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Domain 3-Only Protein Bimand Activation of the Bcl-2 HomologyMacrophages Is Mediated by Up-Regulation Phagocytosis-Induced Apoptosis in

HäckerHäcker, Andreas Villunger, Hubertus Hochrein and Georg Susanne Kirschnek, Songmin Ying, Silke F. Fischer, Hans

http://www.jimmunol.org/content/174/2/671doi: 10.4049/jimmunol.174.2.671

2005; 174:671-679; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Phagocytosis-Induced Apoptosis in Macrophages Is Mediatedby Up-Regulation and Activation of the Bcl-2 HomologyDomain 3-Only Protein Bim1

Susanne Kirschnek,* Songmin Ying,* Silke F. Fischer,* Hans Hacker,† Andreas Villunger,‡

Hubertus Hochrein,* and Georg Hacker2*

Cell death by apoptosis is important in immune cell homeostasis and in the defense against infectious microorganisms. Thephysiological event of uptake and intracellular destruction of bacteria is a powerful apoptotic stimulus to macrophages andneutrophil granulocytes. In this study, we provide a molecular analysis of phagocytosis-induced apoptosis. Apoptosis was blockedby Bcl-2 in a mouse macrophage cell line and in primary mouse macrophages. Analysis of the upstream mechanisms revealed thatapoptosis was triggered by the Bcl-2 homology domain 3-only protein Bim/Bod. Contact with bacteria or bacterial componentsinduced a strong increase in Bim-expression through TLR and MyD88. Inhibition of the MAPK p38 and JNK reduced bothup-regulation of Bim and apoptosis. Phosphorylation of Bim was further observed in mouse macrophages, which appeared to bethe result of TLR-dependent phosphatase inhibition. Although TLR-induced Bim was, unlike Bim in resting cells, not bound tothe microtubuli cytoskeleton, the up-regulation of Bim was not sufficient to cause apoptosis. A second signal was required that wasgenerated in the process of phagocytosis. Phagocytosis-induced apoptosis was strongly reduced in Bim�/� macrophages. Thesedata provide the molecular context of a form of apoptosis that may serve to dispose of terminally differentiated phagocytes. TheJournal of Immunology, 2005, 174: 671–679.

C ell death by apoptosis is a frequent event in infectiousdisease. Apoptosis may occur (or in some cases be in-hibited) in a cell that is either infected or otherwise

brought into close contact with the infectious agent. Additionally,the immune reaction involves proliferation and differentiationevents in several cell types that will also lead to changes in thesusceptibility to apoptosis.

Phagocytosis and intracellular destruction by specialized phago-cytes such as granulocytes and macrophages is an important de-fense mechanism against infections with pyogenic bacteria (suchas Streptococci and Enterobacteriaceae). Phagocytosis can occurupon recognition of conserved bacterial structures, of Abs or ofcomplement components that have been deposited on bacterial sur-faces, and phagocytosis is normally followed by the digestion ofthe phagocytosed bacterium by host cell enzymes. Bacterial de-struction is rapid and is concluded within a few hours. During thisprocess, massive phagocyte activation is seen as a result of thebinding of bacterial components to specialized receptors on thephagocyte. Various scavenger receptors are involved, but a largeshare of cell activation events is the result of the triggering ofreceptors from the TLR family, which play roles in the secretion of

cytokines, the enhanced expression of cell surface molecules, andin the process of phagocytosis itself (1–3).

We and others have recently demonstrated that phagocytosis ofpyogenic bacteria is also a stimulus for the phagocyte to undergoapoptosis (4, 5). In macrophages, apoptosis was observed at �8–16 hafter bacterial ingestion, clearly after the intracellular destruction ofthe bacteria, which is complete at �4 h postingestion. Although thesignificance of this form of apoptosis is not fully understood, it bearsresemblance to the apoptosis regularly found at the end of a T cellresponse: rather than developing back into resting cells, the terminallydifferentiated effector cells are cleared by apoptosis.

Apoptosis is implemented by a set of cellular enzymes referredto as effector caspases, most importantly caspase-3. Caspase-3 canbe activated by either caspase-8 (normally upon triggering of deathreceptors on the cell surface) or by caspase-9 (which is activated ina large signaling complex consecutive to the release of cytochromec from the cellular mitochondria into the cytosol (6). The initialcharacterization of “phagocytosis-induced apoptosis” showed thatit is indeed classical apoptosis (confirmed by analysis of variousparameters such as subG1-staining, annexin V-labeling, nuclearmorphology, blockade by caspase inhibition; Ref. 5). This form ofapoptosis further involved the activation of caspase-9 but notcaspase-8 and it was efficiently blocked by expression of the ap-optosis-inhibitor Bcl-2 in the mouse macrophage cell lineRAW264.7 (RAW) (5). In the study presented here, we undertookan analysis of the events that trigger this form of cell death.

The release of cytochrome c from mitochondria is regulated bymembers of the Bcl-2 protein family. Bcl-2 itself has a broad an-tiapoptotic effect that depends on its ability to bind a number ofproapoptotic Bcl-2 family members known as Bcl-2 homology do-main 3 (BH3)3-only proteins. BH3-only proteins are structurally

*Institute for Medical Microbiology, Immunology and Hygiene, Technical UniversityMunich, Munich, Germany; †Laboratory of Gene Regulation and Signal Transduc-tion, Department of Pharmacology, School of Medicine, University of California, SanDiego, La Jolla, CA 92093; and ‡Department of Pathophysiology, Innsbruck MedicalUniversity, Innsbruck, Austria

Received for publication June 15, 2004. Accepted for publication October 28, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Deutsche Forschungsgemeinschaft, SFB 576 (toS.K. and G.H.) and Ha 2128/8-1 (to G.H.).2 Address correspondence and reprint requests to Dr. Georg Hacker, Institute forMedical Microbiology, Immunology and Hygiene, Technical University Munich,Trogerstrasse. 9, D-81675 Munich, Germany. E-mail address: hacker@lrz.tum.de

3 Abbreviations used in this paper: BH3, Bcl-2 homology domain 3; BMDM, bonemarrow-derived macrophage; Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin; ODN, oligonucleotide; DC, dendritic cell.

