Phagocytosis of ApoptoticInflammatory Cells by Microglia and

Its Therapeutic Implications:Termination of CNS AutoimmuneInflammation and Modulation by

Interferon-BetaANDREW CHAN,1* ROSANNE SEGUIN,2 TIM MAGNUS,1

CHRISTINA PAPADIMITRIOU,1 KLAUS V. TOYKA,1 JACK P. ANTEL,2AND RALF GOLD1

1Department of Neurology, Clinical Research Group for Multiple Sclerosis andNeuroimmunology, Julius-Maximilians-University, Wurzburg, Germany

2Neuroimmunology Unit, Montreal Neurological Institute, Department of Neurology andNeurosurgery, McGill University, Montreal, Quebec, Canada

KEY WORDS experimental autoimmune encephalomyelitis; multiple sclerosis;T-cell apoptosis

ABSTRACT Apoptosis of autoaggressive T-cells in the CNS is an effective, nonin-flammatory mechanism for the resolution of T-cell infiltrates, contributing to clinicalrecovery in T-cell-mediated neuroinflammatory diseases. The clearance of apoptoticleukocytes by tissue-specific phagocytes is critical in the resolution of the inflammatoryinfiltrate and leads to a profound downregulation of phagocyte immune functions. Adulthuman microglia from surgically removed normal brain tissue was used in a standard-ized, light-microscopic in vitro phagocytosis assay of apoptotic autologous peripheralblood-derived mononuclear cells (MNCs). Microglia from five different patients had ahigh capacity for the uptake of apoptotic MNCs in contrast to nonapoptotic target cellswith the phagocytosis rate for nonapoptotic MNCs amounting to only 61.6% of theapoptotic MNCs. A newly described phosphatidylserine receptor, critical in the phago-cytosis of apoptotic cells by macrophages, is also expressed at similar levels on humanmicroglia. The effects of the therapeutically used immunomodulatory agent interferon-beta (IFN�) were investigated using Lewis rat microglia and apoptotic, encephalito-genic, myelin basic protein-specific autologous T-cells. Also, rat microglia had a highcapacity to phagocytose apoptotic T-cells specifically. IFN� increased the phagocytosis ofapoptotic T-cells to 36.8% above the untreated controls. The enhanced phagocytic activ-ity was selective for apoptotic T-cells and was not mediated by increased IL-10 secretion.Apoptotic inflammatory cells may be efficiently and rapidly removed by microglial cellsin the autoimmune-inflamed human CNS. The in vitro increase of phagocytosis by IFN�merits further investigations whether this mechanism could also be therapeuticallyexploited. © 2003 Wiley-Liss, Inc.

Grant sponsor: the Deutsche Forschungsgemeinschaft; Grant number: DFGGo 459/8-3; Grant sponsor: the State of Bavaria.

Tim Magnus’s present address is Department of Neurology, University ofHomburg, Homburg/Saar, Germany.

Christina Papadimitriou’s present address is Department of Neurology, Uni-versity Clinic, Ahepa Hospital, Thessaloniki, Greece.

*Correspondence to: Dr. Andrew Chan, Neurologische Universitatsklinik, Jo-sef-Schneider-Straße 11, D-97080 Wurzburg, Germany.E-mail: andrewhkchan@hotmail.com

Received 13 March 2003; Accepted 14 March 2003

DOI 10.1002/glia.10258

GLIA 43:231–242 (2003)

© 2003 Wiley-Liss, Inc.

INTRODUCTION

Apoptosis of encephalitogenic T-cells is crucial in theresolution of autoimmune T-cell inflammation in theCNS and contributes to clinical recovery from experi-mental autoimmune encephalomyelitis (EAE) (Gold etal., 1997; Pender and Rist, 2001), a model of organ-specific autoimmunity that mimics some importantfeatures of multiple sclerosis (MS). Apoptosis of T-lym-phocytes also occurs in inflammatory human brain le-sions, most prominently in the acute monophasicdisease acute disseminated leukoencephalomyelitis(ADEM) (Bauer et al., 1999) and to a lesser extent inactive MS lesions (Ozawa et al., 1994). The high degreeof T-cell apoptosis in the inflamed rodent (EAE) andhuman CNS (ADEM) with approximately 30–50% ofall invading T-cells undergoing apoptosis highlightsthe importance of the local apoptotic destruction ofthese unwanted inflammatory cells in the terminationof CNS inflammation (Gold et al., 1997; Bauer et al.,2001).

A key event in the resolution of an inflammatoryinfiltrate is the gentle and safe phagocytic clearance ofdying yet intact leukocytes undergoing apoptosis(Fadok et al., 2001a; Savill et al., 2002). In vivo, therapid recognition, uptake, and degradation by tissue-specific phagocytes is the common fate of cells dying byapoptosis, thus preventing the spilling of potentiallyharmful contents and inhibiting secondary immune re-sponses directed toward the dying cells. In addition,the ingestion of apoptotic cells also actively modulatesphagocyte immune responses. In histological sectionsof Lewis rat EAE, phagocytosis of apoptotic lympho-cytes by macrophages/microglia, oligodendrocytes, andastrocytes has been described (Nguyen and Pender.1998). Lewis rat microglia efficiently phagocytoses spe-cifically apoptotic, encephalitogenic CNS autoantigen-specific T-cells in vitro, differentially regulated byTh1/Th2-type cytokines (Chan et al., 2001). The phago-cytosis of apoptotic T-cells by Lewis rat microglia ismore efficient than by other glial cell elements such asastrocytes. Moreover, phagocytosis specifically of apo-ptotic T-cells leads to a profound downregulation ofmicroglial immune functions with a suppression ofproinflammatory cytokines and microglial T-cell acti-vation, thus silencing the microglial phagocyte in theinflammatory context (Magnus et al., 2002).

