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Neurobiology of Disease 22 (2006) 33 – 39

Interferon-g induces microglial-activation-induced cell death:

A hypothetical mechanism of relapse and

remission in multiple sclerosis

Hideyuki Takeuchi,*,1 Jinyan Wang,1 Jun Kawanokuchi, Norimasa Mitsuma,

Tetsuya Mizuno, and Akio Suzumura

Department of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

Received 27 June 2005; revised 19 August 2005; accepted 30 September 2005

Available online 28 December 2005

Relapse and remission are characteristics of multiple sclerosis (MS). The

underlying mechanisms, however, remain uncertain. Interferon-; (IFN-

;) disturbs the immunological privilege of the central nervous system

(CNS) by inducingmajor histocompatibility complex antigen expression

in CNS cells and activating microglia to become antigen-presenting and

effector cells. Thus, IFN-; and microglia are thought to play important

roles in the initiation and development of MS. Here, we show that IFN-;

induces microglial apoptosis as the activation-induced cell death. This

microglial apoptosis was associated with the up-regulation of pro-

apoptosis proteins, especially Bax. Microglial apoptosis was also

observed in peak EAE mice, but not in early EAE mice. Therefore,

IFN-; may act on microglia as part of a self-limiting negative feedback

system. The activation and subsequent death of microglia induced by

IFN-; may play pivotal roles in the mechanism of MS relapse and

remission.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Microglia; Interferon-g; Apoptosis; Activation-induced cell

death; Bax; Multiple sclerosis

Introduction

Multiple sclerosis (MS), an inflammatory demyelinating disease

of the central nervous system (CNS), is often characterized by

periods of relapse and remission (Hemmer et al., 2002; Kieseier et

al., 2005). Helper T cell type 1 (Th1)-dominant autoimmunity is

thought to be involved in the etiology of MS. Previous reports have

suggested that the mechanisms underlying MS remission might be

associated with apoptosis of infiltrating cells or microglia via Fas–

Fas ligand interactions (Spanaus et al., 1998; Frigerio et al., 2000). In

fact, apoptosis of infiltrating cells and microglia has been observed

0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2005.09.014

* Corresponding author. Fax: +81 52 789 5047.

E-mail address: htake@riem.nagoya-u.ac.jp (H. Takeuchi).1 These authors contributed equally to this work.

Available online on ScienceDirect (www.sciencedirect.com).

in active MS plaques (Dowling et al., 1997). Moreover, a recent

report showed that activated microglia and apoptotic oligodendro-

cytes with few infiltrating lymphocytes or phagocytes were mainly

observed in the new symptomatic lesions in relapsing MS (Barnett

and Prineas, 2004). The mechanisms of relapse and remission,

however, remain largely unknown.

The CNS reportedly has an efficient lymphatic drainage, and the

cervical lymph nodes play a significant role in immunological

interaction in the CNS (Harling-Berg et al., 1989; Cserr and Knopf,

1992; Phillips et al., 1997; Lake et al., 1999). However, in a normal

condition, the immunological privilege of the CNS is well-

maintained. This immunological privilege is thought to be associated

with suppression of immunological interaction by cytokines and

other unknown molecules, the lack of major histocompatibility

complex (MHC) antigen expression on CNS cells, and the presence

of the blood–brain barrier, which blocks the invasion of immune

cells and antibodies into the CNS (Suzumura and Silberberg, 1985;

Cserr and Knopf, 1992; Tseveleki et al., 2004). Interferon-g (IFN-g)

has been shown to disturb the immunological privilege of the CNS by

inducing MHC class I antigen expression on CNS cells (Wong et al.,

1984; Suzumura and Silberberg, 1985). IFN-g also induces the

expression of MHC class II and co-stimulatory molecules on

microglia, enabling these cells to function as antigen-presenting

cells (APCs) for Tcells (Suzumura et al., 1987;Williams et al., 1994;

Satoh et al., 1995; Menendez Iglesias et al., 1997). Moreover, IFN-g

activates microglia to act as effector cells that damage CNS cells via

phagocytosis and the release of cytotoxic factors such as glutamate,

nitric oxide, superoxide, and pro-inflammatory cytokines (Schwartz

et al., 2003; Kempermann and Neumann, 2003; Platten and Stein-

man, 2005; Takeuchi et al., 2005). Thus, IFN-g and microglia are

thought to play critical roles in the initiation and development of MS.