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only distantly related to each other and to all other Bcl-2 familymembers. Unlike Bcl-2 and other relatives, they possess only theso-called Bcl-2 BH3-domain but lack the domains BH1, 2, or 4.Nine BH3-only proteins are known at present, and their activationis the earliest known molecular trigger of apoptosis in most cases(7). Activation of BH3-only proteins can involve de novo geneexpression as well as posttranslational modification such as de-phoshorylation or proteolytic cleavage. Two BH3-only proteins,Bim and Bmf, are also known to be activated by the release fromsites of sequestration at the cytoskeleton, Bim at the microtubuliand Bmf at the actin cytoskeleton.

Because phagocytosis-induced cell death was found to beblocked by Bcl-2, it was likely to involve the activation of BH3-only proteins. Therefore, we directed our attention at the variousBH3-only proteins that might transmit this death signal. Becausephagocytosis and its downstream events involve massive restruc-turing of the cytoskeleton, Bim and Bmf were the most obviouscandidates. Intriguingly, contact with bacteria or bacterial compo-nents caused a strong up-regulation of Bim protein that was me-diated by MyD88 and members of the MAPK family. However,the mere up-regulation was not sufficient for the induction of ap-optosis, which required a second signal that originated during theprocess of phagocytosis.

Materials and MethodsCell lines, bacteria, and stimulation of cells

RAW264.7 mouse macrophages were cultured in Low-Tox Click’s RPMI1640 (Biochrom) supplemented with 10% FCS (PAN), 50 �M 2-ME, andantibiotics (100 IU/ml penicillin G and 100 IU/ml streptomycin sulfate).Cells were normally grown in nonculture-coated petri dishes and only forexperiments seeded into culture-coated plates. Escherichia coli K12 strainDH5� was inoculated from a frozen stock into liquid LB medium andgrown overnight at 37°C with shaking. Bacterial cells were collected bycentrifugation, passed through a 5.0-�m disposable filter and resuspendedin PBS to an OD of 2. For heat inactivation, bacteria were then incubatedfor 30 min at 65°C, cooled to room temperature, and used for experiments.In some experiments, RAW cells were stimulated with LPS (1 �g/ml;Sigma-Adrich), CpG oligonucleotide 1668 (1 �M; TibMolBiol), Pam3Cys(1 �g/ml; EMC Microcollection Tubingen). The broad spectrum caspaseinhibitor z-VAD-fmk (Bachem) was used at a concentration of 50 �M.Apoptosis by UV irradiation was induced using a stratalinker 2400 (Strat-agene) at an energy dose of 160 mJ/cm2. The following kinase/phosphataseinhibitors were used: SB203580 (Alexis), PD98059 (Alexis), SP600125(Apotech), Wortmannin, and calyculin A (Calbiochem).

Generation of mouse bone marrow-derived macrophages(BMDM)

MyD88�/� mice were kindly provided by Dr. S. Akira (Department ofHost Defense, Research Institute for Microbial Diseases, Osaka University,Osaka, Japan), Bim�/� mice by Dr. A. Strasser (The Walter and Eliza HallInstitute of Medical Research, Melbourne, Australia), and vav-bcl-2 miceby Dr. J. Adams (The Walter and Eliza Hall Institute of Medical Research).Controls were age-matched C57BL/6 mice or wild-type litter mates.BMDM were generated according to standard protocols. Briefly, mousebone marrow was harvested by rinsing the femurs and tibiae. Bone marrowcells (2 � 107/10 ml) in a nonculture-coated petri dish were cultured in thepresence of 10 ng/ml recombinant mouse M-CSF (R&D Systems). On day3, another 10 ng/ml M-CSF was added. Adherent cells were harvested onday 8 or 9 by accutase treatment (PAA Laboratories), seeded in 12-wellcell culture plates (2 � 105/well), and used after overnight culture forexperiments as described for RAW cells.

Isolation of primary mouse granulocytes from bone marrow

Primary bone marrow-derived granulocytes from C57BL/6 wild-type andvav-bcl-2 transgenic mice were obtained by flow cytometric sorting ofGR1-positive cells from freshly isolated bone marrow as described (8).

Generation of bone marrow-derived dendritic cells (DC)

DC were generated with Flt3 ligand-supplemented bone marrow cell cul-tures as described (9) with slight modifications. Briefly, bone marrow cells

were flushed out of femurs and tibiae, centrifuged, and RBC were lysed for2 min with tris-ammonium chloride at 37°C. After washing, cells wereplated at 1.5 � 106 cells/ml in medium complemented with 35 ng/ml mu-rine Flt3 ligand (kindly donated by Dr. O’Keeffe (The Walter and ElizaHall Institute of Medical Research), and cultured at 37°C/5% CO2 for 8days. For TLR stimulation, 5 � 105 cells/200 �l were seeded in 96-wellplates and stimulated with LPS, Pam3Cys, or CpG-oligonucleotide (ODN)as indicated. A total of 1.5 � 106 cells (three wells combined)/sample werethen further processed for Western blotting as described below.

Generation of RAW-Bcl-2 cells/RAW Myd88-GyrB cells

RAW cells were transfected by electroporation with an expression plasmidof human Bcl-2 containing an endoplasmic reticulum target sequence (Bcl-2-cb5) under the control of the CMV promoter and a neomycin resistancecassette (10). Cells were selected in G418-containing medium, analyzed byintracellular Bcl-2 staining, and subcloned by limiting dilution. Bcl-2 ex-pression was confirmed by Western blotting and cytofluorometric analysis.Two subclones from originally independent clones stably expressing highlevels of Bcl-2 were chosen for the experiments.

RAW MyD88-GyrB cells were established by electroporation of RAWcells with an expression vector containing a neomycin resistance cassetteand an elongation factor-1� promoter-driven fusion construct of full-lengthmouse MyD88 and the coding sequence of the subunit B of streptomycesgyrase. Stable transfectants were established by limiting dilution in G418-containing medium. The GyrB-based dimerization system has been estab-lished by Perlmutter and colleagues (11). Dimerization of GyrB and GyrBfusion proteins is initiated by the bivalent antibiotic coumermycin A (CM).In the case of MyD88-GyrB, coumermycin A-driven dimerization ofMyD88 mimics receptor (TLR) -induced oligomerization of MyD88 andtriggers typical TLR-dependent effector functions like MAPK activationand secretion of TNF-�. A detailed analysis of this system and the de-scribed MyD88-GyrB RAW cells will be published elsewhere. The cDNAof GyrB was a kind gift from Dr. R. M. Perlmutter.

