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

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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 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 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 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 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. Anti- cells and RAG-KO BM-DCs behave identically as those from normal mice. iC3b mAb (clone 2/11. rat IgG1) was purchased from HyCult Biotechnol- To generate BMDCs from C3Ϫ/Ϫ, CR3Ϫ/Ϫ mice and littermate controls, 3020 APOPTOTIC CELLS INHIBIT DC INDEPENDENT OF iC3b

FIGURE 2. Apoptotic cells alter the LPS induced cytokine profile of DCs. DCs were cocultured with apo- ptotic cells (ACs) for 3 h, after which they were stimulated with 100 ng/ml LPSfor5hinthepresence of mo- nensin. Apoptotic cells were pre- treated with a 30 min incubation with no serum, wild type (WT) serum, or C3Ϫ/Ϫ serum to differentially opso- nize them before coculture. Cytokine production was measured by intracel- lular staining and flow cytometry. A, Histograms of IL-12, TNF-␣, and IL-10 staining, representative of three experiments. B, Relative fold increase in median fluorescence intensity on unstimulated DCs, averaged over Downloaded from three experiments.

bone marrow precursors were depleted of using anti-Thy1.2 cific staining was determined by incubating apoptotic cells with the sec- and anti-B220 directly conjugated magnetic beads. ondary Ab only. Cells were collected on a FACSCanto flow cytometer (BD http://www.jimmunol.org/ Biosciences) and the data were analyzed using FlowJo software (Tree Generation of peritoneal Star). Mice were injected i.p. with 2 ml of 3% aged Brewer’s thioglycollate. Three days after injection, mice were sacrificed and cells collected from Immunofluorescent microscopy peritoneal lavage. Peritoneal cells were placed in plastic tissue culture ϫ dishes and nonadherent cells were removed after 1 h. The remaining cells We examined BMDCs under an Axioplan-2 microscope with a 63 oil were Ͼ95% macrophages as assessed by surface marker phenotype. objective and we captured and analyzed digital images including 3-D re- constructions using SlideBook software. Generation of apoptotic thymocytes and BaF3 cells by guest on September 24, 2021 Thymocytes were prepared from 6- to 8-wk-old mice and immediately Statistical analysis irradiated with 500 rads of gamma irradiation to induce apoptosis. Irradi- Two-tailed Student’s t test or ANOVA testing was performed where ap- ated thymocytes were cultured in RPMI 1640 medium with 10% FCS for propriate to determine the statistical significance of our findings. The p 16 h before use. BaF3 cells were grown in RPMI 1640 with 10% FCS values of Ͻ0.05 were considered to represent statistical significance. supplemented with 1 ng/ml of murine IL-3 (PeproTech). BaF3 cells were rendered apoptotic by 48 h of culture in medium lacking IL-3. Both types of apoptotic cells were incubated for 30 min at 37°C with normal C57BL/6 Results mouse serum or serum from C3Ϫ/Ϫ mice (gift of Dr. Wenchao Song, Uni- versity of Pennsylvania) to