Properdin Binds to Late Apoptotic and Necrotic Cells Independently of C3b and Regulates Alternative Pathway Complement Activation This information is current as of September 27, 2021. Wei Xu, Stefan P. Berger, Leendert A. Trouw, Hetty C. de Boer, Nicole Schlagwein, Chantal Mutsaers, Mohamed R. Daha and Cees van Kooten J Immunol 2008; 180:7613-7621; ; doi: 10.4049/jimmunol.180.11.7613 Downloaded from http://www.jimmunol.org/content/180/11/7613 References This article cites 56 articles, 27 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/180/11/7613.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • 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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Properdin Binds to Late Apoptotic and Necrotic Cells Independently of C3b and Regulates Alternative Pathway Complement Activation1,2 Wei Xu,* Stefan P. Berger,* Leendert A. Trouw,† Hetty C. de Boer,*‡ Nicole Schlagwein,* Chantal Mutsaers,* Mohamed R. Daha,* and Cees van Kooten3* Cells that undergo apoptosis or necrosis are promptly removed by phagocytes. Soluble opsonins such as complement can opsonize dying cells, thereby promoting their removal by phagocytes and modulating the immune response. The pivotal role of the com- plement system in the handling of dying cells has been demonstrated for the classical pathway (via C1q) and lectin pathway (via mannose-binding lectin and ficolin). Herein we report that the only known naturally occurring positive regulator of complement, properdin, binds predominantly to late apoptotic and necrotic cells, but not to early apoptotic cells. This binding occurs inde- Downloaded from pendently of C3b, which is additional to the standard model wherein properdin binds to preexisting clusters of C3b on targets and stabilizes the convertase C3bBb. By binding to late apoptotic or necrotic cells, properdin serves as a focal point for local ampli- fication of alternative pathway complement activation. Furthermore, properdin exhibits a strong interaction with DNA that is exposed on the late stage of dying cells. Our data indicate that direct recognition of dying cells by properdin is essential to drive alternative pathway complement activation. The Journal of Immunology, 2008, 180: 7613–7621. http://www.jimmunol.org/ nder steady-state conditions, cells that undergo apopto- plement is involved in removal of dying cells and the immune sis and necrosis can be safely and silently eliminated by regulation associated with this process. Complement-mediated professional phagocytes, that is, immature dendritic clearance of apoptotic cells has been well documented both in vitro U 4 cells and macrophages (M␾) (1–3). Apoptotic cells are a rich (10) and in vivo (11). Nevertheless, the role of the complement source of autoantigens, which are involved in the induction of system in the handling of dying cells has been mostly linked to the self-tolerance and autoimmunity (4). Compelling evidence has classical pathway (via C1q) and lectin pathway (via mannose- emerged that abnormal clearance of apoptotic cells is associated binding lectin (MBL) and ficolin) (10–14). It has been suggested with development of the autoimmune disease systemic lupus ery- that the main product of complement activation, iC3b, facilitates thematosus (SLE) (5, 6). the removal of dead material and mediates peripheral tolerance by guest on September 27, 2021 Soluble factors from the innate immune system such as com- (10, 15, 16). plement or pentraxins can opsonize apoptotic cells, thereby pro- The alternative pathway of complement is thought to be acti- moting their removal by phagocytes (6–8). In humans, homozy- vated following hydrolysis of C3, generation of C3b, and forma- gous deficiency of any of the early components of the classical tion of a positive feedback loop to mount a rapid local response pathway of complement activation (C1q, C1r, C1s, C4, and C2) (17). The alternative pathway was initially recognized to amplify predisposes to the development of SLE (9), suggesting that com- complement activation triggered by the classical pathway. Proper- din, discovered in 1954 (18), is the only known naturally occurring positive regulator of complement activation (19). It was originally *Department of Nephrology, †Department of Rheumatology, and ‡Einthoven Labo- shown that properdin binds to C3b and increases the stability of the ratory for Experimental Vascular Medicine, Leiden University Medical Center, Lei- alternative pathway convertases at least 10-fold on target surfaces den, The Netherlands and immune complexes (20). It has been recently suggested that Received for publication November 6, 2007. Accepted for publication March 21, 2008. properdin could bind directly to microbial targets (21), which is consistent with a proposal made more than 50 years ago (18). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance In the present study, we investigated whether properdin, like with 18 U.S.C. Section 1734 solely to indicate this fact. C1q and MBL, contributes to the recognition and opsonization of 1 This work was supported in part by a grant (C02.2015) from the Dutch Kidney dying cells. We found that properdin binds predominantly to late Foundation. apoptotic and necrotic cells independently of C3b, but not to early 2 W.X., M.R.D., and C.v.K. designed the study; W.X., N.S., and C.M. performed apoptotic cells, leading to alternative pathway-mediated comple- research; W.X., S.P.B., L.A.T., H.d.B., M.R.D., and C.v.K. analyzed data; and W.X., M.R.D., and C.v.K. wrote the paper. All authors reviewed and approved the paper. ment activation. DNA was identified as one of the ligands on dying 3 Address correspondence and reprint requests to Dr. Cees van Kooten, Department cells to which properdin binds. This accounts for a C3b-indepen- of Nephrology, C3-P, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, dent mechanism of properdin-initiated complement activation on Leiden, The Netherlands. E-mail address: [email protected] dying cells. 4 Abbreviations used in this paper: M␾, macrophage; C4ds, C4-depleted serum; dsDNA, double-stranded DNA; EBV-LCL, Epstein-Barr virus-transformed B lym- phoblastoid cell line; MBL, mannose-binding lectin; M-CSF, macrophage colony- Materials and Methods stimulating factors; MFI, mean fluorescence intensity; NHS, normal human serum; Induction of apoptosis and necrosis Pds, properdin-depleted serum; PI, propidium iodide; SLE, systemic lupus erythem- atosus; ssDNA, single-stranded DNA; WT, wild type. Jurkat cells were cultured in RPMI culture medium. Early or late apoptosis was induced in Jurkat cells by exposure to UV-C light (Philips TUV lamp, Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 predominantly 254 nm) at a dose of 50 J/m2, followed by 3 or 30 h culture www.jimmunol.org 7614 PROPERDIN BINDS TO APOPTOTIC AND NECROTIC CELLS FIGURE 1. Complement-mediated phagocytosis of dying cells. A, Early or late apoptosis was induced in Jurkat cells by exposure to UV-C light at a dose of 50 J/m2, followed by 3 or 30 h culture in RPMI serum-free medium, respectively. Necrosis was induced by treating cells at 56°C for 0.5 h. Cells were stained with annexin V and PI by flow cytometry. B, Late apoptotic cells and necrotic cells are scored by light microscopy or fluores- cent microscopy for Hoechst staining on cytospins of these cells. Magnification, ϫ200. C, CFSE-labeled early apoptotic, late apoptotic, or necrotic cells (1 ϫ 105) were first opsonized with or without NHS, then cocul- Downloaded from tured with M␾2 in a 1:1 ratio at 37°C for 0.5 h. M␾2 were stained with a PE-conjugated mAb against CD11b, and uptake was analyzed by two-color flow cytometry. CD11bϩCFSEϩ cells were used as a measure for the percentage of M␾2 that ingested apoptotic cells. D, Rel- ative phagocytosis was calculated as uptake of NHS- http://www.jimmunol.org/ opsonized dying cells vs nonopsonized cells. Data are p Ͻ ,ء .mean Ϯ SEM of three independent experiments 0.01, one-sample paired t test. E, C3 deposition (filled histogram) after NHS opsonization on early, late apo- ptotic, and necrotic cells was detected by flow cytom- etry. Open histograms are the matched isotype controls. by guest on September 27, 2021 in RPMI serum-free medium, respectively. Necrosis was induced by treat- the binding of dying cells to phagocytes. M␾2 were stained with a PE- ing cells at 56°C for 0.5 h or 5 cycles of freeze-thaw from Ϫ80°C to 36°C. conjugated mAb against CD11b (BD Biosciences), and uptake was ana- Both apoptosis and necrosis were confirmed by double staining with FITC- lyzed by two-color flow cytometry. The percentage of CD11b-positive labeled annexin V and propidium iodide (PI, VPS Diagnostics) according cells that stained positive for CFSE was used as a measure for the per- to established methods (22). Additionally, light microscopy and fluorescent centage of M␾2 that ingested and/or bound apoptotic cells.