Coculture of macrophages and bacteria

2 � 105 RAW cells/ml were coincubated with 100 �l of bacterial suspen-sion for 1 h at 37°C. Cells were then washed once with PBS, seeded in12-well plates in 1 ml complete medium, and cultured for the indicatedperiods of time. In some experiments, RAW cells were seeded in tissueculture plates the day before (5 � 105 cells/10-cm plate or 2–5 � 105

cells/12-well plate) and stimulated as above. In case of kinase inhibition,cells were pretreated with the corresponding inhibitors for 15 min beforefurther stimulation.

Assays for apoptosis

For assessment of nuclear morphology, cells were stained with Hoechstdye (Sigma-Adrich), removed from the plate by vigorous pipetting, andscored in UV light under a fluorescence microscope. Assays were done atleast in duplicate as indicated and a minimum of 300 cells were counted persample. Analyses were performed by three investigators in independentexperiments, who obtained very similar results.

In some experiments cells were labeled with annexin V-FITC (BD Bio-sciences) according to the manufacturer’s instructions and analyzed byflow cytometry.

For assay of caspase 3 activity (Asp-Glu-Val-Asp cleavage activity), 1%Triton X-100 extracts prepared as described below were diluted 1/10 inreaction buffer (10 mM HEPES-KOH (pH 7), 40 mM �-glycerophosphate,50 mM NaCl, 2 mM MgCl2, 5 mM EGTA, 1 mM DTT) supplemented with100 �g/ml BSA) containing the caspase substrate acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin (Ac-DEVD-AMC; Bachem) at a finalconcentration of 10 �M. Reactions were performed in triplicate in flat-bottom 96-well plates at 37°C for 1 h. Free 7-amino-4-methyl-coumarinwas then measured by determining fluorescence at 390 nm (excitation) and460 nm (emission) in a Cytofluor 96 reader (Millipore). Values were cal-culated by subtracting background fluorescence.

Western blot analysis

Macrophages were washed once in PBS and harvested in extraction buffer(1% Triton X-100, 50 mM PIPES, 50 mM HEPES, 2 mM MgCl2, 1 mMEDTA) supplemented with 1 mM DTT and complete protease inhibitormixture (Roche). Nuclei and cellular debris were pelleted by centrifugationat 2000 � g for 10 min at 4°C. Aliquots of the supernatants were used fordetermination of protein concentration (Bio-Rad assay). Supernatants wereboiled in Laemmli buffer. Equal amounts of protein were loaded onto12.5% acrylamide gels and resolved by SDS-PAGE. Proteins were thentransferred onto nitrocellulose membranes. Membranes were probed with

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Abs specific for Bim (Sigma-Adrich), �-actin (Sigma-Adrich), �-tubulin(Sigma-Adrich), FoxO3a (Upstate Biotechnology). Secondary HRP-con-jugated anti-rabbit or anti-mouse IgG Abs were obtained from Dianova.Blots were developed using the ECL detection system (PerkinElmer). Fortreatment with calf intestinal phosphatase, cells were lysed in extractionbuffer and then treated with 20 U/50 �l of calf intestinal alkaline phos-phatase for 2 h at 37 °C. Controls were incubated in the same buffer butwithout phosphatase and in the presence of phosphatase inhibitors.

To obtain nuclear extracts, cells were incubated in buffer A (10 mMHEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT,10 mM NaF, 1 mM Na3VO4, and complete protease inhibitor mixture) onice for 15 min, then Nonidet P-40 was added to a final concentration of0.6%, extracts were vigorously vortexed and centrifuged at 6800 rpm for 2min at 4°C. The pellet containing the nuclei was washed in buffer A andresupended in buffer C (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA,1 mM DTT, NaF, Na3VO4, and complete protease inhibitor mixture) andfurther extracted with shaking for 30 min at 4°C. The supernatants obtainedafter centrifugation at 13,000 rpm for 20 min at 4°C in a microfuge wereused as nuclear extracts.

Subcellular fractionation

Subcellular fractionation was performed essentially as described (12).Briefly, cells were lysed in 1% Triton X-100 extraction buffer (1% TritonX-100, 50 mM PIPES, 50 mM HEPES, 2 mM MgCl2, 1 mM EDTA)supplemented with 1 mM DTT and complete protease inhibitor mixture(Roche). Lysates were treated with taxol (80 �M; Sigma-Adrich) and andapyrase (10 U/ml; Sigma-Adrich) for 15 min at 37°C. The lysate was thenloaded onto a 10% sucrose cushion and centrifuged at 40,000 rpm in aSW41TI rotor in a Beckman ultracentrifuge for 16 h at 4°C. Pellets con-taining the microtubule fraction were dissolved in Laemmli buffer, super-natants were concentrated by acetone precipitation. Samples were pro-cessed by Western blotting as described above.

Sucrose gradient centrifugation was performed by loading the taxol-treated extract on top of a 5–20% discontinuous sucrose gradient followedby ultracentrifugation in a SW41TI rotor at 40,000 rpm for 16 h. The pelletfraction was dissolved in Laemmli buffer, either 1-ml or 2-ml fractions ofthe supernatant were obtained and acetone precipitated. Fractions wereresolved by SDS-PAGE and subjected to Western blotting as describedabove.

