Phagocytosis of ApoptoticInflammatory Cells by Microglia andModulation by Different Cytokines:

Mechanism for Removal of Apoptotic Cellsin the Inflamed Nervous System

ANDREW CHAN, TIM MAGNUS, AND RALF GOLD*Department of Neurology, Clinical Research Group for Multiple Sclerosis and Neuroimmunology,

Julius-Maximilians University, Wurzburg, Germany

KEY WORDS T-cell apoptosis; multiple sclerosis; experimental autoimmune en-cephalomyelitis; Th1; Th2

ABSTRACT Apoptosis of autoaggressive T cells in the central nervous system (CNS)is an effective, nonphlogistic mechanism for the termination of autoimmune inflamma-tion in experimental autoimmune encephalomyelitis (EAE). The clearance of apoptoticleukocytes by tissue-specific phagocytes is a critical event in the resolution of theinflammatory attack. To investigate the role of microglia in the removal of apoptotic cellsand potential regulatory mechanisms of microglial phagocytosis, an in vitro phagocyto-sis assay was established, using Lewis rat microglia. Microglia exhibited a high capacityfor the uptake of apoptotic autologous thymocytes, as well as apoptotic encephalitogenicmyelin basic protein (MBP)-specific T cells, in contrast to nonapoptotic target cells.Pretreatment of microglia with interferon-g (IFN-g) raised the proportion of microgliacapable of phagocytosing apoptotic cells to 75% above the untreated controls. Theincreased phagocytic activity was selective for apoptotic target cells and was not depen-dent on phosphatidylserine-mediated recognition mechanisms. In contrast, preincuba-tion of microglia with interleukin-4 (IL-4) inhibited the uptake of apoptotic cells,whereas tumor-necrosis factor-a (TNF-a) and transforming growth factor-b (TGF-b) didnot alter phagocytosis. Phagocytic clearance of apoptotic inflammatory cells by microgliamay be an important mechanism for the termination of autoimmune inflammation inthe CNS. Augmentation of microglial phagocytosis by the Th-1-type cytokine IFN-gsuggests a feedback mechanism for the accelerated clearance of the inflammatoryinfiltrate in the CNS. GLIA 33:87–95, 2001. © 2001 Wiley-Liss, Inc.

INTRODUCTION

Elimination of inflammatory cells by apoptotic celldeath plays a major role in the resolution of inflamma-tory reactions in the nervous system. Active downregu-lation of inflammation by apoptosis, rather than seclu-sion from the systemic immune surveillance, appearsto be responsible for the “immune-privileged” status oftissues with specific immune-defense mechanisms suchas the eye or the central nervous system (CNS) (Gold etal., 1997; Griffith and Ferguson, 1997). A high degree ofapoptosis of pathogenic, inflammatory T cells has been

described in experimental autoimmune encephalomy-elitis (EAE) (Pender et al., 1991; Schmied et al., 1993;Bauer et al., 1998), a model of organ-specific autoim-munity in the CNS that serves as a paradigm for some

Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: DFG Go459/8-1); Grant sponsor: state of Bavaria.

*Correspondence to: Ralf Gold, Neurologische Universitatsklinik, Josef-Schneider-Strasse 11, D-97080 Wurzburg, Germany.E-mail: r.gold@mail.uni-wuerzburg.de

Received 21 June 2000; Accepted 14 September 2000

GLIA 33:87–95 (2001)

© 2001 Wiley-Liss, Inc.

aspects of the human disease multiple sclerosis (MS).In susceptible animals, EAE can be actively inducedeither by immunization with myelin antigens or by theadoptive transfer of activated, autoantigen-specificCD41 T lymphocytes (Gold et al., 2000). In the Lewisrat, EAE follows a monophasic, self-remitting diseasecourse with the peak of T-cell apoptosis that occursduring clinical recovery, in which 30–50% of the infil-trating T cells exhibit morphological signs of apoptosis(Schmied et al., 1993; Gold et al., 1997). Although theextent and the time course of T-cell apoptosis havebeen extensively investigated in different EAE models(Pender et al., 1991; Schmied et al., 1993; Bauer et al.,1998), little is known about the fate of the inflamma-tory cells once they have undergone the apoptotic celldeath program.

A key event in the resolution of an inflammatoryinfiltrate is the nonphlogistic, and thus safe, phagocyticclearance of dying, yet intact, leukocytes undergoingapoptosis (reviewed in Platt et al., 1998; Ren andSavill, 1998). In vivo, the rapid recognition, uptake,and degradation by professional phagocytes (e.g., mac-rophages) or neighboring cells acting as semiprofes-sional phagocytes is the common fate of cells dying byapoptosis. The fast and efficient phagocytic clearance ofapoptotic cells limits direct tissue injury by preventingthe spilling of potentially harmful contents of dyingcells and by inhibiting secondary immune responses tomoieties leaking from apoptotic cells or to apoptoticsurface structures (Savill, 1997; Ren and Savill, 1998).In addition, the ingestion of apoptotic cells also activelysuppresses the secondary generation of secretory in-flammatory mediators and proinflammatory cytokines(Stern et al., 1996; Voll et al., 1997; Fadok et al.,1998a).