In this study, we have extended our investigationsto adult human microglia from normal brain tissueas the principal immune cell of the CNS (Perry, 1998;Becher et al., 2000; Aloisi, 2001; Streit, 2002). Usingan in vitro phagocytosis assay, our data demonstratethat adult human microglia has a high capacity forthe phagocytosis of apoptotic autologous peripheralblood-derived mononuclear cells. Modulation ofphagocytosis may indicate a novel mechanism of ac-tion of IFN�, an agent already in use therapeuticallyin multiple sclerosis.

MATERIALS AND METHODS

All cell culture media and supplements were ob-tained from Gibco-BRL (Eggenstein, Germany) unlessotherwise noted. Recombinant rat IFN� was obtainedfrom the Biomedical Primate Research Center (Ri-jswijk, The Netherlands) (Ruuls et al., 1996). All otherrecombinant rat cytokines were from R&D (Minneap-olis, MN). Human recombinant IFN� was from Bio-Source International (Montreal, Canada). Phospho-L-serine, phospho-D-serine, and RGDS/RGES peptideswere from Sigma (Deisenhofen, Germany). Mafos-famide cyclohexylamine was a generous gift fromASTA Medica, Frankfurt/Main, Germany.

Isolation of Human Adult Microglial Cells

The studies were performed in accordance with theguidelines set by the Biomedical Ethics Unit of McGillUniversity (Montreal, Canada). Primary adult humanglial cells were obtained from surgical resections per-formed for the treatment of nontumor-related intracta-ble epilepsy. Tissue was obtained from regions requir-ing resection to reach the precise epileptic focus andwas distant from the main electrically active site. Dis-sociated cultures of microglia were prepared as previ-ously described, based on the differential adherence ofthe glial cells (Yong and Antel, 1992). Briefly, braintissue was subjected to enzymatic dissociation withtrypsin (0.025%) and Dnase I (25 �g/ml; BoehringerMannheim, Laval, Canada) for 30 min at 37°C, fol-lowed by mechanical dissociation by passage through a132 �m nylon mesh (Industrial Fabrics, Minneapolis,MN). Cells were further separated on a linear 30%Percoll density gradient (Pharmacia LKB, Baie D’Urfe,Canada) and centrifuged at 15,000 rpm at 4°C for 30min. The cells recovered from the interface contained amixed glial cell population consisting of � 65% oligo-dendroglia, 30% microglia, and 5% astrocytes. To en-rich for microglia, the mixed cell population was sus-pended in minimal essential culture medium (MEM),supplemented with 5% FCS, 2.5 U/ml penicillin, 2.5�g/ml streptomycin, 2 mM glutamine, and 0.1% glu-cose (all from Life Technologies, Burlington, Canada)and left overnight in 12.5 cm2 tissue culture flasks(Falcon, Fisher Scientific, Montreal, Canada) in a hu-mid atmosphere at 37°C with 5% CO2. The less adher-ent oligodendroglia were removed by gentle pipettingand the remaining adherent cells, consisting mainly ofmicroglia, were allowed to develop morphologically forapproximately 7 days and then harvested bytrypsinization (0.25%). Microglia were of 95% purity asassessed by immunocytochemistry and flow cytometry(Yong and Antel, 1992; Becher and Antel, 1996). Forthe in vitro phagocytosis assay, microglia were seededin 48-well plates (Falcon).

232 CHAN ET AL.

Isolation of Lewis Rat Microglial Cells

Rat microglial cells were isolated from primarymixed glial cell cultures as described before (Chan etal., 2001). In brief, mixed glial cell cultures were pre-pared from brains of neonatal Lewis rats (P0–P2,Charles River, Sulzfeld, Germany) and cultured over9–14 days [basal medium Eagle’s, 10% fetal calf serum(Sigma), 50 U/ml penicillin, and 50 �g/ml streptomy-cin]. Microglial cells were shaken off the primary mixedbrain glial cell cultures and seeded into 48-well plates(Corning, Corning, NY). Purity of Lewis rat microgliawas consistently � 97% as determined by immunohis-tochemistry [monoclonal antibody ED1, Serotec, Kid-lington, U.K.; polyclonal antiglial fibrillary acidic pro-tein (GFAP) antiserum, Dako, Hamburg, Germany](Chan et al., 2001).

Preparation of Apoptotic Autologous HumanPeripheral Blood-Derived Mononuclear Cells

Autologous mononuclear cells (MNCs) were used astarget cells for human adult microglial phagocytes toexclude recognition and uptake mechanisms otherthan those associated with apoptotic cell surfacechanges. Autologous MNCs were isolated by Ficoll-Paque (Pharmacia LKB) from the peripheral blood ofthe patients undergoing epilepsy surgery (Morse et al.,2001). All surgical patients received dexamethasonepreoperatively (10 mg on the preceding night and onthe morning of surgery). They were maintained on12–16 mg/day dexamethasone orally or intravenouslypostoperatively for 4 days and then gradually taperedover the following 7–10 days. After informed consent,patients donated EDTA blood between days 3 and 8postoperatively. MNCs were seeded at 2 � 106 cells/mlin RPMI 1640 medium (Life Technologies, Grand Is-land, NY), supplemented with 10% heat-inactivatedfetal calf serum (Medicorp, Montreal, Canada), 50 U/mlpenicillin, 50 �g/ml streptomycin, and 2 mM glutamine(all from Life Technologies). In some pilot experiments,also IL-2 (5–10 U/ml) or the anti-CD3 monoclonal an-tibody X35 (0.1 �g/ml; Coulter Immunotech, Unter-schleißheim, Germany) were added. For induction ofapoptosis, MNCs were cultured in the presence of 100�M Mafosfamide-cyclohexylamine (ASTA Medica) for4–7.5 h as described previously (Pette et al., 1995).Adult human monocytes/macrophages from healthy do-nors were obtained by sorting with CD14 magneticbeads according to the manufacturer’s instructions(Dynal, Great Neck, NY) (Morse et al., 2001).

