Apoptotic Cell-Mediated Immunoregulation of Dendritic Cells Does Not Require iC3b Opsonization This information is current as Edward M. Behrens, Yue Ning, Nidal Muvarak, Philip W. of September 24, 2021. Zoltick, Alan W. Flake and Stefania Gallucci J Immunol 2008; 181:3018-3026; ; doi: 10.4049/jimmunol.181.5.3018 http://www.jimmunol.org/content/181/5/3018 Downloaded from References This article cites 42 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/181/5/3018.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Apoptotic Cell-Mediated Immunoregulation of Dendritic Cells Does Not Require iC3b Opsonization1 Edward M. Behrens,2*† Yue Ning,*† Nidal Muvarak,‡ Philip W. Zoltick,‡ Alan W. Flake,‡ and Stefania Gallucci*† A number of recent studies show that activation of CR3 on dendritic cells (DCs) suppresses TLR-induced TNF-␣ and IL-12 production and inhibits effective Ag presentation. Although the proposed physiologic role for these phenomena is immune sup- pression due to recognition of iC3b opsonized apoptotic cells by CR3, all of the aforementioned investigations used artificial means of activating CR3. We investigated whether iC3b opsonized apoptotic cells could induce the same changes reported with artificial ligands such as mAbs or iC3b-opsonized RBC. We explored the kinetics of iC3b opsonization in two models of murine cell apoptosis, ␥-irradiated thymocytes and cytokine deprivation of the IL-3 dependent cell line BaF3. Using a relatively homogenous population of early apoptotic cells (IL-3 deprived BaF3 cells), we show that iC3b opsonized apoptotic cells engage CR3, but this interaction is Downloaded from dispensable in mediating the anti-inflammatory effects of apoptotic cells. TLR-induced TNF-␣ and IL-12 production by bone marrow- derived DCs occurs heterogeneously, with apoptotic cells inhibiting only certain populations depending on the TLR agonist. In contrast, although apoptotic cells induced homogeneous IL-10 production by DCs, IL-10 was not necessary for the inhibition of TNF-␣ and IL-12. Furthermore, because the ability of iC3b opsonization to enhance phagocytosis of apoptotic cells has been controversial, we report that iC3b opsonization does not significantly affect apoptotic cell ingestion by DCs. We conclude that the apoptotic cell receptor system on DCs is sufficiently redundant such that the absence of CR3 engagement does not significantly affect the normal anti-inflammatory http://www.jimmunol.org/ processing of apoptotic cells. The Journal of Immunology, 2008, 181: 3018–3026. ying cells need to be carefully handled by the immune cific way for phagocytes to recognize dying cells and induce the ␣ ␤ system so as not to provoke an immune response against appropriate anti-inflammatory environment. The m 2 integrin D self-Ags. Apoptosis, the programmed mode of cell complement receptor 3 (CR3) recognizes apoptotic cells by death, is carefully orchestrated to be an anti-inflammatory event. binding to the serum protein iC3b bound to their surface. Op- Both the apoptotic cells themselves (1), as well as the cells clearing sonization of iC3b is thought to be driven in part by the specific away the apoptotic debris, contribute to this anti-inflammatory envi- exposure of phosphatidylserine molecules by apoptotic by guest on September 24, 2021 ronment (2). This anti-inflammatory environment comes in the form cells (4). of functional suppression of APCs via inhibition of proinflammatory Three recent genome-wide association studies have found that a cytokine production. This suppression prevents the immune system particular polymorphism of the gene encoding the ␣-chain of CR3, from initiating a response against the multiple neo-self-Ags created ITGAM, is associated with high risk to develop SLE (5–7). The during the apoptosis process. In the archetypal autoimmune disease molecular and functional mechanisms underlying this risk factor 3 systemic lupus erythematosus (SLE), an impaired clearance of apo- still remain to be discovered. ptotic cells, or their failure to induce tolerance to self Ags, are hy- We and others have shown that CR3 ligation suppresses inflam- pothesized as a pathogenic mechanism (3). matory cytokine production by dendritic cells (DCs). However, in There are many receptors for apoptotic cells, found in various each of these experimental systems, the ligand used to produce the combinations depending on cell type, which may provide a spe- CR3 effect has been artificial, either using a mAb against the ␣-chain (8, 9), or RBC that have been opsonized with iC3b (10). *Laboratory of Dendritic Cell Biology, Division