MAPK3 deficiency drives autoimmunity via DC arming

Ivo Bendix�1, Caspar F. Pfueller�1,2, Tina Leuenberger�1,

Nadezhda Glezeva1, Volker Siffrin1, Yasmin Muller1,

Timour Prozorovski1, Wiebke Hansen3, Ulf Schulze Topphoff1,

Christoph Loddenkemper4, Frauke Zipp��1,5 and Sonia Waiczies��1,6

1 Max Delbruck Center for Molecular Medicine, Berlin, Germany2 NeuroCure Clinical Research Center, Charite - University Hospital Berlin, Berlin, Germany3 Immunregulation Group, Institute of Medical Microbiology, University Hospital Essen,

Essen, Germany4 Department of Pathology/Research Center ImmunoSciences, Charite - University Hospital

Berlin, Campus Benjamin Franklin, Berlin, Germany5 Department of Neurology, University Medicine Mainz, Johannes Gutenberg University, Mainz,

Germany6 Department of Anatomy, University of Malta, Msida, Malta

DC are professional APC that instruct T cells during the inflammatory course of EAE. We

have previously shown that MAPK3 (Erk1) is important for the induction of T-cell anergy. Our

goal was to determine the influence of MAPK3 on the capacity of DC to arm T-cell responses

in autoimmunity. We report that DC from Mapk3�/� mice have a significantly higher

membrane expression of CD86 and MHC-II and – when loaded with the myelin oligoden-

drocyte glycoprotein – show a superior capacity to prime naıve T cells towards an inflam-

matory phenotype than Mapk31/1 DC. Nonetheless and as previously described, Mapk3�/�

mice were only slightly but not significantly more susceptible to myelin oligodendrocyte

glycoprotein-induced EAE than WT littermate mice. However, Mapk31/1 mice engrafted with

Mapk3�/� BM (KO-WT) developed a severe form of EAE, in direct contrast to WT-KO mice,

which were even less sick than control WT-WT mice. An infiltration of DC and accumu-

lation of Th17 cells was also observed in the CNS of KO-WT mice. Therefore, triggering of

MAPK3 in the periphery might be a therapeutic option for the treatment of neuroin-

flammation since absence of this kinase in the immune system leads to severe EAE.

Key words: DC . EAE . MAPK3 . T-cell priming

Introduction

In order for living organisms to build up an immune response

against dangerous entities it is fundamental that DC continuously

patrol the periphery and sample their environment for danger

signals [1]. Their distinct capacity to mobilize swiftly to lymphoid

organs and alternate between antigen uptake and presentation

makes DC decision-makers for inducing antigen-specific T-cell

responses [2]. Previously we showed that the activation of

MAPK3 or Erk1 but not its isoform MAPK1 (Erk2) accompanies,

and is necessary for, the induction of T-cell anergy [3]. Since

their identification [4], MAPK3 and its isoforms have been

commonly ascribed analogous downstream functions due to their

�These authors contributed equally to this work.��Joint senior authors

Correspondence: Dr. Sonia Waicziese-mail: sonia.waiczies@charite.de

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2010. 40: 1–10 DOI 10.1002/eji.200939930 Molecular immunology 1

striking similarities. However, it is becoming increasingly clear

that these isoforms – particularly MAPK3 and MAPK1 – have

explicitly different functions. While MAPK1 has a more

pronounced role in cell proliferation and developmental

processes [5–7], MAPK3 does not seem to be required during

development and its deficiency may be compensated for by

MAPK1. In fact while Mapk�/� mice are lethal, Mapk3�/� mice

are viable and develop normally [8]. However, when challenged

with CNS antigen Mapk3�/� mice are more susceptible to

undergo EAE [9, 10]. So far, a deficiency of MAPK3 in the CNS

has been associated with facilitated learning and long-term

memory [11]. In the immune system, MAPK3 has been reported

to play a key role in positive selection during T-cell development

[8, 12] and we have previously shown MAPK3 activation to be

necessary for the induction of T-cell anergy [3].