In this study, we show that IFN-g induces activation-induced cell

death of microglia through the up-regulation of pro-apoptosis

proteins, particularly Bax. The activation and death of microglia

induced by IFN-gmay play pivotal roles in the relapse and remission

of MS.

H. Takeuchi et al. / Neurobiology of Disease 22 (2006) 33–3934

Materials and methods

Animals and reagents

The protocols for animal experiments were approved by the

Animal Experiment Committee of Nagoya University. C57BL/6J

mice were purchased from Japan SLC (Hamamatsu, Japan). The

MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was syn-

thesized and purified by Operon Biotechnologies (Tokyo, Japan).

Incomplete Freund’s adjuvant (IFA) and lipopolysaccharide (LPS)

were obtained from Sigma-Aldrich (St. Louis, MO). Heat-killed

Mycobacterium tuberculosis H37Ra was obtained from Difco

(Detroit, MI). Pertussis toxin was obtained from List Biological

Laboratories (Campbell, CA). Recombinant mouse IFN-g was

obtained from R&D Systems (Minneapolis, MN).

Cell culture

Mouse primary microglia were isolated from primary mixed

glial cell cultures (obtained from newborn C57/BL6 mice) by the

Fshaking off_ method as described previously (Suzumura et al.,

1987). The purity of the cultures was more than 97% as determined

by Fc-receptor-specific immunostaining as described previously

(Suzumura et al., 1987). Cultures were maintained in Dulbecco’s

Fig. 1. IFN-g induces microglial apoptosis. (A) A DNA fragmentation

assay revealed that IFN-g-treated microglia underwent apoptosis. (B)

Phase-contrast micrographs of untreated primary microglia and (C) micro-

glia treated with 100 ng/ml IFN-g for 72 h. TUNEL-positive nuclei were

observed in IFN-g-treated microglia (purple color marked by the arrows in

panel C). LPS, microglia treated with 1 mg/ml LPS for 72 h; IFN-g,

microglia treated with 100 ng/ml IFN-g for 72 h; IFN-g + LPS, microglia

treated with 1 mg/ml LPS and 100 ng/ml IFN-g for 72 h.

Fig. 2. (A) IFN-g induces microglial apoptosis in a dose-dependent manner.

The pan-caspase inhibitor z-VAD-fmk markedly inhibited microglial

apoptosis. LPS, microglia treated with 1 mg/ml LPS for 72 h; IFN-g 1,

microglia treated with 1 ng/ml IFN-g for 72 h; IFN-g 10, microglia treated

with 10 ng/ml IFN-g for 72 h; IFN-g 100, microglia treated with 100 ng/ml

IFN-g for 72 h; IFN-g 100 + LPS, microglia treated with 1 mg/ml LPS and

100 ng/ml IFN-g for 72 h; IFN-g 100 + z-VAD-fmk, microglia treated with

1 mg/ml LPS and 20 AM z-VAD-fmk for 72 h. *P < 0.05 versus control

conditions. .P < 0.05 versus 1 ng/ml IFN-g treatments. The presented

values are the means T SE. (B) IFN-g induces microglial apoptosis only

after longer IFN-g treatments (48–72 h). *P < 0.05 versus control

conditions. .P < 0.05 versus 48 h treatment. The values are the means T SE.

modified Eagle’s minimum essential medium (Sigma-Aldrich)

supplemented with 10% fetal calf serum (JRH Biosciences,

Lenexa, KS), 5 Ag/ml bovine insulin, and 0.2% glucose.