achieve serum opsonization. Apoptotic cells Opsonization of apoptotic cells with iC3b occurs on both early were used immediately after opsonization. For experiments in which track- and late apoptotic cells ing of the apoptotic cells was necessary, BaF3 cells were incubated with It has been previously reported that iC3b opsonization is predom- 12.5 ␮g/ml of the vital dye TAMRA for 30 min at 37°C before apoptosis induction. inately a late apoptotic cell event in human cells (16). We per- formed experiments to examine at which stage iC3b was deposited Apoptotic cell/BMDC coculture on murine apoptotic cells. Gamma irradiation of thymocytes fol- BMDCs were cocultured with apoptotic GFP-BaF3 cells for 3 h and then lowed by 14 h of culture results in a mixed population of apoptotic stimulated cells with LPS (100 ng/ml) in the presence of the Golgi trans- and necrotic cells as identified by Annexin V/7-AAD staining (Fig. port inhibitor monensin (BD Biosciences) for 5 h. After the 5 h stimulation, 1A). Following the convention described by Gaipl et al. (16), we BMDCs were harvested in cold PBS and immediately stained for flow divided cells into live (Annexin Vneg, 7-AADneg), early apoptotic cytometric analysis. For phagocytosis experiments, TAMRA-labeled apo- pos neg pos ptotic BaF3 cells were incubated with BMDCs for the time points shown. (Annexin V , 7-AAD ), late apoptotic (Annexin V ,7- lo,subG1 pos high BMDCs were then harvested and stained with anti-CD11c Ab conjugated AAD ), and necrotic (Annexin V , 7-AAD ). The for- to Alexa 647 (Invitrogen). These cells were then analyzed by flow cytom- ward (FSC) and side scatter profiles of these populations also ϩ ϩ etry, and percent of phagocytosis was calculated as CD11c TAMRA reflected these designations (data not shown). We incubated apo- events/total CD11cϩ events. ptotic thymocytes in mouse serum for 30 min at 37°C after which Flow cytometry we stained with an Ab specific for mouse iC3b and we detected iC3b deposition by flow cytometry on both early and late apoptotic BMDCs were washed in cold PBS, incubated with rat anti-mouse CD16/ CD32 (clone 2.4G2) mAb for 10 min to block Fc␥R, and then stained for cells, while we found no iC3b bound to necrotic or live cells (Fig. 30 min with protein-G purified Ab from supernatants from the N418 hy- 1B). No iC3b deposition was detected on cells that were not in- bridoma (anti-CD11c, American Type Culture Collection) conjugated to cubated in serum, or on cells incubated in serum from C3Ϫ/Ϫ mice, Alexa 647 (Invitrogen). We conducted all stainings on ice. Intracellular confirming the specificity of the Ab used. Late apoptotic cells were staining for TNF-␣ and IL-12 was performed using the Cytofix/Cytoperm uniformly positive, while early apoptotic cells showed a bimodal kit (BD Biosciences) according to the manufacturer’s instructions. Apo- pos ptotic cells were stained with an anti-iC3b Ab for 30 min, followed by distribution of iC3b deposition. Within this early Annexin V , washing and incubation with a FITC-conjugated secondary Ab. Nonspe- 7-AADneg population, the iC3b-negative cells had a FSC ϫ side The Journal of Immunology 3021