In this study we report that MAPK3 acts as a negative regu-

lator of DC that controls their strength to prime T cells towards an

inflammatory phenotype. Mapk3�/� DC expressed significantly

higher levels of CD86 and MHC-II, and were indeed more capable

of priming a myelin antigen-specific T-cell response than

Mapk31/1 DC. Although Mapk3�/� mice were only slightly and

not significantly more susceptible to myelin oligodendrocyte

glycoprotein (MOG)-induced EAE than Mapk31/1 mice,

as previously described [9, 10], Mapk31/1 mice harboring

Mapk3�/� mice BM (KO-WT) developed a severe form of EAE.

On the other hand, Mapk3�/� mice harboring Mapk31/1 BM

(WT-KO) were even less sick than control mice (WT-WT).

While MAPK3 seems dispensable for growth and development –

as clearly shown by the normal phenotype of Mapk3�/� mice –

we conclude here that it plays an essential role in DC that then

maintain a moderate T-cell response during autoimmune reac-

tions.

Results

MAPK3 regulates surface expression of CD86 andMHC-II

We have previously shown that MAPK3 activation is required for

T-cell anergy [3]. In this study we wanted to determine the

influence of MAPK3 on events upstream of the T-cell response,

specifically on the capacity of DC to prime T cells. For this we first

investigated the expression of surface markers on DC differen-

tiated from BM cells of Mapk3�/� C57BL/6 mice and Mapk31/1

littermate mice. Mapk3�/� immature DC (iDC) expressed higher

constitutive levels of IAb (MHC-II) and the B7 family member

CD86 (B7.2) on their cell surface, already prior to a maturation

stimulus; interestingly MAPK3 appears to regulate CD86 and not

CD80 (B7.1) expression (Fig. 1A and B). In some experiments we

also observed a subpopulation of DC expressing higher levels of

CD86 and MHC-II in Mapk3�/� DC apart from an increased mean

fluorescence (Fig. 1A). Importantly, the difference in expression

of CD86 and MHC-II between Mapk3�/� and Mapk31/1 DC did

not remain pronounced upon maturation with LPS (Fig. 1C),

possibly due to the fact that these markers reach maximum

expression levels after the maturation process. In line with these

observations, we did not observe differences of IAb and CD86

expression of mature Mapk3�/� and Mapk31/1 DC in secondary

LN and spleen of naıve C57BL/6 mice (Fig. 1D).

MAPK3-deficient DC are more potent at priminga T-cell response

Since CD86 provides a dominant costimulatory signal in early

T-cell activation [13], we next investigated the capacity of

Mapk3�/� iDC, which express high levels of CD86 and IAb

(Fig. 1A and B), to prime a self-antigen-driven T-cell response.

Although iDC are capable of taking up self-antigen readily they

do not normally present it efficiently to naıve T cells, thus

ensuring immunological tolerance to self-antigen from peripheral

tissue [14]. To determine the capacity of Mapk3�/� iDC to prime

a self-antigen-driven T-cell response, iDC loaded with CNS

antigen MOG35–55 were administered to Rag1�/� mice prior to

the administration of CFSE-labeled naıve 2d2 T cells (expressing

a transgenic TCR for MOG35–55, see the Materials and methods

section). Mapk3�/� DC loaded with MOG35–55 antigen were

much more potent than WT DC at priming naıve 2d2 T cells in

vivo (Fig. 2A). As shown in Fig. 2A, Mapk31/1 iDC were capable

of priming a moderate T-cell response towards MOG35–55 self-

peptide in the draining popliteal LN but not in inguinal,

mesenteric and axillary LN. On the other hand, Mapk3�/� iDC

were not only able to prime naıve T cells to a stronger extent in

the draining popliteal LN but also to a considerable extent in non-

draining LN (Fig. 2A). We also observed an increased T-cell

response when MOG35–55 antigen-loaded Mapk3�/� DC were

incubated with CFSE-labeled naıve 2d2 T cells in vitro (unpub-

lished data). In these in vitro priming experiments we observed a

significant increase in the inflammatory cytokine IL-17 (Fig. 2B).