The high degree of T-cell apoptosis in situ duringclinical recovery from EAE underscores the need for anefficient disposal mechanism in the CNS. Also, localmechanisms that tightly regulate the phagocytic capac-ity for apoptotic cells during the resolution of the in-flammatory reaction must be active (Ren and Savill,1995; Gold et al., 1997). In this study, we investigatedthe role of microglial cells as the principal immune cellsof the CNS (Perry, 1998; Becher et al., 2000) in thephagocytosis of apoptotic cells and its regulation bydifferent cytokines. Using an in vitro phagocytosis as-say, our data demonstrate that microglia has a highcapacity for the phagocytosis of apoptotic thymocytes,as well as encephalitogenic, CNS autoantigen-specificT cells. Enhancement of microglial phagocytosis by theT-helper 1-cytokine interferon-g (IFN-g) may consti-tute a feedback mechanism for the efficient clearance ofapoptotic inflammatory cells in the CNS.

MATERIALS AND METHODSMaterials

All cell culture media and supplements were ob-tained from Gibco/BRL (Eggenstein, Germany), unless

otherwise noted. Recombinant rat cytokines were fromR&D (Minneapolis, MN). Annexin V and trypsinizedpeptide fragments were a kind gift of Blake Pepinsky,Biogen (MA). Bovine annexin V was purified from bo-vine lung with minor modifications to the previouslypublished procedure (Pepinsky et al., 1988). Fortrypsinization, the protein was treated with 3% (w:w)trypsin (Promega, Madison, WI) for 24 h at 37°C. Tryp-sin was added in three aliquots at time 0, after 4 h, andafter an additional 4 h, and the extent of digestionassessed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). Phospho-L-serine andphospho-D-serine were from Sigma (Deisenhofen, Ger-many). Mafosfamide cyclohexylamine was a generousgift by ASTA Medica, Frankfurt/Main, Germany.

Isolation of Lewis Rat Microglial Cells

Rat microglial cells were isolated from primarymixed brain glial cell cultures using a modification of apreviously described method (Giulian and Baker, 1986;Zielasek et al., 1992). In brief, brains from neonatalLewis rats (P0–P2, Charles River, Sulzfeld, Germany)were stripped off their meninges and minced with scis-sors under a dissecting microscope (Wild, Heerbrugg,Switzerland) in phosphate buffer, pH 6.5, containing20 g/L sucrose and 1 g/L glucose. After trypsinization(0.5% trypsin, 10 min, 37° C, 5% CO2), the tissue wasdissociated by trituration. The cell suspension waswashed in culture medium (basal medium Eagle’s[BME] supplemented with 10% fetal calf serum [FCS];Sigma; 50 U/ml penicillin, and 50 mg/ml streptomycin)and cultured at 37°C/5% CO2 in 75-cm2 plastic flasks(Primaria, Falcon, Franklin Lakes, NJ). One-half of themedium was changed after 6 h in culture and twiceweekly thereafter, starting on day 3, for a total culturetime of 9–14 days. Microglial cells were shaken off theprimary mixed brain glial cell cultures (150 rpm, 37°C,7 h) with maximum yields between days 9 and 14. Afteradhesion to the surface of an FCS-coated culture flask(1 h, 37°C, 5% CO2) the nonadherent cells were rinsedoff and the adherent cell fraction was resuspended byvigorous, brief shaking after trypsinization. Cells werethen washed in culture medium and seeded on 48-wellplates (Corning, Corning, NY). An aliquot of the cellswas incubated for 24 h in Labtek 8-well-chamber slides(Miles Scientific, Naperville, IL), fixed (4% PFA/PBS,12 min, RT) and permeabilized (0.025% NP-40/PBS, 20min, RT). The purity of microglial cells was then con-firmed by immunohistochemistry, using the monoclo-nal antibody ED1 (1:50, Serotec, Kidlington, UK) andthe polyclonal anti-glial fibrillary acidic protein(GFAP) antiserum (1:100, Dako, Hamburg, Germany)according to previously published protocols (Zielasek etal., 1992). Microglia with a purity of consistently .95%was used for the phagocytosis assay.