ResultsBcl-2 protects macrophages and granulocytes againstphagocytosis-induced apoptosis

Our previous studies had indicated that Bcl-2 blocked apoptosiswhen expressed in cells from the macrophage cell line RAW264.7(RAW cells), suggesting the involvement of the “mitochondrialleg” of the apoptotic pathway (5). To confirm the involvement ofthe mitochondrial signaling pathway, we generated primary cellcultures from transgenic mice expressing human Bcl-2 throughoutthe hemopoietic compartment (vav-bcl-2 mice; Ref. 13). In pri-mary BMDM, phagocytosis of E. coli led to apoptosis in a largeportion of wild-type macrophages but much less in macrophagesfrom vav-bcl-2 mice. Similarly, high-level expression of humanBcl-2 protected RAW cells efficiently against phagocytosis-in-duced apoptosis (Fig. 1, A and B). Besides macrophages, neutro-phil granulocytes play an important role in the phagocytosis anddestruction of pyogenic bacteria. Granulocytes freshly isolated frommouse bone marrow were also efficient at taking up E. coli bacteria(data not shown). As in macrophages, uptake and digestion of bacteriainduced apoptosis in granulocytes (Fig. 1, C and D).

Expression of the BH3-only protein Bim is up-regulated duringcontact with bacteria

Although this is still somewhat controversial, Bcl-2 probably func-tions on a molecular level by binding and perhaps sequesteringproapoptotic BH3-only proteins (7). The efficient inhibition ofphagocytosis-induced apoptosis by Bcl-2 therefore suggested thatphagocytosis led to the activation of at least one BH3-only protein.The BH3-only proteins Bim and Bmf are, in their inactive state,sequestered to the cytoskeleton. Apoptotic stimuli can induce their

FIGURE 1. Phagocytosis-induced apoptosis of macrophages and gran-ulocytes. A, left, BMDM from C57BL/6 wild-type or vav-bcl-2 transgenicmice were seeded in 12-well plates (2 � 105/well) and then either incu-bated with E. coli bacteria or left untreated. After 1 h, bacteria were washedaway and cells were further incubated for 20 h. Apoptosis was measured byassessing nuclear morphology. f, Wild-type mice; �, vav-bcl-2 mice. Val-ues (mean/SD of duplicate samples of one mouse) are representative of atotal of four wild-type and four vav-bcl-2 transgenic mice. Right, RAWcells (f) or RAW-Bcl-2 cells (stably expressing human Bcl-2, �) weretreated and analyzed as under A. Values are representative of three exper-iments with two separate Bcl-2-expressing clones. B, RAW cells (f) orRAW-Bcl-2 cells (�) were treated with E. coli bacteria or left untreated asin A and 1% Triton extracts were prepared. Effector caspase activity wasmeasured as cleavage activity of the caspase substrate Ac-DEVD-AMC.Results were normalized by setting the highest value as 1. Values � SDwere calculated from duplicate samples (with triplicate measurement ofeach sample) and are representative of three experiments. C, granulocytes(freshly sorted from bone marrow) were exposed to E. coli bacteria or notfor 1 h, washed, and cultured for 24 h. Cells were fixed and nuclear apo-ptosis was measured as subG1-staining with propidium iodide and flowcytometric analysis. D, Granulocytes were exposed to E. coli bacteria ornot for 1 h, washed, and cultured under normal conditions. After 4 h andafter 22 h, aliquots were taken, stained with FITC-labeled annexin V, andanalyzed by flow cytometry. Gray lines, control cells; black lines, cellsincubated with E. coli. Annexin V-positive cells (cells under gate) were(mean/SD of triplicates) 4.7/0.29% (control) and 9.0/1.3% (E. coli) after4 h, and 15.2/1.9% (control) and 30.6/2.4% (E. coli) after 22 h.

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release from this site, upon which they translocate to the mito-chondria and cause the activation of Bax. Cytoskeletal structuresundergo massive rearrangements during the process of phagocy-tosis and the consecutive steps of vesicle transport (1). Bim andBmf were therefore considered likely candidates for the BH3-onlyproteins that are activated during phagocytosis-induced apoptosisand were next investigated. Bmf (bound to the actin cytoskeletonin its inactive state) was only weakly expressed in macrophages asjudged by Western blotting, and no induction of expression orrelease from its subcellular localization was observed upon bacte-rial stimulation (data not shown). Therefore, Bmf does not seem toplay a significant role in phagocytosis-induced apoptosis. The ex-pression of Bim by macrophages was next studied by Westernblotting. A number of isoforms of Bim have been described, ofwhich BimL and BimEL appear to be the most abundant in mostcell types (14). Evidence is accumulating that Bim can be regu-lated not only by release from the cytoskeleton but also by geneinduction (see for instance Refs. 15 and 16). Phosphorylation ofBim has further been described although the significance of this isunclear.

Both BimEL and BimL were easily detectable in resting RAWcells, with BimEL being the prominent isoform (Fig. 2). Upon stim-ulation with bacteria, an increase of Bim-protein expression wasdetectable after 4–6 h. High protein expression was sustained until16–20 h (Fig. 2A, and data not shown), at which time many cellshad already undergone apoptosis. Analysis of primary BMDMgave similar results (Fig. 2B; the size shift in Bim protein was dueto phosphorylation, see below). This up-regulation of Bim expres-sion due to bacterial stimulation correlated with apoptosis induc-tion by live bacteria. However, heat-inactivated bacteria, which aretaken up poorly and cause very limited apoptosis (5), were foundto have the same potential to enhance Bim expression (Fig. 2A).This suggests that the up-regulation of Bim expression is on itsown not sufficient for the efficient induction of apoptosis.

Induction of Bim expression is mediated by TLR signaling viaMyD88

A large part of the signaling events that bacterial components elicitin macrophages is mediated through TLR. Various TLR recognizedifferent components of microbial origin. To understand whetherTLR were involved in the induction of Bim upon contact withbacteria, we tested the effect of several TLR stimuli on the expres-sion of Bim in RAW cells. LPS (which stimulates TLR4), the

bacterial lipopeptide Pam3Cys (TLR2), and CpG-ODN (TLR9) allcaused up-regulation of Bim expression (Fig. 3).

The signal originating at TLR can be conveyed into the cells byseveral adaptor molecules. A large part of this signal is mediatedthrough the adaptor protein MyD88 (17). We found that MyD88was both necessary and sufficient for the induction of Bim. BMDMfrom MyD88�/� mice stimulated with LPS showed no discernibleup-regulation of Bim (Fig 3C). The shift in Bim caused by phos-phorylation was also MyD88-dependent as it did not occur inMyD88�/� cells (Fig. 3C).