FIGURE 3. Serum opsonization does not impact apoptotic cell in- duced cytokine inhibition of DCs. A, Dendritic cells were treated as in Fig. 2. Numbers represent the percentage of BMDCs in each population. B, Re- sults of three experiments normaliz- ing to the LPS treated BMDCs that Downloaded from were not cocultured with apoptotic -Statistically significant dif ,ء .cells ferences between treatments within each population (p Ͻ 0.05, ANOVA test). C, Serum does not affect apo- ptotic cell inhibition of CpG induced

cytokines. Results of three experi- http://www.jimmunol.org/ ments normalizing to the CpG treated BMDCs that were not cocultured with -Statistically signifi ,ء .apoptotic cell cant differences between treatments within each population (p Ͻ 0.05, ANOVA test). by guest on September 24, 2021

scatter profile more consistent with live cells, while the iC3b-pos- shown that CR3 activation can have the same effect. However, in itive population had a lower FSC, consistent with further progres- all of these reports, CR3 activation was obtained with artificial sion in apoptosis (Fig. 1C). This suggests that iC3b deposition is ligands such as mAbs or iC3b coated RBC. Therefore, the phys- an event occurring during the development of early apoptosis, but iologic contribution of CR3 to the inhibition of the proinflamma- not at its initial onset. tory cytokines induced by apoptotic cells still remains to be de- Because we desired a more homogenous apoptotic cell popula- termined. To do so, we used BaF3 cells that were transduced with tion for our experiments, we tested multiple models of apoptotic the GFP protein (GFP-BaF3) to generate apoptotic cells that could cell death. IL-3 deprivation of the IL-3 dependent BaF3 cells (17) be easily differentiated from BMDCs. We cocultured BMDCs for for 48 h resulted in the most homogenous cell death, with ϳ70% 3 h with apoptotic GFP-BaF3 cells that had been previously incu- of cells in the early apoptotic stage (Fig. 1D). Incubation with bated with no serum, wild type serum, or C3Ϫ/Ϫ serum for 30 min. serum resulted in iC3b deposition on both early and late apoptotic We then stimulated these BMDCs with LPS in the presence of BaF3 cells (Fig. 1E). Neither live nor necrotic BaF3 cells fixed monensin for 5 h and analyzed for cytokine production by intra- iC3b to their surface. cellular staining and flow cytometry. Because GFP-BaF3 cells lost Thus, we find in two different models of murine apoptotic cell their GFP positivity as they became apoptotic, likely due to pro- death that iC3b deposition occurs both in early and late apoptotic teolysis of the GFP during apoptosis (data not shown), we used the cells, while it does not occur in necrotic cells. GFP label to gate early apoptotic BaF3 cells out and identified BMDCs as CD11cϩGFPϪ cells within the appropriate forward and Apoptotic cells inhibit TLR-stimulated DC cytokine production side scatter gate for DCs. We found that apoptotic cells inhibited in an iC3b independent manner both LPS-induced TNF-␣ and IL-12 expression by DCs as mea- Apoptotic cells can suppress the LPS-stimulated production of sured by median fluorescence intensity (MdFI) (Fig. 2), confirming proinflammatory cytokines by DCs. We and others (8–10) have previous reports that apoptotic cells inhibit LPS-induced 3022 APOPTOTIC CELLS INHIBIT DC CYTOKINES INDEPENDENT OF iC3b

FIGURE 4. Local C3 production by DCs does not result in opsonization of apoptotic cells with iC3b. A, TAMRA labeled apoptotic BaF3 cells were cocultured with C3ϩ/ϩ or C3Ϫ/Ϫ BMDCs for 3 h and stained for surface iC3b. TAMRAϩCD11cϪ cells were analyzed for iC3b staining using the designated gates for live and apoptotic cells. No iC3b was detected on the surface of cells incubated with C3ϩ/ϩ BMDCs indicating that lo- cal C3 production was not contributing to iC3b opso- nization of the cocultured apoptotic cells. B, No differ- ence in the amount of inhibition of cytokines was seen with apoptotic cell coculture with C3ϩ/ϩ BMDCs com- pared with C3Ϫ/Ϫ BMDCs. Downloaded from