When we restimulated the resulting MOG35–55-specific T cells, we

observed an increased recall response towards MOG35–55 antigen

in effector T cells restimulated with antigen-loaded Mapk3�/�

iDC as shown by an increased IL-17 production (Fig. 2B).

Of note, MAPK3 does not seem to play a direct role in T-cell

signaling during priming processes: Mapk3�/� naıve T cells were

equally capable of expanding independently of antigen presen-

tation as compared with Mapk31/1 naıve T cells (unpublished

data). To determine the impact of Mapk3�/� in T cells during

priming processes in an antigen-specific setting, we administered

CFSE-labeled naıve Mapk3�/� OT-2 T cells or Mapk31/1 OT-2

T cells (expressing a transgenic TCR for OVA323–339) to Rag1�/�

mice that had been administered OVA323–339-loaded iDC before-

hand. Mapk3�/� OT-2 T cells and Mapk31/1 OT-2 T cells

proliferated at an equal rate when primed with DC loaded with

OVA323–339 antigen (Fig. 2C).

We next studied the influence of MAPK3 during a recall

response to myelin antigen. When we isolated draining LN from

Mapk3�/� mice that had been immunized with MOG35–55

and restimulated sorted CD41 T cells with MOG35–55–loaded

Eur. J. Immunol. 2010. 40: 1–10Ivo Bendix et al.2

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Mapk31/1 iDC, we observed an increased inflammatory recall

response to MOG as shown by increased production of inflam-

matory cytokines (Fig. 2D) and increased T-cell proliferation

(Fig. 2E) ex vivo. The increased recall response to autoantigen

could be further enhanced when Mapk3�/� effector T cells from

isolated LN (that had been primed in the presence of Mapk3�/�

APC in vivo) were restimulated with MOG35–55–loaded Mapk3�/�

DC (Fig. 2D and E).

MAPK3 regulates the autoimmune response inneuroinflammation

The above observations indicate an increased capacity of

Mapk3�/� iDC to prime autoantigen-specific naıve T cells in vivo

and to potentiate a recall response towards autoantigen. To

translate these observations into a model of autoimmune

inflammation, we next investigated the role of MAPK3 in the

EAE animal model of MS. In recent years, DC have been

repeatedly demonstrated to play an important role in the

development of CNS lesions [15–17]. Indeed, DC are present in

close proximity to MS lesions [18]. Furthermore, it has been

suggested that DC, which are already present in inflammatory

areas, activate transmigrating naıve T cells to initiate epitope

spreading [19]. Since we had observed that Mapk3�/� DC are

more immunogenic at priming MOG-specific T-cell responses, we

speculated that MAPK3 might regulate the priming of naıve

T cells towards an inflammatory phenotype during the develop-

ment of neuroinflammation in EAE. In line with previous reports

[9, 10], a deficiency of MAPK3 in both CNS and immune cell

compartments (Mapk3�/� mice) resulted in a mild but not

significant increase in EAE severity when compared with the

disease course of Mapk31/1 littermate mice (Fig. 3A). However,

using chimeric mouse models it became clear that MAPK3 has a

pronounced regulatory role in the immune system. Namely,

Mapk31/1 mice harboring Mapk3�/� BM (KO-WT) exhibited a

severe and rapid form of disease and had to be sacrificed at the

peak of disease (day 18, Fig. 3B). While all KO-WT mice

reached a maximum disease score of at least 4

(Mean7SD 5 4.0470.04 and n 5 7), only 57% of WT-WT

mice reached a disease score of at least 2 (Mean7SD 5 2.0770.4

and n 5 7) 18 days following immunization.