The dependency of Bim induction on MyD88 function was fur-ther investigated using RAW cells stably expressing a MyD88 gy-rase B fusion protein. Addition of the cell-permeable dimeric li-gand of gyrase B, coumermycin, causes the dimerization of gyraseB and its fusion partner (18). Coumermycin treatment of RAWcells expressing the MyD88 gyrase B fusion protein causes theknown MyD88-dependent signaling events (see also Materials andMethods). This direct activation of MyD88 led to an up-regulationof Bim expression with similar kinetics as seen when using naturalTLR ligands (Fig. 3D). MyD88-dependent signals are thereforeboth necessary and sufficient to cause the up-regulation of Bim inmacrophages.

TLR-stimulation is a critical signal in the activation and matu-ration of DC. Therefore, we studied whether TLR-ligands alsocaused the up-regulation of Bim in mouse bone marrow-derivedDC. As shown in Fig. 3E, a clear up-regulation of Bim was alsoobserved in these cells during TLR-stimulation.

FIGURE 2. Expression of the BH3-only protein Bim is up-regulatedupon contact with bacteria. A, Detection of Bim by Western blotting inRAW cells treated with live or heat-inactivated E. coli bacteria. RAW cellswere seeded in 12-well plates (5 � 105/well) overnight and then eitherincubated with live or heat-inactivated E. coli bacteria for the indicatedperiods of time or left untreated. Cell extracts were analyzed by Westernblotting. C, Untreated control. Similar results were obtained in three inde-pendent experiments. B, BMDM from a C57BL/6 mouse were treated withlive E. coli and processed as above. Similar results were obtained in threeindependent experiments.

FIGURE 3. Induction of Bim expression is mediated by TLR signalingvia Myd88. A and B, RAW cells (5 � 105/well in 12-well plates) weretreated with LPS (1 �g/ml, A), bacterial lipopeptide (Pam3Cys, 1 �g/ml, B)or CpG-ODN (1 �M, B) for the indicated periods of time. Cells wereextracted and analyzed by Western blotting. Data are representative ofthree similar experiments. C, BMDM from either wild-type C57BL/6 miceor Myd88�/� mice were treated with LPS for the indicated periods of timeand further processed by Western blotting as above. A total of three micewere analyzed. D, RAW-K6 cells stably expressing a Myd88-GyrB fusionprotein were seeded as above and treated with 1 �M coumermycin for theindicated periods of time. Cells were processed as above. Typical results ofthree similar experiments are shown. E, Mouse bone marrow-derived DC(5 � 105 cells/200 �l in 96-well plates, 3 wells/sample) were stimulatedwith LPS (1 �g/ml), Pam3Cys (1 �g/ml), or CpG-ODN (1 �M), and an-alyzed as above.

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BimEL and BimL are phosphorylated upon TLR stimulation ofRAW cells

A shift of Bim protein to higher m.w. in SDS-PAGE was observedduring stimulation with whole bacteria as well as with TLR ligands(Figs. 2 and 3) indicating some form of posttranslational modifi-cation. A phosphorylation of Bim has been described earlier (19,20). To test whether the mobility shift we observed here was dueto phosphorylation, extracts from stimulated cells were digestedwith calf intestinal alkaline phosphatase. The mobility shift couldbe reversed by phosphatase treatment (Fig. 4A), indicating that theobserved modification of Bim was indeed phosphorylation. Anal-ysis of the kinetics showed that phosphorylation of BimEL wasrapid and complete while BimL was phosphorylated with similarkinetics but only partially (Fig. 4B). BimEL appeared to becomephosphorylated at more than one site as bands of different sizeswere observed, while the discrete bands for BimL suggest morelimited phosphorylation events (Figs. 2–4).

The MAPKs p38 and JNK are involved in regulation ofapoptosis and induction of Bim

TLR triggering causes a number of downstream signaling events,including the activation of MAPKs and the classical NF-�B-path-way. To assess the contribution of these signals to phagoytosis-induced apoptosis, various kinase inhibitors were used. Inhibitionof p38 MAPK led to reduction of apoptosis (close to 50% decreasecompared with the control) (Fig 5, A and B). A weak apoptosis-reducing effect was also seen when JNK was inhibited (Fig. 5A).In contrast, inhibition of the ERK-pathway or PI3K had no no-ticeable effect on apoptosis (Fig. 5A). Treatment of resting cellswith the inhibitors alone had no detectable apoptosis-inducing orcytotoxic effects (data not shown).

To understand the molecular background of this reduction inapoptosis by inhibition of p38 and JNK, the influence of kinaseinhibitors on Bim up-regulation and phosphorylation was studied.Intriguingly, inhibition of neither p38 nor JNK blocked TLR-de-pendent phosphorylation (data not shown) while blockade of ERK-activation had a borderline effect (Fig. 5C). However, inhibition ofp38 or (to a smaller extent) JNK but not of ERK-activation re-duced the up-regulation of Bim through TLR (Fig. 5D). Inhibitorsof Src kinase (PP2) or protein kinase C (staurosporine) affectedneither phosphorylation nor induction of Bim (data not shown).Treatment with proteasome inhibitors slightly increased phagocy-tosis-induced apoptosis without affecting Bim-phosphorylation orinduction, making a contribution from NF-�B unlikely (data notshown).