up-regulation of proinflammatory cytokines in human (18) and Local C3 production by DCs is not sufficient to opsonize http://www.jimmunol.org/ murine systems (11). Apoptotic cells exposed to serum in gen- apoptotic cells in vitro eral tended to have a slightly more pronounced effect on the Because DCs are capable of local C3 production (19), we hypoth- ␣ overall MdFI of both TNF- and IL-12 that did not reach sta- esized that lack of C3 in the C3Ϫ/Ϫ serum could have been com- tistical significance, while the presence or absence of C3 in the pensated for by BMDC-derived C3 secretion. This would account serum (and thus iC3b on the surface of the apoptotic cells) did for the lack of differences in cytokine suppression seen between not make a difference. the wild type and the C3Ϫ/Ϫ serum. We cocultured apoptotic The technique of intracellular staining provides the advantage of TAMRA labeled BaF3 cells with C3ϩ/ϩ and C3Ϫ/Ϫ BMDCs for detection of cytokines at a single cell level and we discovered that BMDCs do not activate homogeneously. Four populations of by guest on September 24, 2021 BMDCs were present after LPS stimulation, cells that produced no cytokines, cells that were single positive for either TNF-␣ or IL- 12, and cells that were positive for both cytokines (Fig. 3A, top left panel). Apoptotic cells reduced the IL-12 single-positive and dou- ble-positive BMDCs while leaving the TNF-␣ single positive pop- ulation unaffected (Fig. 3B). The addition of serum did not have a significant effect on the extent of inhibition, suggesting that this was a CR3/iC3b independent event. It remained possible that the lack of a serum effect was specific only to high dose LPS stimulation. We performed similar exper- iments decreasing the dose of LPS over a three log range (1 ng/ml to 100 ng/ml) and found no difference in the ability of serum op- sonized apoptotic cells to inhibit cytokines over nonopsonized ap- optotic cells (data not shown). These data demonstrate that apo- ptotic cells reduce the IL-12 single-positive and double-positive BMDCs while leaving the TNF-␣ single positive population un- affected upon TLR stimulation. We therefore hypothesize that the effect of the apoptotic cells is not unique to a particular cytokine, but rather targets distinct functional subsets of BMDCs. We have previously shown that CR3 ligation with the specific mAb decreases the cytokine production induced by the TLR9 li- gand CpG (8). We have now found that apoptotic cells can also inhibit the CpG-induced production of proinflammatory cytokines in DCs. Serum opsonization did not affect the inhibition of cyto- kines caused by apoptotic cells in CpG stimulated DCs, demon- strating serum independence for multiple TLR ligands (Fig. 3C). FIGURE 5. Apoptotic cells result in a mild reduction of IL-12 and Interestingly, in this case, apoptotic cells inhibited not only the TNF-␣ producing macrophages. Peritoneal macrophages were treated with IL-12 single positive and double positive DCs, but also the TNF-␣ apoptotic cells (ACs) and LPS as in Figs. 2 and 3. Cytokine production was single-positive cells. Thus, apoptotic cells may attenuate different measured using intracellular staining analyzed by flow cytometry. Results DC populations depending on which TLR agonist follows them. representative of three experiments. The Journal of Immunology 3023 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 6. Serum opsonization of apoptotic cells has no effect their ability to elicit IL-10 from BMDCs. A, Dendritic cells were treated with apoptotic cells and LPS as in Fig. 2. IL-10 production was determined by intracellular staining and analyzed via flow cytometry. IL-10 production is expressed as a median fluorescence intensity (MdFI). Apoptotic cells, but not LPS alone, induce IL-10 production. Neither serum opsonization, nor TNF-␣/IL-12 status affects IL-10 production. B, Peritoneal macrophages treated and analyzed as in A. MdFI is shown for all macrophages. Results are representative of three experiments. C, BMDCs were treated with either apoptotic cells or LPS for 5 h and analyzed for IL-10 production via flow cytometry. Only apoptotic cells Statistically significant difference compared with untreated ,ء .induced IL-10 production over baseline. Averages and SEM for three experiments are shown cells (p Ͻ 0.05, t test). D, Blockade of IL-10 receptor did not alter the apoptotic cell induced inhibition of IL-12 and TNF-␣/IL-12 producing BMDCs. BMDCs were treated as in Fig. 3B in the presence of the IL-10 receptor blocking Ab 1B1.3a (10 ␮g/ml) or an control Ab.

3 h. We identified the apoptotic cells as CD11cϪTAMRAϩ events cells, confirming that local C3 production is not playing a role in the and determined the surface levels of iC3b by the MdFI within this cytokine inhibition caused by apoptotic cells (Fig. 4B). population. No differences were seen in iC3b levels in the apopto- tic cells exposed to wild type and C3Ϫ/Ϫ BMDC, suggesting that iC3b opsonization does not increase the inhibition of local C3 production by BMDCs is not playing a role in iC3b opso- LPS-induced cytokine production by apoptotic cells nization of apoptotic cells in our culture system (Fig. 4A). Further- in macrophages more, no differences in the pattern of inhibition of cytokines were seen It has been previously shown that apoptotic cells inhibit the in BMDC sufficient or deficient in C3 exposed to apoptotic BaF3 production of proinflammatory cytokines induced by LPS in 3024 APOPTOTIC CELLS INHIBIT DC CYTOKINES INDEPENDENT OF iC3b