Experimental autoimmune encephalomyelitis (EAE) mice

EAE mice were produced as described previously (Kato et al.,

2004). Briefly, C57BL/6J mice aged 6–8 weeks were immunized

subcutaneously at the base of the tail with 0.2 ml of emulsion

containing 200 Ag MOG35–55 in saline combined with an equal

volume of IFA containing 300 Ag heat-killed M. tuberculosis

H37Ra. Mice were injected with pertussis toxin intravenously on

the day of immunization (25 ng/mouse) and 2 days after

immunization (50 ng/mouse). Mice were assessed daily for clinical

signs of EAE according to the following scale: 0—normal; 1—

limp tail or mild hind limb weakness; 2—moderate hind limb

weakness or mild ataxia; 3—moderate to severe hind limb

weakness; 4—severe hind limb weakness, mild forelimb weakness,

or moderate ataxia; 5—paraplegia with moderate forelimb weak-

ness; 6—paraplegia with severe forelimb weakness, severe ataxia,

or moribundity. Mice developed peak EAE (scale values of 4–5)

Fig. 3. IFN-g-induced apoptosis occurs in an activation-dependent manner. IFN-g induced mild microglial proliferation in a dose-dependent manner (red and

green points). Most of the apoptotic cells (green points) belonged to the population of proliferating cells (red and green points).

Fig. 4. (A) High-throughput immunoblotting analysis of IFN-g-induced

apoptosis-related proteins. IFN-g, microglia treated with 100 ng/ml IFN-g

for 48 h. (B) Apoptosis-related proteins whose expression levels changed

more than 2-fold compared with control samples. The ratio of the

expression levels of each protein in IFN-g-treated samples to the levels

in control samples is indicated on the right. Ten proteins were up-regulated

after IFN-g treatment, including seven pro-apoptosis proteins (Bax, Bid,

Apaf-1, Btf, DAP kinase, IKKg, and TRADD) and three anti-apoptosis

proteins (Bcl-2, Bcl-x, and InB(). Bax was the most markedly up-regulated

protein examined. The values are the means T SE.

H. Takeuchi et al. / Neurobiology of Disease 22 (2006) 33–39 35

approximately 14–20 days after immunization as described

previously (Kato et al., 2004).

The terminal deoxynucleotidyltransferase-mediated UTP

end-labeling (TUNEL) assay

To detect microglial apoptosis, we performed a TUNEL assay

with an In Situ Cell Death Detection Kit (Roche Diagnostics,

Basel, Switzerland) as described previously (Takeuchi et al., 2002).

For in vitro assessment, primary microglia were plated on 4-well

chamber slides coated with poly-l-lysine at a density of 5 � 104

cells/well. Microglia treated with each drug (1, 10, or 100 ng/ml

IFN-g, 1 Ag/ml LPS, or 100 ng/ml IFN-g plus 1 Ag/ml LPS) for 72

h were assessed according to the manufacturer’s protocol. Micro-

glia treated with 100 ng/ml IFN-g for different periods of time

were also assessed (0, 6, 12, 24, 48 and 72 h incubations). If

necessary, a broad caspase inhibitor (20 AM z-VAD-fmk; Peptide

Institute, Osaka, Japan) was added simultaneously with IFN-g. As

a positive control, microglia were incubated with 10 nM

staurosporin for 24 h. More than 200 cells in duplicate wells were

assessed blindly in three independent trials under a conventional

fluorescence microscope as described previously (Takeuchi et al.,

2002). The percentage of cells that were apoptotic (TUNEL-

positive) was calculated.

For in vivo assessment, mice with early EAE or peak EAE

were anesthetized and perfused transcardially with 4% parafor-

maldehyde in 0.1 M PBS. Lumbosacral spinal cords were

immediately removed, postfixed in 4% paraformaldehyde, and

embedded in paraffin. Five-micrometer sections were labeled

with rat monoclonal fluoresceinisothiocyanate isomer-I (FITC)-

conjugated anti-CD11b antibodies (BD Pharmingen, Franklin

Lakes, NJ) at 4-C overnight and subsequently subjected to a

TUNEL assay according to the manufacturer’s protocol. Labeled

sections were observed under a conventional fluorescence

microscope.