Because none of the tested kinase inhibitors had a pronouncedeffect on TLR-dependent phosphorylation of Bim, we considered

FIGURE 5. Inhibition of MAPK P38 or JNK reduces apoptosis and the up-regulation of Bim expression without affecting Bim phosphorylation. A, RAWcells were pretreated for 15 min with various kinases inhibitors specific for p38(SB203580), MAPKK1 (PD98059), JNK (SP600125), or PI3 kinase (wortman-nin), and then coincubated with E. coli for 20 h. Apoptosis was measured byassessment of nuclear morphology as above. Values represent mean/SD of dupli-cate samples. Similar results were obtained in three independent experiments. Theinhibitors alone did not induce apoptosis under these conditions (apoptosis rateswere (mean/SD of duplicates) control 1.89/0.44%, 10 �M SB203580 1.72/0.43%(experiment 1); control 2.06/0.57%, PD98059 10 �M 2.06/0.15%, SP600125 5�M 2.13/0.55% (experiment 2)). B, RAW cells were pretreated for 15 min withSB203580 at the indicated concentrations and coincubated with E. coli for 15 h.Apoptosis was quantified by annexin V/propidium iodide staining followed byflow cytometric analysis. Values give annexin V-positive, propidium iodide-neg-ative cells. Shown are typical results of three independent experiments. C, Effect ofMAPK inhibitors on Bim phosphorylation. RAW cells were pretreated with theindicated kinase inhibitors for 15 min and then stimulated with LPS for 1 h. Cellextracts were analyzed by Western blotting. Data are representative of at least threesimilar experiments. D, Effect of MAPK inhibitors on Bim induction. RAW cellswere pretreated with the indicated kinase inhibitors for 15 min and then stimulatedwith LPS for 16 h. Cell extracts were analyzed by Western blotting. Data arerepresentative of at least three similar experiments. E, Phosphatase inhibitioncauses phosphorylation of Bim in resting RAW cells. RAW cells were either leftuntreated or treated with 50 nM calyculin A for 1 h. Cells were lysed and analyzedby Western blotting as above. Typical results representative of three independentexperiments are shown. F, Nuclear translocation of the forkhead transcription fac-tor FoxO3a. RAW cells were in duplicates stimulated with LPS or not. Nuclearextracts were prepared and analyzed by Western blotting for FoxO3a (100 kDa).�, Nonspecific band. Data are representative of two independent experiments.

FIGURE 4. TLR-stimulation causes the phosphorylation of Bim. A, 1%Triton X-100 extracts of RAW cells stimulated as indicated were split andincubated either with calf intestinal alkaline phosphatase or in the samebuffer without phosphatase plus phosphatase inhibitors for 2 h at 37°C.Samples were analyzed by Western blotting. Results are representative ofthree similar experiments. B, RAW cells were stimulated with LPS (1�g/ml) for the indicated periods of time, lysed, and analyzed by Westernblotting. Shown are results representative of three similar experiments.

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the possibility that the effect of TLR-stimulation was not the in-duction of kinase activity but the reduction of phosphatase activity.We tested this hypothesis by treating resting RAW cells with thebroad-spectrum inhibitor of phosphatases calyculin A. As shownin Fig. 5E, this treatment caused a prominent shift of BimEL and,to a lesser extent, of BimL in unstimulated RAW cells. These datapoint toward the possibility that, in macrophages, Bim is con-stantly phosphorylated and dephosphorylated by as yet unidenti-fied enzymes.

The induction of Bim through extracellular signals has, in neu-rons and in T cells, been found to be under the control of themember of the forkhead transcription factor family, FoxO3a. Ourobservations so far indicate that in RAW cells, FoxO3a translo-cates into the nucleus upon TLR stimulation (Fig. 5F). This sug-gests that FoxO3a is also activated upon TLR-signaling and con-tributes to the induction of Bim also in macrophages.

Bim is activated upon phagocytosis-induced apoptosis

Because BimEL and BimL are normally sequestered to the dyneinmotor complex on the microtubuli cytoskeleton, transcriptional in-duction may not be sufficient for its activation but rather sensitizethe cell for a second, possibly independent, signal to apoptosis.Indeed, treatment of RAW cells with either heat-killed bacteria orLPS, both of which are only weak inducers of apoptosis under thisprotocol (Ref. 5, and see below) induced the expression of Bim ina way comparable to live E. coli bacteria. However, phagocytosisof live E. coli is a strong stimulus to undergo apoptosis. Thissuggested that Bim was induced but not activated by LPS or by

heat-killed bacteria. As release of Bim from microtubuli is knownto be involved in regulation of apoptosis, we next measured therelease of Bim from the microtubuli cytoskeleton in RAW cellsthat had phagocytosed E. coli and were undergoing apoptosis. Theattachment of Bim to microtubuli was assessed by density frac-tionation over a sucrose gradient. As shown in Fig. 6A (left), thegreat majority of Bim was found in the pellet fraction in untreatedcells, together with the marker of microtubuli, �-tubulin. However,upon phagocytosis of bacteria a significant portion of Bim wasfound in the fractions of lower m.w. indicating its release from thelarge microtubular complex (Fig. 6A, right). This shift was muchmore noticeable for BimL than for BimEL. Similar results wereobtained when cell lysates were separated over a 10% sucrosecushion: again BimL but only very little BimEL was found in thesupernatant where released Bim is expected (Fig. 6B). Surpris-ingly, LPS (which failed to induce apoptosis) also caused an ac-cumulation of BimL in the low m.w. fraction (Fig. 6B). Theseresults suggest that, although release of Bim from the cytoskeletonis a step in its activation, additional regulatory mechanisms existthat govern the induction of apoptosis by Bim.

These data suggest that TLR-signaling can increase the levels ofBim protein while a second stimulus is required to activate Bimand to cause apoptosis. To test this model, RAW cells were stim-ulated with LPS and then subjected to UV-irradiation, a stimulusthat is known to activate Bim (at least in human epithelial cells,Ref. 12). Prestimulation with LPS enhanced UV-induced apoptosisin an overadditive manner as predicted (Fig. 6, D and E). Analysisof Bim-release indicated that UV-irradiation caused the release of