Preparation of Apoptotic Lewis RatThymocytes and Myelin Basic

Protein-Specific T-Cells

To obtain apoptotic target cells, freshly prepared au-tologous Lewis rat thymocytes were treated with 0.1

�g/ml methylprednisolone (Hoechst, Frankfurt/Main,Germany) for 5 h, 37°C/5% CO2 as described before(Chan et al., 2001). Thymocytes kept on ice for 5 h afterpreparation were used as nonapoptotic control cells(Chan et al., 2001). The generation of the CD4-positiveLewis rat myelin basic protein (MBP)-specific T-cellline MBP13 specific for the dominant encephalitogenicMBP epitope spanning amino acids 68–88 of guineapig MBP complexed to RT1.B1 class II MHC moleculeshas been reported in detail before (Chan et al., 1999).All immunizations were performed according to Bavar-ian state regulations. For induction of apoptosis, cellswere cultured in the absence of T-cell growth factorswith the addition of 0.1 �g/ml methylprednisolone for3 h at 37°C/5% CO2 (Chan et al., 2001).

Flow Cytometric Analysis of Cell Death ofTarget Cells for In Vitro Phagocytosis Assay

For each phagocytosis experiment, the human orrodent target cells were characterized by flow cytom-etry using fluorescein-conjugated annexin V (an-nexin V FITC) and propidiumiodide (PI), character-istic of early apoptotic or late apoptotic/necroticchanges, respectively (Vermes et al., 1995). Stainingwas performed according to the manufacturer’s in-struction (Roche Diagnostics Boehringer Mannheim,Mannheim, Germany).

In case of human autologous MNC, additional sub-population analyses were performed for three patients.MNC were double-stained using phycoerythrin (PE)-conjugated anti-CD3, -CD14, or -CD19 monoclonal an-tibodies (Becton Dickinson, Mountain View, CA) andannexin V FITC; 5 � 105 cells were resuspended in 100�l PBS/2% FCS and incubated with 10 �l of the PE-conjugated monoclonal antibodies (15 min, ice). Afterwashing with binding buffer (10 mM HEPES-NaOH,pH 7.4, 140 mM NaCl, 5 mM CaCl2), cells were incu-bated with 2.5 �l annexin V FITC in 100 �l bindingbuffer (15 min, ice). Pilot experiments had shown alower proportion of annexin V-positive cells if the cellswere first stained for annexin V and subsequently withthe PE-conjugated monoclonal antibodies. Cells wereanalyzed by flow cytometry with 10,000 eventscounted.

Modes of induction of apoptosis were modified toresult in a high degree of apoptosis with a low propor-tion of secondary necrotic cells in the mafosfamide-treated MNC and a low degree of spontaneous apopto-sis in the nonmafosfamide-treated MNC. The targetautologous MNC induced to undergo apoptosis bymafosfamide treatment on average showed 40.8% �6.5% (mean � SEM; range, 32–66.7%) annexin V-pos-itive and 26.5% � 7.8% PI-positive cells. The annexinV-positive cells were in majority CD3-positive, with aminor proportion of CD19-positive cells and only sparseCD14-positive cells (Ann�/CD3� 50.9% � 4.7%, Ann�/CD19� 7.1% � 0.03%, Ann�/CD14� 3.4% � 1.6%).Nonmafosfamide-treated MNC were 8% � 2.6% (range,

233MICROGLIAL PHAGOCYTOSIS OF APOPTOTIC CELLS

4.3–18%) annexin V-positive and 2.5% � 1.1% PI-pos-itive. Viability as assessed by trypan blue stainingwas � 86.5% in the mafosfamide-treated MNC (trypanblue-positive cells: mean, 13.5% � 8.4%; median, 8%)and � 99% in the nonmafosfamide-treated cells.

In the Lewis rat system, cortisone-induced apoptoticthymocytes used as target cells had a proportion of49.3% � 2.8% annexin V-positive cells with 7.3% �2.1% PI-positive cells, similar to values reported before(Chan et al., 2001). Control thymocytes used as nega-tive controls stored on ice after preparation showed10.9% � 2.8% annexin V-positive and a proportion of3.2% � 0.8% PI-positive cells. In case of cortisone-treated, apoptotic encephalitogenic MBP13 T-cells,32% were annexin V-positive and 7% PI-positive (Chanet al., 2001). Noncortisone-treated MBP13 showed aproportion of 16% annexin V-positive and 5% PI-posi-tive cells. The proportion of viable thymocytes orMBP13-cells as assessed by trypan blue exclusion wasconsistently � 97%.

In Vitro Phagocytosis Assay

The standardized, microscopically quantified in vitrophagocytosis assay of microglial uptake of autologousapoptotic cells has been described and illustrated indetail before (Chan et al., 2001); 400 �l of a 0.75 �106/ml suspension of either adult human or Lewis ratmicroglial cells per well were seeded in 48-well plates(Costar) and cultured overnight at 37°C/5% CO2. Trip-licate wells of microglial cells were then incubated withthe respective concentrations of cytokines or culturemedium for 24 h. For heat inactivation, the cytokineswere boiled for 10 min in a water bath. Before additionof the target cells, microglial wells were briefly washedwith the respective serum-free culture medium; 500 �lof a 10 � 106/ml (MEM) suspension of autologous hu-man MNC were added to each microglia well. Due tolow MNC numbers, in one patient (HA 231) 500 �l of a4 � 106/ml MNC suspension was added. In the Lewisrat system, 500 �l of a 20 � 106/ml (BME) suspensionof apoptotic or nonapoptotic rat thymocytes or 500 �l ofa 10 � 106/ml suspension of apoptotic or nonapoptoticMBP-specific T-cells were added to each well, respec-tively. Pilot experiments in the human and rodent sys-tem as well had shown no further increase in the pro-portion of phagocytosing microglia when the amount ofapoptotic target cells was raised. Microglia and thetarget cells were cocultured at 37°C/5% CO2 for 2 h andthe interaction was terminated by vigorous washing ofthe wells with cold PBS (4°C). The microglial cell mono-layer was then trypsinized, and a separate cytocentri-fuge preparation was prepared for each well, whichdislodged target cells that were attached but not in-gested (Hughes et al., 1997; Chan et al., 2001). Thecytocentrifuge preparations were stained with May-Giemsa (Merck, Darmstadt, Germany). The proportionof microglial cells containing apoptotic cells and theamount of phagocytosed target cells was counted by

light microscopy with an average of 500 microglial cellsper slide being counted in a blinded fashion. In someexperiments, the result was expressed as the percent-age of the mean of the control wells for that particularexperiment. In some experiments, data were addition-ally given as the phagocytic index, which reflects thephagocytic capacity of individual microglial cells and isderived by multiplication of the percentage of phagocy-tosing microglia by the average number of ingestedtarget cells per microglia (Fadok et al., 1992).