we found that IL-10 was produced equally by all four populations of BMDCs (Fig. 6A). These results suggest that the capacity to produce IL-10 is independent of the state of production of inflam- matory cytokines. It is noteworthy that IL-10 was induced specif- ically by the apoptotic cells, because at this early time point (5 h) LPS did not yet induce any IL-10, while apoptotic cells without LPS resulted in IL-10 production (Fig. 6C). These data are con- sistent with the previously described kinetics of cytokine produc- tion in DCs (23). Opsonization with C3ϩ/ϩ or C3Ϫ/Ϫ serum did not affect the induction of IL-10 by apoptotic cells, either in BMDCs or in peri- FIGURE 7. Serum opsonization does not enhance phagocytosis of ap- toneal macrophages (Fig. 6, A and B). These results indicate that in optotic cells. TAMRA-labeled apoptotic BaF3 cells were opsonized with serum as previously described and were cocultured with either BMDCs or the murine system, apoptotic cells induce IL-10 production both in peritoneal macrophages for the time points shown. Cells were then ana- dendritic cells and macrophages through a mechanism independent lyzed by flow cytometry. Percentage of BMDCs phagocytosing was cal- from serum . culated as number of TAMRAϩCD11cϩ events divided by number of Importantly, the IL-10 induced by apoptotic cells was not re- CD11cϩ events. CD11b was used in a similar fashion for macrophages. As sponsible for the inhibition of TNF-␣ and IL-12 because pretreat- a negative control, BMDCs were cocultured with apoptotic cells at 4°C. ment with an IL-10 receptor blocking Ab did not affect TNF-␣/

Results representative of three experiments. IL-12 inhibition induced by apoptotic cells with or without iC3b Downloaded from opsonization (Fig. 6D). The efficacy of IL-10 receptor blockade is demonstrated by the increase in baseline LPS induced TNF-␣/ macrophages (20). We tested whether the opsonization with mouse IL-12 production in the IL-10 receptor blocked DCs compared serum, deficient or not of C3, affected this inhibition. Peritoneal with isotype Ab-treated cells. macrophages responded more homogeneously to activation than BMDCs, because the large majority produced TNF-␣ upon LPS Serum opsonization does not enhance apoptotic cell http://www.jimmunol.org/ stimulation (Fig. 5); only a small percentage of them (5%) pro- phagocytosis by either DCs or macrophages duced IL-12, confirming previous reports that macrophages do not Because there are conflicting reports in the literature about the role have the same potential to produce IL-12 as DCs (21). Apoptotic of serum opsonins and iC3b especially in facilitating phagocytosis cells completely obliterated IL-12 production in macrophages, and of apoptotic cells, we further investigated whether serum opso- they also induced a small reduction of percentage of TNF-␣-pro- nization of apoptotic cell enhances the extent or kinetics of phago- ducing macrophages. Serum opsonization did not alter macro- cytosis in mouse macrophages and DCs. We incubated apoptotic phage LPS induced cytokines (Fig. 5). These results show that TAMRA labeled BaF3 cells in serum as described above. We serum opsonization is also dispensable for apoptotic cell mediated cocultured apoptotic cells with either BMDCs or peritoneal mac- inhibition of cytokine production. rophages and measured phagocytosis at various time points (Fig. by guest on September 24, 2021 7). No differences were seen in either the amount or the rate of iC3b opsonization of apoptotic cells does not alter their ability phagocytosis when we used serum opsonized apoptotic cells or to induce IL-10 apoptotic cells without serum. Furthermore, phagocytes from mice ␣ Ϫ/Ϫ The anti-inflammatory cytokine IL-10 is secreted by phagocytes deficient in CR3 (integrin m ) showed no defect in phagocy- upon the ingestion of apoptotic cells (22). In our hands, the expo- tosis (data not shown). These results are in agreement with Ren et sure to apoptotic cells caused an increase in IL-10 production in al. (36) suggesting that complement opsonization of apoptotic cells both BMDCs and peritoneal macrophages, but this effect was not does not enhance phagocytosis in otherwise normal phagocytes. enhanced by serum opsonins (Figs. 2 and 6, A and B). Although we have shown that ligation of CR3 via mAb actually inhibits IL-10 iC3b opsonized apoptotic cells interact with surface CR3 on production (8), no difference was seen in IL-10 levels when wild dendritic cells upon internalization type or C3Ϫ/Ϫ serum was used. When we analyzed BMDCs for Given the lack of effect of iC3b opsonization on cytokine produc- expression of IL-10 with respect to IL-12 and TNF-␣, surprisingly tion and phagocytosis, we confirmed that iC3b on the surface of