88 CHAN ET AL.

Preparation of Autologous ApoptoticThymocytes

To obtain apoptotic target cells freshly prepared au-tologous Lewis rat thymocytes were treated with 0.1mg/ml methylprednisolone (Aventis, Frankfurt amMain, Germany) for 5 h, 37°C/5% CO2 as described byChan et al. (1998). Thymocytes kept on ice for 5 h afterpreparation or freshly prepared thymocytes were usedas nonapoptotic control cells. For flow cytometric quan-tification of apoptotic and necrotic changes, cells weredouble-stained using fluorescein-conjugated annexin V(Roche Diagnostics Boehringer-Mannheim, Mann-heim, Germany) and propidium iodide (PI); 5 3 105

cells were suspended in 100 ml binding buffer (10 mMHepes-NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) andincubated for 15 min on ice with 2.5 ml/ml fluorescein-conjugated annexin V and PI according to the manu-facturer’s instructions and Chan et al. (1998) andVermes et al. (1995). Analyses were performed on aFACScan, using Cell Quest software (Becton Dickin-son, San Jose, CA). At each time point, 5,000 cells wereanalyzed. Cell debris was excluded by setting appropri-ate light scatter gates and quantitative analysis wasdone by setting region markers for positive cells in theappropriate channels (Chan et al., 1998). The propor-tion of viable cells as assessed by Trypan blue exclusionwas consistently .97%.

Preparation of Apoptotic Myelin BasicProtein-Specific T Cells

The generation of the myelin basic protein-specificT-cell line MBP 13 has been reported in detail (Chan etal., 1999). This CD41 Lewis rat T-cell line is specific forthe dominant encephalitogenic MBP epitope spanningamino acids (aa) 68–88 of guinea pig MBP, complexedto RT1.B1 class II MHC molecules. In brief, the T-cellline was established from lymph nodes of female Lewisrats immunized in the hind footpad with 100 mg ofMBP emulsified in complete Freund’s adjuvant (CFA)(Difco, Detroit, MI). All immunizations were performedaccording to Bavarian state regulations. Antigen-spe-cific T cells were selected by repeated cycles of propa-gation in medium with 7.5% supernatant of concanava-lin A (Con A)-treated mouse spleen cells and 5% FCS,followed by antigen-specific restimulation using irradi-ated thymic antigen-presenting cells. T cells were thenpropagated in interleukin-2 (IL-2)-containing medium.In parallel experiments, aliquots of the same cell prep-arations used for phagocytosis experiments were en-cephalitogenic, producing clinically mild to severeadoptive transfer EAE. For induction of apoptosis, cellswere cultured in the absence of T-cell growth factorswith the addition of 0.1 mg/ml methylprednisolone for3 h at 37°C/5%CO2. The proportion of viable cells asassessed with Trypan blue was .97%.

In Vitro Phagocytosis Assay

We used a modification of a previously described,microscopically quantified in vitro phagocytosis assayof uptake of apoptotic cells (Fadok et al., 1992a,b; Savillet al., 1989a,b, 1992); 400 ml of a 0.75 3 106/ml suspen-sion of microglial cells per well was seeded in 48-wellplates (Costar) and cultured overnight at 37°C/5% CO2.Triplicate wells of microglial cells were then incubatedwith the respective concentrations of cytokines or cul-ture medium for 24 h. For heat inactivation, the respec-tive cytokines were boiled for 10 min in a water bath.After the cells were washed with basal medium Eagle’s(BME), 500 ml of a 20 3 106/ml suspension of apoptoticthymocytes or nonapoptotic control cells was added toeach well resulting in a ratio of microglia to target cellsof approximately 1:30. In the case of MBP-specific Tcells (T-cell line MBP 13), 500 ml of a 10 3 106/mlsuspension of apoptotic or nonapoptotic cells wasadded, respectively. Pilot experiments had shown nofurther increase in the proportion of phagocytosing mi-croglia when the amount of apoptotic target cells wasraised. Microglia and the target cells were coculturedat 37°C/5% CO2 for 2 h, and the interaction was termi-nated by vigorous washing of the wells with cold PBS(4°C). The microglial cell monolayer was thentrypsinized, and a separate cytocentrifuge preparationwas prepared for each well. The washing step andsubsequent trypsinization dislodged target cells thatwere attached, but not ingested (Savill et al., 1989b,1992). The cytocentrifuge preparations were stainedwith May-Giemsa (Merck, Darmstadt, Germany). Theproportion of microglial cells containing apoptotic cellsand the amount of phagocytosed target cells werecounted by light microscopy, with a minimum of 500cells per slide counted in a blinded fashion. The resultwas expressed as the percentage of the mean of thecontrol wells for that particular experiment. In someexperiments, data were also given as the phagocyticindex, which reflects the phagocytic capacity of individ-ual microglial cells and is derived by multiplication ofthe percentage of phagocytosing microglia by the aver-age number of ingested target cells per microglia(Fadok et al., 1992b). For inhibition experiments withphospho-L-serine and phospho-D-serine, apoptotic thy-mocytes were preincubated with the reagents dilutedat 1 mM and 2 mM in BME for 15 min at 4°C. Apoptoticthymocytes were then co-incubated with microglia inBME in the presence of 1 mM or 2 mM phospho-L-serine or phospho-D-serine, respectively. For inhibitionexperiments with purified bovine lung annexin V, ap-optotic thymocytes were preincubated (15 min, 4°C)with 1mM annexin V or trypsinized control peptidefragments in 10 mM Hepes/NaOH, pH 7.4, 10 mMNaCl, 5 mM CaCl2. After washing, apoptotic thymo-cytes were then interacted with microglia in BME con-taining 1mM annexin V or the respective trypsinizedpeptide control.