FIGURE 6. Activation of Bim during phagocytosis and upon UV-irradiation. A and B, Release of Bim from microtubuli upon phagocytosis. Cells wereeither left untreated or treated with bacteria or with LPS for 16 h in the presence of z-VAD-fmk. 1% Triton cell extracts were prepared, and microtubuleswere polymerized with taxol. A, Samples were then fractionated by sucrose gradient centrifugation. Fractions of decreasing sucrose content and pelletfraction (P) were obtained, acetone-precipitated, and analyzed by Western blotting. B, Cell extracts of either untreated cells or cells stimulated with LPSor E. coli for 16 h were prepared as above and loaded on a 10% sucrose cushion. The microtubule fraction was pelleted by ultracentrifugation and thenonsedimented fraction was precipitated. Samples were separated by SDS-PAGE analyzed by Western blotting. Data are representative of three independentexperiments. C, Translocation of Bim to low m.w. fractions upon LPS and UV-treatment. RAW cells were stimulated with LPS for 22 h or left unstimulated.Cells were then UV-irradiated and harvested after an additional 8 h. Cell extracts were prepared as above and fractionated by sucrose gradient centrifugation.The pellet fraction P and fractions of decreasing sucrose content were obtained, actetone-precipitated, and analyzed by Western blotting. Similar resultswere obtained in three independent experiments. D and E, TLR stimulation sensitizes RAW cells for apoptosis-induction by UV-irradiation. D, RAW cellswere stimulated with LPS for 22 h or left unstimulated. Cells were then UV-irradiated, and apoptosis was measured after an additional 8 h by assessmentof nuclear fragmentation. Values are mean/SD of at least duplicate samples. Shown are typical results of three similar experiments. E, RAW cells werestimulated as in D and extracted in 1% Triton X-100. Effector caspase activity was measured as cleavage activity of the caspase substrate Ac-DEVD-AMC.Results were normalized by setting the highest value as 1. Data are presented as means � SD of free 7-amino-4-methyl-coumarin generated by one lysateof RAW cells. Similar results were obtained in three separate experiments.

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at least some Bim-protein in RAW cells (Fig. 6C). LPS-stimula-tion again up-regulated the expression of Bim, and the combina-tion of LPS and UV increased the proportion of free Bim (Fig. 6C).Taken together, these results indicate that TLR-stimulation leads tothe up-regulation of Bim in macrophages, and although this Bim isnot firmly attached to the microtubular structure it does not acti-vate the apoptotic pathway until a second signal is provided. Asdescribed previously, heat-inactivated bacteria cause in RAW cellsall activation events investigated but fail to induce apoptosis (5).The only apparent difference is that these bacteria are taken upvery poorly by the macrophages. Furthermore, Yersinia, whichpossess virulence factors that prevent uptake by phagocytes (21)stimulate RAW cells but also fail to induce apoptosis (our unpub-lished data). These data suggest that phagocytosis itself (or eventsconsecutive to and triggered by phagocytosis) provides the secondstimulus that activates Bim.

Bim�/� mice are protected against phagocytosis-inducedapoptosis

To confirm the role of Bim in phagocytosis-induced apoptosis,BMDM from Bim-deficient mice were tested. Bim�/�-BMDMwere as efficient as BMDM from normal mice at phagocytosing E.coli bacteria as assessed with GFP-expressing bacteria by flowcytometry (data not shown). However, the induction of apoptosismeasured as both nuclear fragmentation and the binding of an-nexin V/uptake of propidium iodide was strongly reduced in Bim-

deficient cells as compared with wild-type cells (Fig. 7). Therefore,it is clear that Bim acts to transmit the apoptotic signal of phago-cytosis-induced apoptosis, a mechanism that may act to kill phago-cytes after they have fulfilled their function.

DiscussionPhagocytosis and intracellular digestion of bacteria is a principaleffector function of cells of the innate immune system (1). We andothers have shown that, consecutive to the process of phagocyto-sis, macrophages and granulocytes die by apoptosis (4, 5). In thisstudy, we provide the molecular context for this form of apoptosis.In macrophages dying by “phagocytosis-induced cell death,” theBH3-only protein Bim is activated and makes a major contributionto this form of apoptosis. At the same time, Bim is up-regulatedand phosphorylated via a signal pathway originating from TLR.

TLR have over the past few years been recognized as arguablythe most important sensors of microbial presence within the im-mune system. Upon recognition of conserved molecular structuresby TLR, adaptor molecules convey a signal into the cell that causesa broad line of downstream events such a MAPK activation, ac-tivation of NF-�B and the expression of a large number of genes.Evidence has been presented that TLR-signaling can act as a mod-ulator of apoptosis, although the derived models are not entirelyconsistent with each other. On one hand, TLR-induced NF-�B-activity probably has an antiapoptotic effect (22). On the otherhand, TLR2 and TLR4 have been described to have the potentcapacity to induce apoptosis, and both adaptor molecules MyD88and TRIF have been found independently to transmit this signal(23, 24). We found that MyD88-dependent signaling was a pow-erful stimulus to up-regulate the expression of Bim. The strongestdetectable contribution to this increase in Bim expression camefrom the MAPK p38, and the inhibition of p38 reduced phagocy-tosis-induced apoptosis. Although the inhibitor used (SB203580)may also, to a smaller degree, affect JNK, an inhibitor with greaterspecificity toward JNK (SP600125) had a smaller effect, suggest-ing that p38 is the kinase more important for up-regulation of Bim.It is therefore conceivable that the contribution from p38 consistsin the induction of Bim.

BH3-only proteins like Bim are likely to be involved in themajority of instances of apoptosis (7). Bim has been recognized tobe responsible for or at least to contribute to apoptosis upon UV-irradiation, taxol treatment, and anoikis (cell death upon loss ofintercellular contact) but also to cell death as a consequence offactor withdrawal in neurons and during the contraction phase of aT cell response when Ag-activated T cells die by apoptosis (12, 16,25–27). Notably, Bim-deficient mice have increased cell numbersnot only of the lymphatic but also of the myeloid blood cell lineage(for instance, blood monocyte and spleen granulocyte levels areincreased by a factor of �3 (28), suggesting that Bim is involvedin lifespan-defining apoptosis in granulocytes and macrophages.The induction of Bim through TLR in macrophages and DC maytherefore be one determinant of the susceptibility to apoptosis inthese cells. However, the mere induction is not sufficient to causeapoptosis. This is what one would expect: the straight induction ofapoptosis through TLR upon encounter of microbial componentswould not be productive and indeed does not take place (as evi-denced by a host of studies in vitro). Such sensibilization of amacrophage or a DC to a later apoptotic stimulus (such as growthfactor withdrawal) could well serve to regulate survival in the in-nate immune system. Little is known about life and death of thesecells. In addition to the cellular shifts in Bim�/� mice mentioned,it has been found that Bcl-2 expression enhances the number of

FIGURE 7. Bim�/� mice are protected against phagocytosis-inducedapoptosis. A, BMDM from wild-type or Bim�/� mice were seeded in 12-well plates (2 � 105 cells/well) and exposed to E. coli for 20 h. Apoptosiswas quantified by assessing nuclear morphology. f, Control-treated; �, E.coli-treated samples. The data shown are representative of four indepen-dent experiments that included a total of six wild-type and six Bim�/�

mice. B, BMDM were stimulated as above, and apoptosis was quantified byannexin V/propidium iodide staining followed by flow cytometric analysis.Results obtained from one wild-type and one Bim�/� mouse are shown andare representative for a total of four wild-type and four Bim�/� mice an-alyzed. Numbers give percentages of cells in quadrants.