For inhibition experiments in the Lewis rat systemwith phospho-L-serine and phospho-D-serine, apopto-tic thymocytes were preincubated with the reagentsdiluted at 1 and 2 mM in BME for 15 min at 4°C.Apoptotic thymocytes were then coincubated with mi-croglia in BME in the presence of 1 or 2 mM phospho-L-serine or phospho-D-serine, respectively. Inhibitionexperiments with RGDS and RGES peptides (1–2 mM)were performed identically.

Lewis Rat Microglial Cytokine Secretion

After the 2-h interaction of microglia with the targetcells, the wells were carefully washed with 37°C warmmedium to remove nonadherent cells in order to avoidan influence by secondary necrotic cells and subse-quently medium was added. To increase cytokine se-cretion, microglia was stimulated with 10 ng/ml LPS asdescribed before (Magnus et al., 2001). As control, su-pernatants of LPS-stimulated apoptotic and nonapop-totic target cells were also analyzed. Supernatantswere collected after 24 h, centrifuged to remove partic-ulate debris, and stored in aliquots at �70°C. CytokineELISAs (rat tumor necrosis factor-alpha, TNF; rat-interleukin-12, IL-12; rat interleukin-10, IL-10; trans-forming growth factor-beta 1, TGF�1) were performedaccording to the manufacturer’s instructions (R&DSystems).

Flow Cytometric Determination ofPhosphatidylserine Receptor Expression onAdult Human Microglia and Macrophages

A total of 0.3 � 106 human microglial cells or mac-rophages were incubated with the mouse antihumanphosphatidylserine receptor (PSR) antibody mAb 217(IgM isotype, 100 �g/ml; Cascade Bioscience, Winches-ter, MA) according to Fadok et al. (2000). Mouse IgM(Dako Diagnostics Canada, Mississauga, Canada) wasused as isotype control. Secondary antibody was fluo-rescein-conjugated goat antimouse IgM (1:50; Pharm-ingen Canada, Mississauga, Canada); 5,000–10,000cells were analyzed by flow cytometry on an FACS scanusing Cell Quest software (BD Biosciences, Missis-sauga, Canada).

234 CHAN ET AL.

Statistical Analysis

All values are expressed as mean � SEM. Statisticalsignificance (defined as P value 0.05) was evaluatedusing Student’s t-test (Graph Pad Software, San Diego,CA).

RESULTSHuman Adult Microglia Phagocytoses

Autologous Apoptotic PeripheralBlood-Derived MNCs

Adult human microglia was isolated from nontumor-ous tissue obtained during epilepsy surgery. As targetcells for phagocytosis, autologous peripheral blood-de-rived mononuclear cells were added that were inducedto undergo apoptosis using the alkylating agent mafos-famide (Pette et al., 1995).

The phagocytosis assay used is a modification of astandardized assay of the uptake of apoptotic cells,which has been employed on different cell types andoptimized for glial cells (Hughes et al., 1997; Chan etal., 2001). Figure 1B depicts the typical light-micro-scopic appearance of human microglial phagocytosis ofautologous apoptotic, mafosfamide-treated MNC.While usually phagocytosing microglia with one or twoingested apoptotic target cells were observed, occasion-ally multiple phagocytosed apoptotic cells were de-tected. As depicted in Figures 1 and 2, human micro-glia cocultured with autologous MNC preferentiallyingested the mafosfamide-treated, apoptotic targetcells in comparison with the nonmafosfamide-treatedcontrol cells. On average, the phagocytosis rate fornonmafosfamide-treated MNC reached only 61.6% ofthe phagocytosis rate for mafosfamide-treated apopto-tic cells (range, 35.4–76.1%; see absolute numbers inFig. 2). Also, the phagocytic capacity of the individualmicroglial cells as reflected by the phagocytic index inthe nonmafosfamide-exposed MNC amounted to only64.5% in comparison with the apoptotic target cell pop-ulation. Baseline microglial phagocytosis of nonmafos-famide-treated MNC can at least partly be explainedby the varying proportions of apoptotic cells inevitablypresent in the MNC of the corticosteroid-treated pa-tients, which were prone to undergo apoptosis sponta-neously (Leussink et al., 2001).

The exposure of the phospholipid phosphatidylserine(PS) on the outer plasma membrane early during apo-ptosis has recently been suggested to represent a crit-ical requirement for the removal of apoptotic cells bydifferent phagocytes (Fadok et al., 2001b). A recentlyidentified stereospecific PS receptor (PSR) expressedby different professional and semiprofessional phago-cytes appears to be coupled to the phagocytosis and thesubsequent functional downregulation of differentphagocyte immune functions (Fadok et al., 2000). PSRexpression on adult human microglia was assessed byflow cytometry using a recently described monoclonalantibody (Mab 217) (Fadok et al., 2000). Figure 3 shows

a representative FACS analysis of the PSR cell surfaceexpression on unstimulated human adult microgliaand macrophages. The level of PSR expression on un-stimulated human microglia was similar to the expres-sion level on macrophages.