89MICROGLIAL PHAGOCYTOSIS OF APOPTOTIC CELLS

Statistical Analysis

All values are expressed as mean 6SEM. Statisticalsignificance (defined as P , 0.05) was evaluated usingStudent’s t-test (Graph Pad Software, San Diego, CA).

RESULTSMicroglial Phagocytosis of Apoptotic

Autologous Thymocytes

The method used is a modification of a standardizedassay of the uptake of apoptotic cells, which has beenemployed on different cell types and has been describedand illustrated in detail before (Newman et al., 1982;Fadok et al., 1992a,b; Savill et al., 1989a,b, 1992).Moreover, using peritoneal macrophages in our assaysystem, engulfment and subsequent uptake of apopto-tic thymocytes could also be demonstrated by confocalmicroscopy (data not shown). Figures 1A and 3A/Bdepict the typical light microscopic appearance of mi-croglial phagocytosis of apoptotic, glucocorticosteroid-treated autologous thymocytes. While usually phago-cytosing microglia with one or two ingested apoptoticcells were observed, occasionally multiple phagocy-tosed apoptotic cells and rarely microglial cells withmore than five phagocytosed thymocytes were detected(Fig. 1A). After 2-h interaction, different stages of ap-optotic morphology of the phagocytosed target cells werevisible, with nuclear chromatin condensation, apoptoticbodies, and digested thymocytes (Figs. 1A, 3A/B).

For each phagocytosis experiment performed, thetarget cells offered for microglial uptake were analyzedfor signs of cell death by cytofluorometry with annexinV and PI. Apoptotic, cortisone-induced thymocytesused as target cells had a proportion of 48.3 6 1.5%(mean 6SEM) annexin V-positive cells with 7.7 6 0.8%PI-positive cells. Nonapoptotic control thymocytesstored on ice for 5 h after preparation used as negativecontrols showed 9.8 6 0.6% annexin V-positivity and aproportion of 3.2 6 0.4% PI-positive cells. The propor-tion of microglial cells phagocytosing cortisone-treatedapoptotic thymocytes varied between different experi-ments (47.0 6 4.8% (mean 6SEM), range 32.8–67.9%)as is known from other phagocytosis systems of apo-ptotic cells (Savill et al., 1989b). When the phagocytosisrate was expressed as the percentage of the mean of thecontrol replicates in that experiment, a high reproduc-ibility was found (Fig. 1B), as described previously(Hughes et al., 1997; Savill et al., 1989b). Microgliathat were co-incubated with autologous thymocyteswere able to discriminate between apoptotic and non-apoptotic target cells (Fig. 1B). The phagocytosis ratefor nonapoptotic thymocytes reached only 51.1 6 4.7%(mean 6SEM) of the apoptotic control cells (P ,0.0001). Microglial phagocytosis of nonapoptotic thy-mocytes stored on ice for 5 h reached essentially thesame background phagocytosis level as for freshly pre-pared thymocytes used as target cells with 6.5% and7.4%, respectively of the total microglia phagocytosing.

Microglial Phagocytosis of ApoptoticEncephalitogenic MBP-Specific T Cells

To investigate whether also CNS autoantigen-spe-cific, encephalitogenic T cells undergoing apoptosis arephagocytosed by microglia, the MBP-specific Lewis rat

Fig. 1. A: Lewis rat microglial cell phagocytosis of autologous cor-tisone-induced apoptotic thymocytes. Photomicrograph after 2-h in-teraction of microglial phagocytes and autologous apoptotic thymo-cytes, followed by washing with cold phosphate-buffered saline (PBS)trypsinization to detach microglia from the plate and to dissociateattached, but noningested, target cells; cytocentrifuge preparation;and staining with May-Giemsa. The nuclei of the ingested thymocytesshow typical apoptotic morphology with condensed chromatin. Manyapoptotic cells (arrowheads) are found within phagolysosomes sur-rounded by a characteristic halo (arrow). B: Microglial phagocytosis ofautologous thymocytes. Microglial phagocytosis rate is given as per-centage of the mean of controls (1) 1SEM. Microglia has a highcapacity for the uptake of cortisone-treated, apoptotic (1) vs. non-apoptotic (2) thymocytes (***P , 0.0001). In the experiments shown,47 6 4.8% (range 32.8–67.9%) of the total microglia were capable ofphagocytosing cortisone-treated apoptotic thymocytes. Three inde-pendent, representative experiments, each performed in triplicate.Scale bar 5 10 mM in A.