To further dissect the specificity of MAPK3 in regulating the

immune system, we performed EAE in Mapk3�/� mice harboring

Mapk31/1 BM (WT-KO) and compared the disease course with

that from Mapk31/1 mice harboring Mapk31/1 BM (Fig. 3C). As

a further indication for a specific role for MAPK3 in regulating the

immune response during inflammation, we observed a contrary

Figure 1. Surface expression of CD86 and MHC-II on iDC is regulated by MAPK3. (A) BM-derived Mapk31/1 or Mapk3�/� iDC were stained for MHC-II(IAb) and costimulatory markers (CD80, CD86) and measured by FACS. (B) Pooled data from five individual experiments (performed as in (A)) areshown. The geometric mean for unstained controls was subtracted from that of stained samples and bars represent mean (1SD). �po0.05,���po0.001, t-test. (C) BM-derived Mapk31/1 or Mapk3�/� iDC were treated with LPS for 18 h, stained for MHC-II (IAb) and costimulatory markers(CD80, CD86) and measured by FACS. Shown are pooled data from five individual experiments. (D) LN and spleens were extracted from Mapk31/1 orMapk3�/� C57BL/6 mice. Single-cell suspensions were stained for CD11b1CD11c1 DC and anti-CD80, -CD86 and -MHC-II (IAb) antibodies andanalyzed by FACS. Pooled data from three individual experiments are shown, represented as geometric means (1SD).

Eur. J. Immunol. 2010. 40: 1–10 Molecular immunology 3

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role for this MAPK in the CNS during neuroinflammation. As

shown in Fig. 3C, WT-KO mice acquired a significantly milder

course of disease than WT-WT mice, suggesting a role for this

molecule in promoting neuronal damage in the CNS. Indeed, the

same mice have been previously shown to exhibit increased

synaptic plasticity and enhanced behavioral responses [11].

In line with the severity of disease in KO-WT mice

(Fig. 3B), histological examination revealed increased

inflammatory lesions scattered throughout the brain

stem and spinal cord of KO-WT mice as shown by H&E staining

and increased CD31 T-cell infiltration (Fig. 3D). Strikingly,

the CNS of KO-WT mice showed dense accumulation

of CD11c-positive cells close to areas of neuroinflammation

(Fig. 3D).

When we isolated immune cells from brains of sacrificed EAE

animals that had hosted a Mapk3�/� immune system (KO-WT),

Figure 2. MAPK3-deficient iDC are more potent at priming naıve CD41 T cells and at promoting a recall response. (A) BM-derived Mapk31/1 orMapk3�/� iDC were loaded with MOG35–55 peptide and injected i.c. into T-cell-deficient Rag1�/� mice. CFSE-labeled naıve CD41 2d2 T cells wereinjected i.v. 24 h thereafter. The in vivo T-cell proliferation following 5 days is shown, represented by a decrease in CFSE fluorescence intensitymeasured by FACS (the percent of proliferating and non-proliferating cells is depicted above the CFSElo and CFSEhi cell populations, respectively).One experiment representative of three individual experiments is shown. (B) Cytokine secretion following in vitro priming of CFSE-labeled 2d2naıve CD41 T cells (priming) and in vitro activation of 2d2 effector T cells (recall) in a 5-day T cell:iDC (20:1) co-culture. Soluble cytokineconcentrations of IFN-g and IL-17 in the culture supernatant were measured on a Luminex 100 IS analyzer using a Milliplex MAP Mouse Cytokine/Chemokine Panel kit. Fluorescent intensities were converted into cytokine concentrations by using a standard curve. Bars represent mean (1SD)out of three individual experiments analyzed statistically by unpaired two-tailed t-test (�p-value o0.05). (C) BM-derived WT iDC were loaded withOVA323–339 peptide and injected i.c. into T-cell-deficient Rag1�/� mice. CFSE-labeled naıve CD41 T cells from OT-2 Mapk3�/�or OT-2 Mapk1/1 wereinjected i.v. 24 h thereafter. The in vivo T-cell proliferation after 5 day culture is shown, represented by a decrease in CFSE fluorescence intensitymeasured by FACS (the percent of proliferating and non-proliferating cells is shown above the corresponding populations). One experimentrepresentative of three individual experiments is shown. (D) Mapk31/1 and Mapk3�/� C57BL/6 mice were s.c. immunized with MOG35–55 peptide.Ten days later, CD41 T cells were sorted from cell suspensions isolated from draining LN and restimulated with MOG35–55-peptide–loaded Mapk31/1