IFN� Increases Lewis Rat MicroglialPhagocytosis of Apoptotic Autologous

Thymocytes and EncephalitogenicMBP-Specific T-Cells

After the proof of principle in the human system,further phagocytosis experiments were performed us-ing Lewis rat microglia with apoptotic autologous T-cells as described before (Chan et al., 2001). Also, therat microglia exhibited a high capacity to phagocytoseapoptotic thymocytes or CNS autoantigen-specific, en-cephalitogenic T-cells in contrast to nonapoptotic tar-get cells as reported before (Chan et al., 2001) and asdepicted in Figure 5. Figure 4A and B shows represen-tative photomicrographs of the phagocytosis of apopto-tic thymocytes by untreated microglia (Fig. 4A) or afterpreincubation with recombinant rat IFN� (300 U/ml;Fig. 4B). As depicted in Figure 4C, recombinant ratIFN� not only enhanced the proportion of phagocytos-ing Lewis rat microglia (162% � 15.6% of unstimulatedcontrol microglia; P � 0.0007), but also the phagocyticindex was increased to a greater relative extent(183.8% � 22% of unstimulated controls; P � 0.0001).This indicated that IFN� did not only recruit addi-tional microglial cells into the phagocytic subpopula-tion, but also enhanced the phagocytic capacity of theindividual microglial cell. The increase of phagocytosiswas dose-dependent and reached a plateau with 300U/ml IFN� (phagocytosis rate with 30 U/ml IFN�:123.7% � 5.4% of unstimulated control microglia; P �0.0008). Higher concentrations of IFN� (600–3,000U/ml) did not further increase microglial phagocytosis.Heat-inactivated IFN� had no effect, demonstratingthat the enhancement of phagocytosis was dependenton the intact cytokine (data not shown).

To investigate whether IFN� also increased thephagocytosis of apoptotic CNS autoantigen-specific, en-cephalitogenic T-cells, the MBP-specific T-cell lineMBP13 was used as target cell population (Fig. 5).IFN� increased the microglial phagocytosis rate forapoptotic MBP13 T-cells by 36.8% � 5.4% above theuntreated controls (P 0.0001).

To test whether the phagocytosis-promoting effect ofIFN� on microglia was specific for apoptotic targetcells, untreated and IFN�-pretreated microglia werealso offered nonapoptotic MBP13 T-cells (Fig. 5). Mi-croglial phagocytosis of the nonapoptotic MBP-13 T-cells reached only 65.3% � 6.5% of the phagocytosisrate for apoptotic target cells (P 0.0001). No differ-ence in the uptake of nonapoptotic MBP13 T-cells wasobserved between untreated and IFN�-stimulated mi-croglia (72.4% � 8.6%; P � not significant). This indi-cated that the phagocytosis-promoting effect of IFN�

235MICROGLIAL PHAGOCYTOSIS OF APOPTOTIC CELLS

was specific for apoptotic target cells and not an unspe-cific stimulation of the microglial phagocytic potentialfor any target cell population.

First experiments performed in the human systemindicated that recombinant human IFN� also in-

creased the phagocytosis specifically of apoptotic,mafosfamide-treated MNC by adult human microglia(patient HA 297). Thus, pretreatment with IFN� (500U/ml) increased the baseline phagocytosis rate for ap-optotic cells from 27.2% � 4% to 34.9% � 0.5% (mean �SEM); also, the phagocytic index was increased from39.8 � 6.8 (untreated microglia) to 52.6 � 3.1 (IFN�-pretreated microglia). No major difference could be ob-served in the phagocytosis of nonmafosfamide-treated

Fig. 2. Phagocytosis of autologous peripheral blood-derived mono-nuclear cells by human microglia from five different individuals (HA231-HA 300). Microglia had a higher capacity for the uptake of mafos-famide-treated, apoptotic (�) MNCs than for nonmafosfamide-treated(�) MNCs. Each experiment was performed in triplicates, except HA299 (duplicates). Phagocytosis rate is given as mean � SEM (mean �SD in case of HA 299). Phagocytic indexes as measure of the phago-cytic capacity of individual microglial cells for individual patients (�vs. �, mean � SEM; mean � SD in case of HA 299): HA 231: 29.7 �5.1 vs. 9.8 � 2.7; HA 296: 55.9 � 2.5 vs. 38.5 � 7.4; HA 297: 39.8 � 6.8vs. 24.3 � 3; HA 300: 56.1 � 2.8 vs. 36.2 � 3.7; HA 299: 61.1 � 17.2vs. 57.9 � 18.4.

Fig. 1. Phagocytosis of autologous peripheral blood-derived mono-nuclear cells by human adult microglia. Photomicrographs after 2-hinteraction of microglial phagocytes (open arrows) with nonmafos-famide-treated autologous MNCs (A) or with mafosfamide-treatedapoptotic autologous MNCs (arrowheads; B). The interaction wasfollowed by washing with cold PBS, trypsinization to detach microgliafrom the plate and to dissociate attached but noningested target cells,cytocentrifuge preparation, and staining with May-Giemsa. The nu-clei of the ingested apoptotic MNCs show typical apoptotic morphol-ogy with condensed chromatin (arrowheads). Bar � 10 �m. [Colorfigure can be viewed in the online issue, which is available atwww.interscience.wiley.com].

236 CHAN ET AL.

MNC by untreated or IFN�-pretreated microglia, re-spectively (phagocytosis rate, 18.1% � 1.9% vs. 16.2%� 0.9%; phagocytic index, 24.3 � 3 vs. 22.1 � 2.1).Overnight pretreatment with IFN� (500 U/ml) didnot modulate the PSR expression on human micro-glia as determined by flow cytometry (data notshown). However, due to the limited supply of humancells, more detailed studies of IFN�-effects on phago-cytosis and PSR expression using different IFN� con-centrations over varying time points could not beperformed.