DNA fragmentation assay

To detect microglial apoptosis, we also employed a DNA

fragmentation assay using a Quick Apoptotic DNA Ladder

Detection Kit (BioVision, Mountain View, CA). Microglia treated

with each drug (1, 10, or 100 ng/ml IFN-g, 1 Ag/ml LPS, or 100

H. Takeuchi et al. / Neurobiology of Disease 22 (2006) 33–3936

ng/ml IFN-g plus 1 Ag/ml LPS) for 72 h were assessed according

to the manufacturer’s protocol.

Flow cytometry

To assess which population of microglia was apoptotic,

microglial proliferation and caspase-3 activation were detected

with flow cytometry. 5 � 104 microglia plated on 24-well

multidishes were treated with 1, 10, or 100 ng/ml IFN-g for 72

h. The cells were then harvested and fixed with a Cytofix/

Cytoperm Kit (BD Pharmingen) according to the manufacturer’s

protocol. Cells were treated with RNase and were subsequently

labeled with 10 Ag/ml propidium iodide (PI; Molecular Probes,

Eugene, OR) and rabbit polyclonal FITC-conjugated anti-cleaved

caspase-3 antibodies (BD Pharmingen) at room temperature for 1

h. Fluorescence signals were measured with a flow cytometer

(Cytomics FC500, Beckman Coulter, Fullerton, CA).

High-throughput immunoblotting analysis

Primary microglia samples (untreated or treated for 48 hwith 100

ng/ml IFN-g) were lysed in TNES buffer (50mMTris–HCl, pH 7.5,

150 mM NaCl, 1% NP-40, 2 mM EDTA, 0.1% SDS, and protease

inhibitor cocktail; Complete Mini EDTA-free; Roche Diagnostics).

Samples were processed by PowerBlot analysis using an apoptosis

array (BD Pharmingen), which measured the expression levels of

270 different apoptosis-related proteins using a combination of

SDS-PAGE (5–15% gradient), immunoblotting with specific

monoclonal antibodies, detection of bound antibodies with goat

anti-mouse horseradish-peroxidase-conjugated secondary antibod-

ies, acquisition of chemiluminescence data with a CDD camera, and

computerized processing of densitometric data in triplicate after

normalization to mean protein expression levels. Expression levels

that change by more than two-fold were well above background

variations, which are generally less than 1.5-fold in these assays

(Castedo et al., 2002; http://www.bdbiosciences.com/pharmingen/).

Fig. 5. Apoptotic microglia were observed in the spinal cord of a peak EAE mouse

TUNEL-positive cells in the dorsal column of the spinal cord (red). (C) Overlay o

panels A, B, and C, respectively.

Statistical analysis

Results were analyzed by one-way analysis of variance

(ANOVA) with a Tukey–Kramer post-hoc test using Statview

software version 5 (SAS Institute Inc., Cary, NC, USA).

Results

IFN-c induces microglial apoptosis

A DNA fragmentation assay demonstrated that IFN-g induced

primary microglial apoptosis (Fig. 1A). Treatment with LPS alone

did not induce microglial apoptosis in this study (Figs. 1A and 2A).

LPS treatment, however, tended to enhance IFN-g-induced micro-

glial apoptosis (Fig. 2A, data not shown). In vitro TUNEL assays

also revealed that IFN-g induced microglial apoptosis (Fig. 1C,

arrows). Furthermore, IFN-g induced microglial cell death in a

dose-dependent manner (Fig. 2A). A pan-caspase inhibitor, z-

VAD-fmk, markedly inhibited microglial apoptosis (Fig. 2A).

Thus, the observed microglial apoptosis most likely occurred

through caspase activation. A time course of microglial apoptosis

showed that IFN-g induced cell death only after long incubations

(Fig. 2B, 48–72 h).