of Rheumatology, Joseph Stokes, Jr. Although apoptotic cells have certainly been demonstrated to sup- Research Institute, Children’s Hospital of Philadelphia, Philadelphia, PA 19104-4318; †Division of Rheumatology, Department of Pediatrics, University of Pennsylvania press DC inflammatory cytokine production (11), the physiological School of Medicine, Philadelphia, PA 19104-4318; ‡Department of Surgery, Chil- contribution of CR3/iC3b interactions toward this effect remains in dren’s Hospital of Philadelphia, Philadelphia, PA 19104-4318 question. Furthermore, there are multiple conflicting reports on the Received for publication February 29, 2008. Accepted for publication June 20, 2008. ability of CR3/iC3b interactions to enhance the phagocytosis and The costs of publication of this article were defrayed in part by the payment of page internalization of apoptotic cells (10, 12–14). Because apoptosis is charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. an asynchronous event, it is difficult to generate homogenous pop- ulations of apoptotic cells. Using an IL-3-dependent cell line, we 1 E.M.B. was supported by the National Institute of Health (NIH Grant T32- HD0043021) and an Arthritis Foundation Post-Doctoral Fellowship, and S.G. by the are able to generate a relatively homogenous population of apo- Lupus Foundation Southeastern Pennsylvania Chapter, Arthritis Foundation (Innova- ptotic cells that can be opsonized with serum proteins. Using this tive Grant). system, we show that although apoptotic cell coculture results in 2 Address correspondence and reprint requests to Dr. Edward M. Behrens, Children’s Hospital of Philadelphia, 3615 Civic Center Boulevard, ARC 1102, Philadelphia, PA cytokine suppression, this effect does not require CR3/iC3b inter- 19104-4318. E-mail address: [email protected] actions. We furthermore present data that CR3/iC3b interactions 3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; DC, dendritic do not contribute to the amount or kinetics of apoptotic cell phago- cell; FSC, forward scatter; MdFI, median fluorescence intensity; BMDC, bone mar- cytosis. Understanding the physiologic role of CR3 in apoptotic row derived DCs; CR3, complement receptor 3. cell processing will help us to evaluate its potential pharmacologic Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 utility. www.jimmunol.org The Journal of Immunology 3019 FIGURE 1. iC3b deposition occurs on both early and late apoptotic cells. A, An- nexin V/7-AAD staining of thymocytes after 16 h of culture post 500 rads of gamma ir- radiation. Small numbers represent percent- age of cells within each population. B, Thy- Downloaded from mocytes prepared as in A were incubated with no serum, wild type serum, or serum from C3-deficient mice for 30 min and stained for iC3b on their surface. Panels 1–4 refer to the populations 1–4 in A. Cells stained only with secondary Ab are shown to http://www.jimmunol.org/ establish baseline staining. Vertical lines are provided for reference to the median inten- sity of this baseline. C, Forward and side scatter profiles of the iC3b positive and iC3b negative populations from the early apopto- tic thymocytes (population 2). Small num- bers represent percentage of cells within each population. The forward and side scat- ter profile of the live thymocytes (population 1) is shown for comparison. D, Apoptotic by guest on September 24, 2021 BaF3 after 48 h of IL-3 deprivation stained as in A. E, Apoptotic BaF3 cells stained and plotted as in B. Results representative of two to three experiments. Materials and Methods ogy and FITC-anti-Rat IgG1 (BD Biosciences) was used as a secondary ␮ Mice and Abs Ab. IL-10 receptor blocking Ab 1B1.3A was used at 10 g/ml. C57BL/6, C57BL/6-Rag1Ϫ/Ϫ, and ITGAMϪ/Ϫ (deficient in CR3) mice Ϫ Ϫ were purchased from Jackson ImmunoResearch Laboratories. C3 / mice Bone marrow derived DC (BMDC) generation were generously provided by Dr. Wenchao Song (University of Pennsyl- vania, Philadelphia, PA). All mice were bred and maintained in accordance Bone marrow-derived DCs were generated as previously described (15). In Ϫ Ϫ with guidelines of the Institutional Animal Care and Use Committee of The brief, bone marrow precursors from Rag1 / mice were cultured for 6 days Children’s Hospital of Philadelphia, an American Association for the Ac- in complete IMDM containing 3.3 ng/ml GM-CSF (BD Biosciences). Gen- creditation of Laboratory Animal Care accredited facility. Anti-TNF-␣, erating DCs from RAG-KO BM does not require depletion of T- and B- IL-12, IL-10, and CR3 Abs were purchased from BD Biosciences.