IFN� Treatment Does Not Alter Lewis RatMicroglial Cytokine Secretion After

Phagocytosis of Apoptotic Inflammatory Cells

Phagocytosis of apoptotic thymocytes by Lewis ratmicroglia downregulates LPS-induced secretion ofproinflammatory cytokines without an effect on thesecretion of potentially downregulating cytokines(Magnus et al., 2001). We investigated alterations ofthe LPS-induced Lewis rat microglial cytokine secre-tion after pretreatment with IFN� (Table 1). We firstanalyzed alterations of the LPS-induced Lewis rat mi-

croglial cytokine secretion after pretreatment withIFN�. While IL-10 secretion in untreated microgliawas below detection levels, there was an increase in theIFN�-pretreated microglia (114 � 25 pg/ml, mean �SEM). Also, for TNF-secretion, an increase could beobserved after IFN� pretreatment. For IL-12 andTGF�1, there was no major change of the secretionlevels after pretreatment with IFN�. We next investi-gated LPS-stimulated microglial cytokine secretion af-ter phagocytosis of apoptotic thymocytes or after inter-action with nonapoptotic cells, respectively. Formicroglial TNF and IL-12 secretion, there was a cleardownregulation after phagocytosis of apoptotic cells incomparison to microglia that had interacted with non-apoptotic cells (% reduction: TNF 71%, IL-12 50%).Under the phagocytosis-promoting effect of IFN�, thereduction of TNF and IL-12 secretion was still appar-ent. Whereas there were no major changes in the gen-erally low secretion levels of IL-12, the reduction ofmicroglial TNF secretion appeared to be less pro-nounced (48%). The phagocytosis of apoptotic thymo-cytes had no major effect on microglial TGF�1 andIL-10 secretion. Likewise, there was no additional ef-fect seen after pretreatment of the microglia withIFN�.

Fig. 3. Surface expression of the stereospecific phosphatidylserinereceptor on adult human microglia or macrophages as detected byflow cytometry. Binding of monoclonal mouse anti-PSR Mab 217(black line) to unstimulated human microglia in comparison to un-

stimulated human macrophages. As controls, the isotype control(light-gray line, mouse IgM) or the secondary antibody only (dark-gray line, goat antimouse, fluorescein-conjugated) were used. Histo-gram plots of representative stainings.

237MICROGLIAL PHAGOCYTOSIS OF APOPTOTIC CELLS

Figure 4.

238 CHAN ET AL.

Increased Phagocytosis of ApoptoticThymocytes by IFN�-Stimulated Lewis Rat

Microglia Is Not Mediated by IL-10 and CannotBe Inhibited by Phospho-L-Serine or

RGDS Peptides

Since IFN� induced microglial IL-10 secretion in ourin vitro assay, we also investigated the effects of IL-10on phagocytosis of apoptotic thymocytes by Lewis ratmicroglia. Recombinant rat IL-10 in a concentrationrange from 1 to 10 ng/ml did not have an influence onmicroglial uptake of apoptotic thymocytes (phagocyto-sis rate expressed as percentage of the mean of un-treated controls � SEM: 97.1% � 3.7% at 1 ng/ml and102.9% � 2.6% at 2 ng/ml, respectively). This indicatedthat the phagocytosis-promoting effect of IFN� is notmediated by increased IL-10 secretion in vitro.

To investigate whether increased uptake of apoptoticthymocytes by IFN�-stimulated microglia was associ-ated with PS-dependent recognition mechanisms onapoptotic cells, we performed inhibition experimentsusing the stereospecific, structural PS-derivative phos-pho-L-serine (Fadok et al., 1992). Although phospho-L-serine appeared to inhibit the increased phagocytosisof IFN�-stimulated microglia by about 27%, this inhi-bition was not stereospecific as compared with thephospho-D-serine control (14% inhibition, P � 0.12, 2mM phospho-L/D-serine). Similarly, inhibitory pep-tides (RGDS) of the vitronectin receptor (VnR) knownto be involved in the recognition of apoptotic cells bymacrophages (Savill et al., 1990) did not specificallyinhibit the increased phagocytosis of IFN�-pretreatedmicroglia in comparison to control peptides (RGES, 1–2mM).

DISCUSSION

Inactivation of inflammatory T-cells by apoptosis is apotent mechanism in the clearance of an autoimmuneinflammatory infiltrate from the CNS leading to theelimination of bystander T-cells as well as autoantigen-specific T-cells (Gold et al., 1997; Bauer et al., 2001;Pender and Rist. 2001). Here we describe the phagocy-tosis of apoptotic, peripheral blood-derived mononu-clear cells by human adult microglia, the brain-specificphagocyte. Moreover, IFN�, an effective immunomodu-latory agent in the treatment of MS, may contribute tothe clearance of apoptotic inflammatory cells.

Human microglia exhibited a high capacity for thespecific uptake of autologous apoptotic inflammatorycells in vitro, possibly based on the expression of thephosphatidylserine receptor, which is critical in thephagocytosis of apoptotic cells. After the proof of prin-ciple in the human system, further experiments wereperformed in the rodent system, which circumventedthe limited supply of human adult microglia and autol-ogous MNC and allowed more thorough control of as-say conditions. Pretreatment of Lewis rat microgliawith IFN� led to an increase of the microglial phago-

Fig. 5. Phagocytosis of encephalitogenic myelin basic protein-spe-cific T-cells by untreated and IFN� (300 U/ml)-pretreated Lewis ratmicroglia. (�) indicates T-cell growth factor-deprived, corticosteroid-treated apoptotic T-cells; (�) indicates nonsteroid-treated, interleu-kin-2 (IL-2)-exposed T-cells. The phagocytosis rate [given as percent-age of the mean of untreated controls phagocytosing apoptotic T-cells(�) � SEM] for apoptotic MBP-specific T-cells is raised in IFN�-pretreated cells (P 0.0001). No difference in phagocytosis betweenuntreated and IFN�-pretreated microglia is observed with non-steroid-treated, IL-2-exposed target cells (�). In the experimentsshown, 32.3% � 0.9% (mean � SEM) of the untreated microglia werecapable of phagocytosing apoptotic T-cells. Three independent exper-iments, performed in duplicates or triplicates.