FIGURE 8. BMDC CR3 is colocalized specifically with iC3b opsonized apoptotic cells upon phagocytosis. TAMRA-labeled BaF3 cells were opsonized with serum as previously described and cocultured with BMDCs for 30 min. BMDCs were then permeabilized and stained for CR3 (FITC). A, BMDCs not interacting with apoptotic cells showed a rim, surface staining pattern for CR3. B, BMDCs phagocytosing nonopsonized apoptotic cells retained their surface staining for CR3 (two representative cells shown from different fields). C, BMDCs phagocytosing serum opsonized apoptotic cells internalized CR3 surrounding the vesicle containing the ingested apoptotic cell (two representative cells shown from different fields). The Journal of Immunology 3025 apoptotic cells does interact with CR3 on dendritic cells. We have absence of C3, does not affect their in vitro cytokine inhibition or previously observed that triggering of CR3 with a specific Ab phagocytosis also suggests that serum factors in general are re- causes CR3 internalization (8). We used immunofluorescence mi- dundant for apoptotic cell processing by DCs. croscopy and 3-D reconstruction to determine whether iC3b-op- The heterogeneous nature of cytokine secretion by CD11cϩ sonized apoptotic cells could induce the same CR3 internalization, BMDCs was surprising because it suggests that there may be dif- as sign of CR3/iC3b interaction. To do so, we cocultured ferent subsets of DCs with varying capacity to respond to TLR TAMRA-labeled apoptotic BaF3 cells, with or without iC3b op- stimulation in a population that otherwise would appear fairly ho- sonization, and BMDCs for 1 h. BMDCs were then harvested, mogenous. These same subsets would appear to also respond dif- permeabilized, and stained for CR3. BMDCs that had not inter- ferently to apoptotic cell exposure with respect to suppression of acted with an apoptotic cell showed a rim, surface staining pattern inflammatory cytokines because in the context of LPS, TNF-␣ of CR3 (Fig. 8A). BMDCs were able to internalize nonopsonized single producing cells are less affected by apoptotic cells than apoptotic cells, however CR3 remained on the surface of the IL-12 single producers or TNF-␣/IL-12 double producers. Inter- BMDC and did not colocalize with the vesicle containing the ap- estingly, all the three subpopulations of cytokine producing DCs optotic cell (Fig. 8B). In contrast, phagocytosis of iC3b opsonized were inhibited by apoptotic cells upon stimulation with CpGs, sug- apoptotic cells resulted in the internalization of CR3 from the sur- gesting that these functionally different subsets of BMDCs have face, colocalizing around the vesicle containing the apoptotic cells different sensitivity to apoptotic cells depending on which TLR (Fig. 8C). Thus, iC3b on apoptotic cells does interact with CR3 agonist is used. In contrast, the effect of apoptotic cells on the and induces its internalization even though it does not enhance production of IL-10 was independent of the TNF-␣/IL-12 status of phagocytosis or cytokine suppression. the BMDC, suggesting that some apoptotic cell responses occur Downloaded from homogenously within DCs. Further characterization of these DC Discussion subsets may reveal new factors that determine TLR responsiveness Much attention has recently been given to CR3 activation as an as well as participate in apoptotic cell processing. anti-inflammatory event. These data, including our own, have Our data show in two different murine systems of apoptosis that mostly been generated using artificial means of activating CR3 to iC3b deposition occurs both on early and late apoptotic cells. This

