IgM-Dependent Phagocytosis in Microglia Is Mediated by Complement Receptor 3, Not Fc α/µ Receptor This information is current as Jonathan R. Weinstein, Yi Quan, Josiah F. Hanson, Lucrezia of September 25, 2021. Colonna, Michael Iorga, Shin-ichiro Honda, Kazuko Shibuya, Akira Shibuya, Keith B. Elkon and Thomas Möller J Immunol 2015; 195:5309-5317; Prepublished online 23 October 2015; doi: 10.4049/jimmunol.1401195 Downloaded from http://www.jimmunol.org/content/195/11/5309 Supplementary http://www.jimmunol.org/content/suppl/2015/10/23/jimmunol.140119 Material 5.DCSupplemental http://www.jimmunol.org/ References This article cites 56 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/195/11/5309.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology IgM-Dependent Phagocytosis in Microglia Is Mediated by Complement Receptor 3, Not Fca/m Receptor Jonathan R. Weinstein,*,1 Yi Quan,*,1 Josiah F. Hanson,* Lucrezia Colonna,† Michael Iorga,* Shin-ichiro Honda,‡ Kazuko Shibuya,‡ Akira Shibuya,‡ Keith B. Elkon,† and Thomas Mo¨ller* Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement re- ceptors triggering phagocytosis. In this study, we investigated the role of Fca/mR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fca/mR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fca/mR in IgM-mediated phagocytosis, we performed experiments in microglia from Fca/mR2/2 mice. Surprisingly, we found Downloaded from that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fca/mR2/2 mice. We hypoth- esized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincu- bation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD182/2 and C32/2 mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fca/mR, in IgM- http://www.jimmunol.org/ mediated phagocytosis of S. aureus by microglia. The Journal of Immunology, 2015, 195: 5309–5317. hagocytosis is a multistep and receptor-mediated process. expressed on B cells and macrophages, where it has been shown to It is initiated by particle recognition and can be separated mediate uptake of IgM-Ag immune complexes (8, 9). In addition to P experimentally into two steps, as follows: 1) particle at- opsonization by Abs, phagocytic objects (microorganisms or cells) tachment to the cell surface and 2) particle ingestion by cells (1). can be opsonized by complement and recognized by complement Phagocytosis is mediated by a wide variety of cell surface receptors receptors, including complement receptor 1, complement receptor 3 that bind directly or indirectly, through opsonins, to particles (2). Fc (CR3), complement receptor 4, and C1qR(P) (2, 11). by guest on September 25, 2021 receptors, specifically the Fcg receptor subtypes that recognize IgG, Microglia, the resident tissue macrophages of the CNS, are active are well studied for their role in phagocytosis (3, 4). However, other sensors and versatile effector cells in the normal and pathologic brain Igs, including IgM (5, 6) and IgA (7), are also capable of opsonizing (12). Microglia shift activity states depending on the surrounding pathogens and playing a role in phagocytosis. A recently discovered microenvironment. Under normal conditions, they are characterized phagocytosis-related Fc receptor known as Fca/mR (CD351) rec- by a small cell body with fine, ramified processes and low expres- ognizes IgM and IgA, but not IgG (8–10). It is a type 1 trans- sion of surface Ags. In response to brain injury, ischemia, and in- membrane protein with an extracellular Ig-like domain. Fca/mRis flammatory stimuli, microglia rapidly transform into an activated phenotype associated with morphological changes, proliferation, migration to the site of injury, elaboration of both neurotoxic and *Department of Neurology, School of Medicine, University of Washington, Seattle, WA neurotrophic factors, as well as increased phagocytosis (12–14). In- 98195; †Division of Rheumatology, Department of Medicine, School of Medicine, University of Washington, Seattle, WA 98195; and ‡Department of Immunology, vading pathogens require opsonization by Ig and complement fixa- Institute of Basic Medical Sciences, Graduate School of Comprehensive Human tion for efficient recognition and phagocytosis by Fc and complement Sciences, University of Tsukuba, Ibaraki 305-8575, Japan receptors in microglia (2, 15). Microglial Fc and complement re- 1J.R.W. and Y.Q. contributed equally to this work. ceptors have been implicated in the pathophysiology of bacterial ORCIDs: 0000-001-6080-9758 (J.R.W.); 0000-0003-2572-8893 (Y.Q.). brain abscesses (16). Fc and complement receptors also represent Received for publication May 9, 2014. Accepted for publication September 29, 2015. potential molecular targets for pharmacologic therapy in multiple This work was supported by American Heart Association Grant-In-Aid 0750078Z (to sclerosis (3), Alzheimer’s (17–19), and Parkinson’s disease (20). T.M.), National Institutes of Health/National Institute of Arthritis and Musculoskel- Although IgG-mediated phagocytosis (via Fcg receptors) in the etal and Skin Diseases Grant F32AR065837 (to L.C.), and National Institutes of Health/National Institute of Neurological Disorders and Stroke Grant NS065008 CNS is well described (3), there is little characterization to date of (to J.R.W.). IgM-mediated phagocytosis in brain. In this study, we investigate Address correspondence and reprint requests to Prof. Jonathan R. Weinstein, Depart- IgM-induced phagocytosis of the bacterial pathogen Staphylo- ment of Neurology, School of Medicine, University of Washington, Box 356465, coccus aureus in microglia and characterize the role of Fca/mRin 1959 NE Pacific Street, Seattle, WA 98195. E-mail address: [email protected]. edu this process. The online version of this article contains supplemental material. Abbreviations used in this article: CR3, complement receptor 3; MSFM, macrophage Materials and Methods serum-free medium; pMG, primary microglia; P/S, penicillin/streptomycin; qRT- Solutions and reagents PCR, quantitative real-time PCR; WT, wild-type. Fluorescein-labeled S. aureus was obtained from Invitrogen. Anti-S. au- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 reus mAbs (IgM clone 11-248.2 and IgG3 clone 11-232.3) were purchased www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401195 5310 CR3 MEDIATES IgM-DEPENDENT PHAGOCYTOSIS IN MICROGLIA from QED Bioscience (San Diego, CA). Anti-CD11b mAb as well as S. aureus IgM, 2) isotype control IgM, 3) anti-S. aureus IgG, or 4) isotype isotype controls for both anti-S. aureus and anti-CD11b Abs were obtained control IgG, were added to the cells to assess phagocytosis. from BD Biosciences (San Jose, CA). Recombinant mouse GM-CSF, IFN-g, We also used microscopy to confirm phagocytosis in serum-starved and IL-4 were purchased from R&D Systems (Minneapolis, MN). All mouse pMG. After 30-min incubation with each of the four S. aureus/ solutions were freshly prepared from frozen stock solutions or lyophilized Ab reaction mixes, extracellular fluorescence was quenched with trypan preparations. All materials were handled in a sterile manner using blue. Primary microglia were fixed, counterstained with DAPI, and then endotoxin-free microfuge tubes (Eppendorf/Fisher Scientific, Santa Clara, imaged and photographed under both fluorescent and differential inter- CA), polypropylene tubes (BD Labware, Franklin Lakes, NJ), polystyrene ference contrast illumination using a Zeiss Axiovert 200M microscope. culture vessels (BD Labware), serological pipettes (Costar/Corning, Corning, NY), precision pipette tips (Rainin Instruments, Oakland, CA), Quantification of cell surface expression of Fca/m receptor water (Associates of Cape Cod), and PBS (Life Technologies/Invitrogen, Carlsbad, CA). To quantify Fca/mR cell surface protein expression, pMG or N9 cells were cultured on poly-ornithine