Fig. 4. (Continued) Phagocytosis of autologous apoptotic thymo-cytes by untreated and IFN�-pretreated Lewis rat microglia. Pho-tomicrographs of untreated (A) and IFN� (300 U/ml)-pretreated mi-croglia (B), phagocytosing apoptotic thymocytes, May-Giemsastaining. Note the increase in the proportion of microglia phagocytos-ing apoptotic thymocytes (open arrows) as well as the increased num-ber of ingested cells (arrowheads) per phagocyte after IFN� pretreat-ment. Bar � 10 �m. C: Bar graphs showing phagocytosis ofautologous apoptotic thymocytes by untreated microglia and IFN�(300 U/ml)-pretreated microglia. Microglial phagocytosis rate andphagocytic index are given as percentage of the mean of untreatedcontrols � SEM. IFN� pretreatment raised the microglial phagocyto-sis rate for apoptotic thymocytes � SEM (P � 0.0007). The phagocyticindex, which represents the phagocytic capacity of individual micro-glial cells, is increased to an even greater relative extent (P � 0.0001).In the experiments shown, 38.6% � 4.7% (mean � SEM) of theuntreated microglia were capable of phagocytosing apoptotic thymo-cytes. Three independent experiments, each performed in triplicates.[Color figure can be viewed in the online issue, which is available atwww.interscience.wiley.com].

239MICROGLIAL PHAGOCYTOSIS OF APOPTOTIC CELLS

cytosis rate and the phagocytic capacity of the individ-ual microglial cell for apoptotic autologous T-cells. Thisphagocytosis-promoting effect of IFN� was specific forapoptotic autologous encephalitogenic CNS autoanti-gen-specific T-cells as compared to nonapoptoticT-cells. Similar results could also be obtained in pilotexperiments in the human system. Whereas microglialIL-10 secretion was increased after IFN� pretreat-ment, recombinant IL-10 did not enhance the micro-glial phagocytosis of apoptotic cells. Pretreatment ofmicroglia with IFN� did not further change cytokinesecretion after phagocytosis of apoptotic cells.

The high capacity of human phagocyte populationsfrom different sources to phagocytose apoptotic inflam-matory cells as well as several in vivo studies under-scores the importance of this process in the safe andpotent resolution of inflammation in different organsand tissues (Fadok et al., 2001a; Savill et al., 2002).The high proportion of apoptotic inflammatory cells inthe autoimmune-inflamed human and rodent CNShighlights the need for an efficient and tightly regu-lated removal mechanism for these unwanted cells insitu. The data presented here suggest that in the adulthuman CNS, microglial phagocytosis may provide anefficient clearance mechanism for these dying yet stillintact inflammatory cells. The proportion of approxi-mately a third of the human microglial cells phagocy-tosing apoptotic MNC in vitro corresponds well to thephagocytosis rate observed for rodent microglia withapoptotic encephalitogenic, autoantigen-specific T-cells(Chan et al., 2001). Similar phagocytosis rates havealso been observed in other well-established in vitrophagocytosis assays of different human phagocytes andapoptotic inflammatory leukocytes (Hart et al., 1997;Hughes et al., 1997). In addition to microglia, hema-togenous monocyte-derived macrophages might alsocontribute to the phagocytic clearance of the apoptoticinflammatory infiltrate in situ. An unequivocal assess-ment of the relative contribution of both cell types tophagocytosis would, for instance, require the use ofbone marrow chimeric animals where hematogenouscells express a transgenic marker (Kiefer et al., 2001).

Since the uptake of apopototic cells may be mediatedby mechanisms other than the phagocytosis of necroticcells (Hirt et al., 2000; Henson et al., 2001) and apo-ptotic cells in vitro eventually undergo secondary ne-crosis, the modes of induction of apoptosis were modi-

fied to achieve a high degree of apoptotic changes withonly a low proportion of secondary necrotic cells. Beforeeach phagocytosis experiment, the respective targetcell population was analyzed for apoptotic and necroticchanges by flow cytometry. For the Lewis rat T-cells,consistently stable proportions of apoptotic cells withonly little necrotic cells were obtained (Chan et al.,2001). In the human MNCs, the CD3-positive T-cellsalso preferentially underwent mafosfamide-inducedapoptosis. The higher variability of early apoptotic andlate apoptotic/necrotic cellular changes in the humanMNCs was to be expected and can at least partly beexplained by the glucocorticosteroid treatment thateach patient had received perioperatively before blooddonation (Leussink et al., 2001). Additional attempts toyield more stable proportions of viable and dyingMNCs, e.g., by addition of IL-2 or stimulation of theT-cell receptor, did not result in significant changes. Ina heterologous approach with MNCs from differentnonglucocorticosteroid-pretreated healthy donors usedin parallel with autologus MNC target cells, we ob-served similar microglial phagocytosis rates. Yet, toformally exclude possible recognition and uptakemechanisms other than those related to apoptotic cellchanges, we adhered to autologous MNCs as phagocy-tosis target cells.

The expression of the aminophospholipid PS on theouter apoptotic cell membrane in conjunction with theselective and stereospecific recognition by a newlycloned PSR present on the phagocyte has been sug-gested to be essential for the ingestion of apoptotic cells(Fadok et al., 2001b). In addition, engagement of thePSR leads to the suppression of inflammatory media-tors in vitro and in vivo and has thus been implied as acrucial molecular switch in the noninflammatory clear-ance of apoptotic cells (Henson et al., 2001). Recently,expression of PSR mRNA has been demonstrated onrat microglia and, additionally, rat microglial secretionof proinflammatory mediators was downregulated inthe presence of PS liposomes (De Simone et al., 2002).As shown here, the surface expression of the PSR alsoon adult human microglia indicates that this CNS im-mune cell indeed possesses critical key molecules nec-essary for the swift and safe uptake of apoptotic cells.