90 CHAN ET AL.

T-cell line MBP13 was used as target for phagocytosis.MBP-specific T cells induced to undergo apoptosis byT-cell growth factor deprivation and addition of glu-cocorticosteroids for 3 h showed 31.6 6 1.4% annexinV-positive and 7.2 6 1,1% PI-positive cells. MBP13 Tcells maintained in IL-2-containing medium were16.5 6 3.5% annexin V-positive and 5.2 6 0.2% PI-positive. Although the difference between the two tar-get populations is not as marked as with thymocytes,pilot experiments had shown a large increase of PI-positive, necrotic cells with longer incubation times ofthe steroid-treated cells. Other modes of induction ofapoptosis, e.g., treatment with the alkylating agentmafosfamide (Pette et al., 1995), also led to a rapiddevelopment of necrotic changes.

As depicted in Figure 2, microglia interacted withMBP13 T cells preferentially ingested the steroid-treated, apoptosis-enriched target cells. The phagocy-tosis rate for non-steroid-treated, IL-2-exposed cellsreached only 64.4 6 4% of the phagocytosis rate forapoptotic cells (P , 0.0001). The phagocytic capacity ofthe individual microglial cells as reflected by thephagocytic index in the non-steroid-treated, IL-2-ex-posed cells amounted to only 55 6 4.8% in comparisonwith the apoptotic cell population (P , 0.0001).

Modulation of Microglial Phagocytosis ofApoptotic Cells by Th1-Type Cytokines: IFN-g,

But Not TNF-a, Increases Phagocytosis

Figure 3 shows representative photomicrographs ofthe phagocytosis of apoptotic thymocytes by untreated

microglia (Fig. 3A) or after preincubation with recom-binant rat IFN-g (30 IU/ml) (Fig. 3B). IFN-g increasedthe proportion of microglia ingesting apoptotic thymo-cytes already at a concentration of 30 IU/ml (Fig. 3C).Heat-inactivated IFN-g had no effect, demonstratingthat the enhancement of phagocytosis was dependenton the intact cytokine. Higher concentrations (300 and500 IU/ml IFN-g) did not further augment phagocyto-sis (data not shown). IFN-g not only enhanced thephagocytosis rate (175.5 6 3.5% of unstimulated con-trols, P , 0.0001), but increased the phagocytic indexto a greater relative extent (230.6 613.1% of unstimu-lated controls, P , 0.0001). This indicated that IFN-gacted not only by recruiting additional microglial cellsinto the phagocytic subpopulation, but also by enhanc-ing the phagocytic capacity of the individual microglialcell. To test whether the phagocytosis-promoting effectof IFN-g on microglia was specific for apoptotic targetcells, untreated and cytokine-stimulated microgliawere also offered nonapoptotic cells (Fig. 3D). As com-pared with untreated microglia, the uptake of nonapop-totic thymocytes by IFN-g-pretreated microglia wasnot increased. This indicated that the IFN-g-mediatedpromoting effect on microglial phagocytosis was spe-cific for apoptotic cells, and not an entirely unspecificstimulation of the phagocytic potential for any targetcell population. We then investigated the effect of theTh1-type cytokine TNF-a on microglial phagocytosis.Within a concentration range of 30–300 IU/ml, TNF-adid not have an influence on microglial uptake of apo-ptotic thymocytes (phagocytosis rate expressed as per-centage of the mean of untreated controls: 107.5 6 6.3at 30 IU/ml and 99 6 5.3% at 300 IU/ml, respectively).

Phagocytosis of Apoptotic Thymocytes byUnstimulated and IFN-g-Stimulated Microglia

Is Not Mediated by Phosphatidylserine-Dependent Mechanisms

Exposure of the phospholipid phosphatidylserine(PS) on the outer plasma membrane leaflet occurs earlyduring apoptosis and serves as a specific recognitionmechanism for the uptake of apoptotic cells by differentphagocytes (Savill, 1997; Chan et al., 1998; Fadok etal., 1998b, 2000). To investigate whether microglialuptake of apoptotic thymocytes was mediated by a PS-dependent mechanism, we performed inhibition exper-iments using the stereospecific, structural PS-deriva-tive phospho-L-serine. In concentrations known toinhibit PS-mediated recognition in other model sys-tems (Fadok et al., 1992a,b), increased phagocytosis ofIFN-g-stimulated microglia was not inhibited by phos-pho-L-serine (Fig. 4). Also, no specific effect of phospho-L-serine on the phagocytosis of apoptotic thymocyteswas seen with untreated microglia. In addition, in com-parison with trypsinized peptide fragments used ascontrol, the PS-binding protein annexin V did not in-hibit phagocytosis of apoptotic thymocytes by eitherIFN-g-treated microglia or unstimulated microglia