IFN-c-induced microglial apoptosis is activation-induced

cell death

We next examined the relationship between microglial activa-

tion and apoptosis with flow cytometry. Microglia treated with

different concentrations of IFN-g were double-labeled with PI and

anti-cleaved caspase-3 antibodies. PI signal intensity correlates

with the amount of DNA per cell; a cell at the G2/M stage displays

a PI-related fluorescent signal that is twice that of a cell at the G0/

G1 stage. PI labeling revealed that IFN-g induced mild microglial

proliferation (activated the microglia) in a dose-dependent manner

. (A) Microglia in the dorsal column of the spinal cord (CD11b, green). (B)

f panels A and B. Panels C, D, and E are higher magnification pictures of

H. Takeuchi et al. / Neurobiology of Disease 22 (2006) 33–39 37

(Fig. 3, red and green points). Labeling the cells with anti-cleaved

caspase-3 antibodies demonstrated that the percentage of apoptotic

microglia also increased dose-dependently as was previously

shown in Fig. 2. Interestingly, most of the apoptotic microglia

belonged to the G2/M population of cells (Fig. 3, green points).

Thus, a subpopulation of the proliferating microglia seemed to

undergo apoptosis, i.e. activation-induced cell death. Therefore,

IFN-g-induced microglial apoptosis can be considered activation-

induced cell death.

Fig. 6. A hypothetical mechanism for the involvement of microglia in the develo

microglia drive inflammatory cascades in the CNS, which causes MS pathologies

cascades through a negative feedback mechanism, which remits the MS pathologie

type 1; Tc, cytotoxic T cell.

Up-regulation of Bax is involved in IFN-c-induced microglial

apoptosis

We next carried out high-throughput immunoblotting analysis

for the expression levels of 270 different apoptosis-related proteins

(Fig. 4A). Proteins whose expression levels were up-regulated to

more than twice the levels observed in control samples or were

down-regulated to less than half the levels in control samples were

selected for further analysis. The expression levels of most proteins

pment and remission of MS. IFN-g is released from T cells, and activated

(top panel). IFN-g-induced microglial apoptosis inhibits the inflammatory

s (bottom panel). Mi, microglia; Th0, naive helper T cell; Th1, helper T cell

H. Takeuchi et al. / Neurobiology of Disease 22 (2006) 33–3938

did not change significantly. Among the ten proteins that were up-

regulated after IFN-g treatment, seven were pro-apoptosis proteins,

Bax, Bid, apoptotic protease activating factor-1 (Apaf-1), Bcl-2-

associated transcription factor (Btf), IFN-g-induced death-associ-

ated protein (DAP) kinase, InB kinase (IKK) g, and TNF recep-

tor-associated death domain protein (TRADD), and three were

anti-apoptosis proteins, Bcl-2, Bcl-x, and InB( (Fig. 4B). In par-

ticular, Bax was markedly up-regulated (more than 10-fold). We

did not uncover any proteins that were down-regulated due to the

IFN-g treatment.

Microglial apoptosis occurs in the spinal cord of peak EAE mice

Immunohistochemical analysis revealed that microglia in the

spinal cord of a peak EAE mouse were apoptotic (Fig. 5).

Amoeboid-shaped activated microglia were mainly observed in

the dorsal column (Figs. 5A and D). A small population of

these activated microglia underwent apoptosis (Figs. 5B and E).

In contrast, apoptotic microglia were hardly detected in an early

stage EAE mouse (data not shown). Thus, the microglial

apoptosis seemed to occur after the inflammation had advanced.

Discussion

Here, we provide evidence that IFN-g induces microglial-

activation-induced cell death. Additionally, this microglial apopto-

sis was associated with the up-regulation of pro-apoptosis proteins,

especially Bax. It is thought that the expression ratio of Bax/Bcl-2

regulates apoptosis (Shimizu et al., 1999). We suggest that the

activated microglia underwent apoptosis because the expression

levels of the pro-apoptosis proteins including Bax were over-

whelming when compared with those of the anti-apoptosis proteins

such as Bcl-2/x (Fig. 4B). A previous series of reports mentioned

that simultaneous stimulation with LPS and IFN-g induced

microglial apoptosis (Lee et al., 2001a,b, 2003), whereas our

study demonstrated that treatment with IFN-g alone caused

microglial apoptosis in a primary culture model. We propose that

IFN-g directly up-regulates microglial pro-apoptosis proteins,

which leads to subsequent microglial apoptotic cell death.