model the presumed effects of iC3b opsonized apoptotic cells bind- is in contrast to an earlier report in human cells that described iC3b http://www.jimmunol.org/ ing to the receptor. Data examining the role of iC3b/CR3 interac- binding as a predominantly late apoptotic event (16). Certainly this tions in the phagocytosis of apoptotic cells is conflicting (10, 12– may reflect both differences in the organisms (human vs mouse) 14). In this report, we provide evidence suggesting that CR3 is not and cell types (polymorphonuclear cells and peripheral blood lym- necessary for either apoptotic cell phagocytosis or apoptotic cell phocytes vs thymocytes and a leukemic cell line). Because in vivo mediated DC cytokine suppression. The BaF3 model provides an clearance of apoptotic cells usually occurs before the apoptotic excellent means to study early, apoptotic cells in a homogenous cells reach the late stage (reviewed in Ref. 32), only early depo- population, something that is not easily accomplished using other sition of iC3b would allow the binding of iC3b-opsonized apopto- systems we attempted, such as irradiated thymocytes or splenic B tic cells to CR3 receptors in physiologic conditions. Moreover, the cell, and ceramide treated fibroblasts (data not shown). Because it absence of iC3b deposition on necrotic cells suggest that this mol- by guest on September 24, 2021 was the early apoptotic population of BaF3 that had the highest ecule could be partially responsible for the distinct effects that levels of iC3b deposition (Fig. 1), this was an ideal model for apoptotic vs necrotic cells have on the cells of the immune system identifying the optimal iC3b effect. Of course, caution should be (33). applied in generalizing to other apoptotic cell models where more Clearly, CR3 can have a major effect on the maintenance of heterogeneous death is seen, and other cell specific factors may be as evidenced by the strong association of CR3 in effect. polymorphisms with the development of SLE (5–7). Interestingly, The lack of iC3b effect is likely due to the redundancy of the there are also many associations of defective apoptotic cell pro- apoptotic cell receptor system, which includes other molecules cessing and the development of in humans and mice also known to inhibit inflammatory cytokine production including (3, 34–36). However, given our data, it would appear that the mer (24) and CD36 (25), as well as many different phagocytic functional absence of CR3 would be unlikely to result in any sig- receptors (26–28). Indeed, C1q is the more important mediator in nificant deficits in apoptotic cell processing. Furthermore, in the the in vivo clearance of apoptotic cells compared with C4/C3, report of Nath et al. (7), the functional variant of ITGAM associ- particularly in the absence of inflammation (29). It is also possible ated with SLE is not in the iC3b binding region of CR3, again that CR3 triggering induces unique and as of yet uncharacterized suggesting that the association of CR3 with SLE may not be de- factors important in the anti-inflammatory response that we are pendent on apoptotic cell processing. It is possible that other phys- simply unable to assay, such as directing the apoptotic cell to a iologic ligands of CR3, for example fibronectin (37) or ICAM-1 particular endosomal compartment for alternate processing (30). (38), may also result in cytokine suppression and that these inter- Finally, because of the results of the microscopy experiments, we actions are more physiologically relevant for maintaining immune exclude the possibility that, in our culture system, iC3b opsoniza- tolerance than its role as an apoptotic cell receptor. It is also pos- tion simply does not trigger CR3 on DCs. We speculate that phys- sible that the role of CR3 in SLE is related to its many other roles: iologic triggering of CR3 by iC3b opsonized apoptotic cells does adhesion (39), respiratory burst (40), or NO production (41). Fur- not result in the same DC inhibition induced by ligation with a ther study of CR3 in SLE models may help distinguish these mAb due to differences in either the strength or qualitative nature possibilities. of the signals. In conclusion, it would appear that CR3 is dispensable in the In vitro studies of serum opsonization have been limited to phagocytosis of apoptotic cells and in the suppression of cytokines phagocytosis (10, 12–14) and offer conflicting reports. Apoptotic that they induce. Nonetheless, our data do not in any way diminish cells in serum-free conditions have been recently been shown to be the possibility of using artificial ligation of CR3 as a means of equally effective as apoptotic cells with serum opsonins in their manipulating DC function, because supraphysiologic activation of ability to inhibit LPS induced IL-12 (31). Our observation that CR3 can clearly modulate DCs toward an anti-inflammatory phe- serum opsonization of apoptotic cells, regardless of the presence or notype (8–10). Data already exist to suggest that the activation of 3026 APOPTOTIC CELLS INHIBIT DC CYTOKINES INDEPENDENT OF iC3b

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