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DC in vivo (29). Therefore, apoptosis appears to regulate ho-meostasis in this compartment, and the up-regulation of Bimthrough TLR may contribute to this.

Originally, the regulation of Bim was described to occur by therelease from the microtubuli cytoskeleton upon a trigger from anumber of apoptotic stimuli. Several recent studies have suggestedthat phosphorylation may also be involved but the physiologicalimportance of this is still not clear. Direct phosphorylation of Bimby JNK has been shown in vitro and has been proposed to be a wayby which Bim is released (20, 30), and ERK-mediated phosphor-ylation of BimEL has been suggested to cause its turnover by en-hancing proteasomal destruction (31, 32). During MyD88-medi-ated TLR-signaling, the MAPK p38, JNK and ERK, are all rapidlyactivated (33, 34). However, only the inhibition of ERK activationhad a detectable (albeit small) inhibitory effect on Bim phosphor-ylation. Intriguingly, the phosphatase inhibitor calyculin A (abroad-spectrum inhibitor with preference for serine/threoninephosphatases) caused the rapid phosphorylation of Bim. Thispoints toward the possibility that, rather than being phosphorylatedby kinases activated during TLR-signaling such as MAPK, Bim isconstantly phosphorylated and dephosphorylated by constitutivelyactive enzymes. In this scenario, TLR-mediated phosphatase inhi-bition may be the reason for the appearance of phosphorylatedforms of Bim. The biological significance of this balance of mod-ifications remains to be determined but may involve aspects like achange in activity (perhaps by modifying the ability to translocateto mitochondria or to activate Bax) or stability of the protein.

As at least one important step in Bim activation, the protein isreleased from the microtubuli cytoskeleton (12). This release isprobably not a result of direct Bim targeting by the apoptotic pro-cess, as the dynein L chain LC8 (which directly binds Bim andattaches it to the dynein motor complex) is also released in theabsence of Bim (12). It was surprising that the release of BimL andBimEL appeared different upon phagocytosis, in that a much largerportion of BimL was found in the fraction unbound to microtubulithan of BimEL. Because both isoforms are probably bound viaLC8, it is not obvious how their release is differentially regulated.The binding of the two isoforms may lead to an association withdifferent microtubuli substructures, or the LC8-release machinerymay be differentially affected by the isoforms. It is also possiblethat additional as yet unidentified interaction sites of BimEL existwith cytoskeletal components.

One observation of our study that is molecularly unexplained isthat LPS generates free Bim in a way similar to the phagocytosisof bacteria but is much less active at inducing apoptosis. The sim-plest model predicts that the release from its site of sequestrationis sufficient for Bim to proceed and cause Bax to become activatedand to translocate to mitochondria. Although Bim is clearly up-stream of Bax and somehow causes its activation, direct associa-tion between Bim and Bax is at least uncertain. A number of reg-ulatory steps may exist that govern the activity of Bim even afterit has been released, and phagocytosis vs TLR-signaling may dif-ferentially impact on these steps. The finding that BimL andBimEL, which have similar affinities to LC8, have different apop-tosis-inducing activities upon overexpression (BimL is a more po-tent killer, Ref. 14), supports the model of such additional regu-latory steps. It is further possible that the reduction of apoptosis byp38 inhibition impacts at this step, as p38-activity is known to beinvolved in microtubular processes. Another possibility is that,during TLR-stimulation in the absence of phagocytosis, the tran-scriptionally induced Bim does not reach microtubules/LC8, itssite of sequestration. Under nonapoptotic conditions, it must some-how be made sure that de novo synthesized Bim is not active while

assembling with microtubuli, and TLR-induced Bim may still be atthat stage.

The evidence is clear that, upon uptake and subsequent to deg-radation of bacteria, mouse phagocytes die by apoptosis. The phys-iological importance of this process has not been determined, butthere are at least three aspects that should be taken into accountwhen considering its relevance. First, although especially granu-locytes are very short lived in any case, activation of the cells uponcontact with bacteria is potentially harmful, as production and un-controlled release of mediators of inflammation by the activatedgranulocyte may cause unnecessary inflammation that may be pre-vented by apoptosis and subsequent uptake of the apoptotic cell byother phagocytes. Second, apoptotic cells are rapidly cleared invivo, at least in part by uptake through DC, and this uptake hasbeen shown to be able to initiate a T cell response to microbial Agcontained within the apoptotic cell (35, 36). Phagocytosis-inducedapoptosis may therefore be relevant as a means of conveying bac-terial Ag to the adaptive immune system. Third, it appears as aplausible speculation that apoptosis is the “normal” way of dis-posing of terminally differentiated cells that have fulfilled theirfunction. Numerous studies demonstrate that such is the fate ofactivated T cells: resting T cells become activated and T effectorcells die at the end of an immune response in a Bim-dependentmanner (27). Although this to our knowledge has not been ex-plored, it appears plausible that the same principle applies tophagocytes. During and upon phagocytosis, these cells becomemaximally activated and fulfill their function in the disposal ofbacteria. Consecutive to that, the cells could either regress to be-come resting macrophages or, alternatively, die by apoptosis. Theresults shown in this study suggest the latter: that activation ofphagocytes is a one-way road leading to Bim-dependent apoptosis.

AcknowledgmentsWe thank Drs. S. Akira, A. Strasser, and J. Adams for the kind gift ofgenetically modified mice. We thank C. Hilpert for expert technicalassistance.

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