Fig. 2. Microglial phagocytosis of encephalitogenic myelin basicprotein (MBP)-specific T cells. Microglial phagocytosis rate andphagocytic index are given as percentage of the mean of controls (1)1SEM. Microglia has a high capacity for the phagocytosis of T-cellgrowth factor-deprived cortisone-treated apoptotic (1) vs. non-ste-roid-treated interleukin-2 (IL-2)-exposed (2) T cells, as reflected inthe microglial phagocytosis rate and the phagocytic index, whichrepresents the phagocytic capacity of the individual microglial cell(***P , 0.0001). In the experiments shown, 37.3 6 1.8% (range28.2–43.6%) of the total microglia were capable of phagocytosingcortisone-treated apoptotic MBP13 T cells. Three independent exper-iments, each performed in triplicate.

91MICROGLIAL PHAGOCYTOSIS OF APOPTOTIC CELLS

(data not shown). This indicated that the increasedphagocytosis of apoptotic cells by IFN-g stimulated mi-croglia and the uptake by unstimulated microglia wasnot mediated by PS-dependent mechanisms.

Modulation of Microglial Phagocytosis ofApoptotic Cells by Th2-Type Cytokines: IL-4,

But Not TGF-b, Inhibits Phagocytosis

Microglia was preincubated with IL-4 in concentra-tions known to influence microglial and macrophage

phagocytosis of different cellular and noncellular tar-gets in other model systems (Ren and Savill, 1995; vonZahn et al., 1997; Smith et al., 1998). Recombinant ratIL-4 inhibited microglial phagocytosis of apoptotic thy-mocytes (Fig. 5). Uptake of apoptotic cells by IL-4 (1ng/ml)-pretreated microglial cells was decreased to69.2 6 3% of the respective untreated control cells (P ,0.0001). This inhibitory effect was exerted in a dose-dependent manner, as shown in Figure 5. At the high-est concentration of 18 ng/ml IL-4 investigated, thephagocytosis rate was reduced to 56.4 62.6% of con-trols. Again, heat-inactivated cytokine had no effect on

Fig. 3. Interferon-g (IFN-g) increases microglial phagocytosis ofapoptotic thymocytes. Photomicrographs of untreated (A) and IFN-g(30 IU/ml)-pretreated microglia (B), phagocytosing apoptotic thymo-cytes, May-Giemsa staining. Note the increase in the proportion ofmicroglia phagocytosing apoptotic thymocytes, as well as the in-creased number of ingested cells (arrows) per phagocyte after IFN-gpretreatment. C: Microglial phagocytosis of apoptotic thymocytes byuntreated microglia (control), after IFN-g (30IU/ml) treatment andafter preincubation with heat-inactivated IFN-g for 24 h. IFN-g pre-treatment raised the microglial phagocytosis rate for apoptotic thy-mocytes, expressed as percentage of the mean of the untreated con-

trols 1SEM (***P , 0.0001), whereas heat-inactivated cytokine didnot have any effect. Three independent, representative experiments,each performed in triplicate. D: Phagocytosis of apoptotic (1) vs.nonapoptotic (2) thymocytes by untreated microglia (control) andIFN-g pretreated microglia. The phagocytosis rate (given as percent-age of the mean of untreated controls phagocytosing apoptotic thymo-cytes 1SEM) for apoptotic cells is raised in IFN-g pretreated cells(***P , 0.0001). No difference in phagocytosis between untreated andcytokine-pretreated microglia is observed with nonapoptotic targetcells. Two independent experiments, n 5 9. Scale bar 5 10 mm in A,B.

92 CHAN ET AL.

the phagocytic uptake (data not shown). With nonapop-totic cells offered as target cells, no difference in phago-cytic uptake between IL-4-pretreated and naive micro-glia was observed, arguing against a nonspecific ortoxic effect of the cytokine. We then tested the effects ofthe Th2-type cytokine TGF-b. TGF-b in concentrationsof 0.1–200 ng/ml did not alter microglial phagocytosisof apoptotic thymocytes (phagocytosis rate expressedas percentage of mean of untreated controls: 98.1

61.3% at 10 ng/ml and 102.8 62.7% at 50 ng/mlTGF-b).