Several factors have been shown to potentiate phago-cytosis of apoptotic cells in vitro (Ren and Savill, 1998).A phagocytosis-promoting effect of in vivo adminis-

TABLE 1. LPS-Induced Microglial Cytokine Secretion of Unstimulated or IFN�-Pretreated Microglia*

Microglia (�) (�)IFN�

microglia IFN� (�) IFN� (�)

957 � 813 444 � 227 1,520 � 826 1,351 � 691 794 � 361 1,529 � 801 TNF(pg/ml � SEM)

10.1 � 2.7 10.8 � 3.9 21.6 � 7.7 7.1 � 2.3 8.2 � 2.5 23.4 � 8.3 IL-12(pg/ml � SEM)

507 � 104 537 � 40 592 � 70 455 � 77 503 � 67 478 � 38 TGF�1(pg/ml � SEM)

ND 44 � 23 54 � 49 114 � 25 44 � 43 78 � 3 IL-10(pg/ml � SEM)

*After the phagocytosis of apoptotic thymocytes (�) or interaction with nonapoptotic thymocytes (�). Values of two independent experiments, each performed induplicates.ND: below detection level.

240 CHAN ET AL.

tered GM-CSF on monocytes and polymorphonuclearleukocytes has been demonstrated in cancer patients(Galati et al., 2000). We have previously demonstrateda phagocytosis-promoting effect of the Th1-type cyto-kine IFN� on Lewis rat microglial phagocytosis specif-ically of apoptotic inflammatory cells in vitro, whichsuggests a feedback mechanism for the acceleratedclearance of the inflammatory infiltrate in a proinflam-matory milieu (Chan et al., 2001). Here we could alsodemonstrate a phagocytosis-promoting effect of thetype-1 interferon IFN�, which is one of the currentmainstays in the immunomodulatory therapy of MS(Rolak, 2001). The phagocytosis-promoting effect ofIFN� on microglia appeared to be rather selective forapoptotic encephalitogenic T-cells as compared to non-apoptotic T-cells.

The mechanisms responsible for the increased up-take by IFN�-stimulated microglia are currently un-known. IFN� pretreatment did not modulate the PSRexpression on human microglia. Inhibition experi-ments with ligands for phosphatidylserine- or integrin-associated recognition mechanisms did not lead to con-clusive results in our system. However, inhibitionexperiments with competitive ligands of differing affin-ity have to be interpreted with caution (Giles et al.,2000; Hoffmann et al., 2001). Moreover, the interactionof “eat me” signals such as phosphatidylserine withmultiple receptors, e.g., through the association withdifferent bridging molecules, argues for a complex reg-ulation of the pleiotropic adhesion, recognition, andphagocytosis mechanisms involved (Savill et al., 2002).

IFN�-induced microglial IL-10 production in vitroand significant upregulation of IL-10 also in vivo aftertherapeutic IFN� treatment have been demonstrated(Rudick et al., 1998). The lack of an effect of addedrecombinant IL-10 on microglial phagocytosis in oursystem indicates that the phagocytosis-promoting ef-fect of IFN� is not mediated by IL-10. Since in our invitro system only microglia and not the target T-cellpopulations were treated with IFN�, an IFN�-medi-ated increase of T-cell apoptosis as a mechanism foraugmented phagocytosis can be excluded.

The ingestion of apoptotic cells by phagocytes ac-tively suppresses inflammatory responses by down-regulating proinflammatory cytokines. Whereasphagocytosis of apoptotic cells downregulated the LPS-stimulated secretion of TNF and IL-12 by microglia,pretreatment of microglia with IFN� did not furtherchange the secretion of different pro- and anti-inflam-matory cytokines after phagocytosis. Interestingly, in amodel of contact-dependent but antigen-independentT-cell microglia interaction, IFN� increased IL-10 anddecreased certain proinflammatory cytokines (Chabotand Yong, 2000). The degree of potential apoptotic celldeath in the T-cell population as well as possiblephagocytic uptake was not formally analyzed in thatstudy.

While the exact mechanism of action of IFN� in MSis still unknown, numerous putative mechanisms havebeen proposed (Hohlfeld, 1997; Yong et al., 1998).

Whether the therapeutic benefit of IFN� is also basedon the induction of apoptosis in inflammatory cells isstill controversial (Kaser et al., 1999; Zipp et al., 2000;Schmidt et al., 2001). The complex in vivo milieu in theinflamed CNS argues for a cautious interpretation ofour results obtained in vitro. In theory, IFN� couldhelp to engulf and eliminate T-cells driven into apopto-sis by other therapeutically used agents. In this hypo-thetical scenario, T-cells in a first step would be in-duced to undergo apoptosis, e.g., by corticosteroids(Schmidt et al., 2000; Leussink et al., 2001), and wouldin a second step be eliminated through IFN�-promotedphagocytosis. These two steps could constitute poten-tially synergistic mechanisms in the decrease of acuteinflammation, providing a hypothesis for a combinedtreatment approach. However, further studies are war-ranted to prove this hypothetical scenario also in vivo.

In conclusion, our data add a new facet to the grow-ing recognition of the microglial cell as an importantelement in neuroinflammatory processes with differentand complex regulatory functions (Perry, 1998; Becheret al., 2000; Aloisi, 2001; Streit, 2002). In the humanCNS, the microglial cell as the principal CNS residentimmune cell may play a major role in the local down-regulation of the autoinflammatory attack once it hasencroached. The safe clearance of apoptotic inflamma-tory cells by microglia and its differential modulationby agents such as IFN� and polyclonal immunoglobu-lins (IVIg) in vitro (Chan et al., 2003) merit furtherinvestigations as to whether these mechanisms mayeventually also gain therapeutic significance in auto-immune diseases of the CNS.

ACKNOWLEDGMENTS

The authors thank Annette Horn for excellent tech-nical support, Dr. John Savill for many stimulatingdiscussions and helpful comments and suggestions,and ASTA Medica (Frankfurt/Main, Germany) fortheir generous gift of mafosfamide.

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