IFN-g and microglia are key players in the initiation and

development of MS pathology via a positive-feedback mechanism

(Fig. 6, top panel). IFN-g activates microglia, causing these cells to

function as APCs for naive Th cells in the CNS. Simultaneously,

activated microglia secrete IL-12, IL-18, IL-23, and IL-27, which

cause naive Th cells to differentiate into Th1 cells (Stalder et al.,

1997; Aloisi et al., 1997; Suzumura et al., 1998; Conti et al., 1999;

Gran et al., 2002; Becher et al., 2003; Sonobe et al., 2005). Mature

Th1 cells then produce IFN-g to activate microglia. Recently, we

demonstrated that activated microglia themselves can also produce

IFN-g (Kawanokuchi et al., submitted for publication). In MS, these

immunological cascades exponentially expand inflammation in the

CNS. A recent report supported the hypothesis that activated

microglia initiate inflammation in cases of MS (Barnett and Prineas,

2004). Conversely, if the number of activated microglia declines,

the inflammatory positive-feedback cascades collapse and inflam-

mation may be reduced (Fig. 6, bottom panel). In fact, inhibition of

microglia has been shown to improve the symptoms of EAE and

MS (Hall et al., 1997; Popovic et al., 2002). The present study raises

the possibility that IFN-g acts on microglia not only during positive

feedback but also during self-limiting negative feedback.

Flow cytometric analysis revealed that IFN-g induced mild

microglial proliferation in a dose-dependent manner and that most

of the apoptotic microglia belonged to the population of

proliferating cells (Fig. 3). Thus, IFN-g-induced microglial

apoptosis can be considered activation-induced cell death. Activa-

tion-induced cell death is well documented in T cells (Green et al.,

2003), where Fas and Fas ligand are suggested to be involved in

the process. A previous report proposed that infiltrating T cells

might undergo apoptosis via Fas ligand expression on microglia

(Frigerio et al., 2000). Another study reported that activated

microglia expressed Fas and were susceptible to Fas-ligand-

induced apoptosis (Spanaus et al., 1998). High-throughput

immunoblotting analysis, however, demonstrated that pro-apopto-

sis proteins were up-regulated, whereas the levels of Fas and Fas

ligand did not change in this study. Interestingly, TRADD, IKKg,

and InB( were also up-regulated (Fig. 3B). Tumor necrosis factor-

a (TNF-a) signaling has also been suggested to be involved in

microglial apoptosis. Recently, we documented that activated

microglia produce TNF-a and that TNF-a acts on microglia in

an autocrine manner (Kuno et al., 2005). It is possible that

inflammatory cytokines, including TNF-a released from microglia

activated by IFN-g, may affect microglia in an autocrine manner,

and these cytokines signals may lead to microglial apoptosis.

Further studies are needed to resolve this issue.

In this study, microglial apoptosis in EAE mice was

observed in the advanced stage, but not in the early stage of

the disease. Thus, microglial apoptosis in vivo seemed to occur

after inflammation had advanced, which agrees with the

activation-induced cell death theory. Although it is well known

that treatment with IFN-g exacerbates MS symptoms (Panitch et

al., 1987), however, it is possible that well-timed administration

of IFN-g may reduce acute MS symptoms. This hypothesis is

still far away from elucidation.

In conclusion, we demonstrated that IFN-g induces activa-

tion-induced cell death of microglia through the up-regulation of

apoptosis-related proteins, including notably Bax. We propose

that IFN-g acts on microglia not only during an early-phase

positive-feedback cascade, but also during a late-phase self-

limiting negative feedback cascade. The activation and subse-

quent death of microglia induced by IFN-g may play critical

roles in the relapse and remission of MS.

Acknowledgments

We thank Dr. Tsuyoshi Yoshihara for technical assistance.

This work was supported by grants from the Ministry of Health,

Labor, and Welfare of Japan, a young scientists grant and a

Center of Excellence grant from the Ministry of Education,

Culture, Sports, Science, and Technology of Japan.

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