DISCUSSION

The fast and efficient phagocytic elimination of apo-ptotic inflammatory cells protects tissues from leakageof noxious contents of the dying cells and leads to anefficient clearance without inciting secondary proin-flammatory secretory responses (Ren and Savill, 1998;Voll et al., 1997). Our study is the first in vitro demon-stration of the phagocytic clearance of apoptotic thymo-cytes and encephalitogenic T cells by the brain-specificphagocyte, the microglial cell, and its modulation bydifferent cytokines. Microglia exhibited a high capacityfor the specific uptake of apoptotic cells. The level ofphagocytosis of autologous apoptotic thymocytes wassimilar to the extent of ingestion of CNS autoantigen-specific, encephalitogenic MBP-reactive T cells. Pre-treatment of microglia with the T-helper 1-type cyto-kine IFN-g led to an increase of the proportion ofmicroglial cells ingesting apoptotic cells and to an en-hanced phagocytic capacity of the individual microglialcell. The phagocytosis-promoting activity was specificfor apoptotic target cells, since ingestion of nonapop-totic cells was not altered by IFN-g pretreatment. Thisincreased uptake of apoptotic cells by naive or IFN-g-stimulated microglia was not mediated by phosphati-dylserine-dependent recognition mechanisms. In con-trast, the T-helper 2-type cytokine IL-4 decreasedmicroglial phagocytosis of apoptotic cells, whereasTNF-a and TGF-b did not exert an effect in our system.

Cells that die by apoptosis are characterized by spe-cific surface changes that label these cells for phago-cytic disposal. These cell surface changes include alter-ations in charge, glycosylation and surface lipids thatin conjunction with specific receptors on phagocytesmediate the recognition and subsequent uptake of ap-optotic cells (reviewed in Platt et al., 1998; Savill,1997). Since the uptake of cells dying by apoptosis maybe mediated by mechanisms other than the phagocyto-sis of necrotic cells (Hirt et al., 2000; Ren and Savill,1998; Stern et al., 1996), and apoptotic cells in vitroeventually proceed into secondary necrosis, the modesof induction of apoptosis were modified to achieve ahigh level of apoptotic target cells with only a smallproportion of secondary necrotic cells. This was of par-ticular importance for the permanent, encephalitogenicT-cell lines that were likely to enter a state of necrosisrapidly. Viability of the different apoptotic cell popula-tions used as phagocytic targets measured by Trypanblue exclusion exceeded 97% in our study, which issimilar to other well-established systems of phagocyto-sis of apoptotic cells (Fadok et al., 1992b; Hughes et al.,1997).

Although the evidence to date implies that selectivityof the recognition mechanisms employed may predom-inantly reside on the receptor usage of the phagocyte,more recent data indicate that the phagocytosis mech-

Fig. 4. Inhibition experiments of interferon-g(IFN-g) enhanced mi-croglial phagocytosis with phospho-L-serine and phospho-D-serine.Phagocytosis of apoptotic thymocytes by untreated microglia (control),IFN-g-pretreated microglia (IFN-g) and IFN-g stimulated microgliain the presence of phospho-L-serine or phospho-D-serine in the givenconcentrations, respectively. Whereas the phagocytosis rate (given aspercentage of the mean of untreated controls phagocytosing apoptoticthymocytes 1SEM) for apoptotic cells is raised in IFN-g-pretreatedcells (***P 5 0.0005), no specific inhibition is seen with phospho-L-serine in comparison to the phospho-D-serine control. Two indepen-dent experiments, n 5 5.

Fig. 5. Interleukin-4 (IL-4) inhibits microglial phagocytosis of apo-ptotic cells. Phagocytosis rate for apoptotic thymocytes in untreatedmicroglia (control) and IL-4 pretreated microglia. IL-4 pretreatmentdecreased the phagocytosis rate given as percentage of the mean ofuntreated controls 1SEM in a dose-dependent fashion (***P , 0.0001for 1 ng/ml IL-4). Two independent, representative experiments, eachperformed in triplicate.

93MICROGLIAL PHAGOCYTOSIS OF APOPTOTIC CELLS

anisms may not be completely independent of the na-ture of the apoptotic cell (Fadok et al., 1992a; Hart etal., 1997). Ligation of the cell surface glycoproteinCD44 on human monocyte-derived macrophages bybivalent antibodies selectively augmented the phago-cytosis of apoptotic granulocytes, whereas phagocytosisof apoptotic lymphocytes was not altered (Hart et al.,1997). Thus, rather than to solely depend on the recep-tor usage of the phagocyte, the uptake mechanismsused may also be influenced by the expression of cell-type-specific markers on the apoptotic cell. Therefore,in addition to apoptotic thymocytes, we also tested thephagocytic uptake of CNS-autoantigen specific, en-cephalitogenic apoptotic T cells. Microglia has a highcapacity for the selective phagocytosis of steroid-treated, apoptotic MBP-reactive T cells, as comparedwith non-steroid-treated control cells maintained inIL-2-containing medium. The levels of uptake of thedifferent apoptotic inflammatory cells by microglia cor-responded well to the phagocytosis rates seen in othermodel systems with professional and semiprofessionalphagocytes, such as the uptake of autologous and het-erologous apoptotic cells by human and murine macro-phages or human glomerular mesangial cell phagocy-tosis of apoptotic neutrophils (Fadok et al., 1992a;Hughes et al., 1997).

