward the inhibitory Fcgamma receptor IIb is associated with prevention of monocyte activation in rheumatoid arthritis. Arthritis Rheum 50: 3878–3887. 10.1002/art.20672 [doi]. 38. Tripp CS, Beckerman KP, Unanue ER (1995) Immune complexes inhibit antimicrobial responses through interleukin-10 production. Effects in severe combined immunodeficient mice during Listeria infection. J Clin Invest 95: 1628–1634. 10.1172/JCI117837 [doi]. PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35994