Several factors that potentiate the phagocytosis ofapoptotic cells have been identified in vitro. These in-clude glucocorticosteroids (Liu et al., 1999), ligation ofthe CD44 cell surface molecule (Hart et al., 1997), andCD36-gene transfer to semiprofessional phagocytes(Ren et al., 1995). Also, different cytokines have beenshown to regulate the uptake of apoptotic cells by hu-man monocyte derived macrophages (Ren and Savill,1995) and semiprofessional phagocytes (Walsh et al.,1999). Recently, a phagocytosis-promoting effect of invivo administered GM-CSF on monocytes and polymor-phonuclear leukocytes has been demonstrated in can-cer patients (Galati et al., 2000). The dependence ofcytokine effects on phagocyte function upon stimula-tion conditions, the source and nature of the phago-cytes, the activation state, and the species differencesargue for a cautious interpretation of effects observedin vitro. Moreover, the simplified conditions with singlecytokines in culture may not mirror the complex net-work of inflammatory cytokines in vivo, where cyto-kines may rather act in concert. Taken together, theopposing effects of IFN-g and IL-4 on microglial uptakeof apoptotic cells observed in this study may indicatethe extent, to which microglial function could be regu-lated in vivo and thus underscore a putative major roleof microglia in the removal of dying inflammatory cells.Increased microglial phagocytosis of apoptotic cells me-diated by the proinflammatory Th1 cytokine IFN-gmay help to adapt the local phagocytic response to theamount of apoptotic cells and may thus constitute aform of feedback control of inflammatory cells in thediseased CNS.

The early exposure of phosphatidylserine (PS) on theouter plasma membrane leaflet serves as a specific

recognition mechanism for apoptotic cells by differentphagocytes (Fadok et al., 1992a, 1992b, 2000; Savill,1997). In our system, neither the structural PS-deriv-ative phospho-L-serine nor the PS-binding protein an-nexin V exhibited a clear inhibitory effect on the up-take of apoptotic thymocytes by either naive or IFN-g-pretreated microglia, arguing against PS-mediatedrecognition mechanisms. PS exposure has been impliedin the uptake of apoptotic hippocampus-derived neuro-nal HN2-5 cells by mouse microglia, although in thisstudy no PS inhibition experiments were performed(Adayev et al., 1998). Recently, the involvement of PS-dependent recognition mechanisms, as well as lectin-and integrin-mediated uptake has been demonstratedin the phagocytosis of apoptotic cerebellar granule neu-rons by rat microglia (Witting et al., 2000). Compo-nents of the complement system have also been impli-cated in the uptake of apoptotic target cells bymacrophages (Mevorach et al., 1998). Since all experi-ments in our study were conducted in serum-free con-ditions, and the serum used for cultivation of the mi-croglia was heat-inactivated, microglial phagocytosis ofapoptotic cells and its enhancement by IFN-g did notdepend on complement factors.

Inactivation of inflammatory T cells by apoptosis hasbeen identified as a potent mechanism for the clear-ance of the inflammatory infiltrate in different EAEmodels, regardless of the activation state or antigenspecificity of the T cells (Bauer et al., 1998; Gold et al.,1997; Pender et al., 1991). The high degree of apoptosisin the inflamed rodent as well as human CNS with upto 30-50% of all T cells undergoing apoptosis highlightsthe need for an efficient and tightly regulated removalmechanism for the dying cells (Bauer et al., 1999).Although phagocytosis of apoptotic lymphocytes bymacrophages/microglia, oligodendrocytes, and astro-cytes has been described in histological sections ofLewis rat EAE (Nguyen and Pender, 1998), it remainsunclear which phagocytic cell type predominates invivo. Moreover, thus far in vitro phagocytosis of apo-ptotic cells by glial cells and possible regulatory mech-anisms have not been formally demonstrated. Our dataindicate that the microglial cell as the principle CNS-resident immune cell may play a major role in thedownregulation of the autoinflammatory attack, con-sisting of autoantigen-specific as well as bystander Tcells undergoing apoptosis and that this process may betightly regulated by cytokines. The safe clearance ofapoptotic inflammatory cells and modulation of phago-cytic uptake may suggest a novel approach to therapyfor inflammatory reactions in the nervous system.

ACKNOWLEDGMENTS

The authors thank Cornelia Heitzig and AnnetteHorn for excellent technical support. We are indebtedto Dr. John Savill for many stimulating discussionsand for critically reading the manuscript. We thank Dr.Jack Antel for critical discussions on microglial immu-

94 CHAN ET AL.

nobiology and Dr. K.V. Toyka for continuous supportand critical discussion of the manuscript. We are grate-ful to Dr. R.B. Pepinsky for the supply of annexin V. Wegreatly acknowledge the generous gift of mafosfamideby ASTA Medica (Frankfurt/Main, Germany).

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