or Mapk3�/� iDC (as indicated in a criss-cross setup) to determine the extent of antigen recall. The level of cytokine secretion in co-culturesupernatants was measured as described in (B). Bars represent mean (1SD) out of three individual experiments and were statistically analyzedwith Bonferroni’s correction for multiple comparison �po0.05, ��po0.01, ���po0.001. (E) The same CD41 T cells isolated in (C) were labeled withCFSE, and the extent of T-cell response to recall antigen was analyzed by determining T-cell proliferation following 5 days of culture with Mapk31/1

or Mapk3�/� DC (as indicated). The in vitro proliferation of CFSE-stained T cells was measured by FACS as in (A) (the percent of proliferating andnon-proliferating cells is shown above the corresponding populations). Data are representative of three experiments.

Eur. J. Immunol. 2010. 40: 1–10Ivo Bendix et al.4

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CD41 T cells expressed higher amounts of IL-17, in contrast to

T cells in the peripheral LN (Fig. 4A). Since the development of

pathogenic Th17 effector cells and FoxP3-expressing regulatory

Th cells is interrelated [20], we next studied the frequency of

CD41CD251FoxP31 T cells among lymphocytes isolated from

brains and draining LN of non-chimeric Mapk3�/� and Mapk31/1

mice during the EAE course (Fig. 3A), both at peak (day 18) and

remission (day 34). We did not observe any differences in CD41

CD251FoxP31 T-cell frequency in either organ when measuring

both during peak of disease (10.8% in Mapk31/1 CNS versus

9.71% in Mapk3�/� CNS) and during remission (23.7% in

Mapk31/1 CNS versus 22.6% in Mapk3�/� CNS) (Fig. 4B),

suggesting no evidence for a role of MAPK3 in influencing the

development or maintenance of Treg cells during inflammation.

We also observed no differences in FoxP31CD41CD251 T-cell

frequencies or in the regulation of CTLA-4 expression between

T cells isolated from naıve Mapk31/1 and Mapk3�/�

mice (unpublished data). The apparent increase in FoxP3-

expressing cells close to areas of inflammation in the CNS

of EAE animals holding an Mapk3�/� immune system (KOWT)

(Fig. 3D) that were severely sick at this time point (day 18,

Fig. 3B) is probably the result of an increased number of

inflammatory cells altogether and perhaps a failed attempt of

these regulatory cells to compensate for the inflammatory

scenario [21].

Discussion

After several years of research on MAPK3 and its isoforms, our

understanding of their biological role has changed considerably.

It is now clear that despite their strong sequence homology, these

isoforms are functionally not as similar as originally thought and

do play different roles in cellular growth, homeostasis and

development. While it is evident that MAPK1 is necessary for

developmental processes [5–7], MAPK3 does not have any

significant impact on the survival of living organisms since

Mapk3�/� mice are viable and develop normally [8]. However,

from the present report it becomes clear that MAPK3 does play an

important role in controlling the magnitude of an autoimmune

Figure 3. MAPK3 deficiency increases EAE severity. (A) EAE was induced in Mapk31/1 and Mapk3�/� C57BL/6 mice (n 5 6) by immunization withMOG35–55 peptide. Depicted are the mean clinical EAE scores (7SD). (B) To dissect the role of MAPK3 in the immune system duringneuroinflammation, EAE was induced in BM chimeras of WT C57BL/6-CD45.1 mice that received BM transplants from C57BL/6-CD45.2 Mapk31/1 orC57BL/6-CD45.2 Mapk3�/�mice (n 5 7). Depicted are the mean clinical EAE scores (7SD). �po0.05, ��po0.01, Mann–Whitney U-test). (C) BM chimeraswere generated (as described in the Materials and methods section) using Mapk31/1 and Mapk3�/� recipient mice (n 5 8) that were reconstituted withMapk31/1 BM. The mean clinical EAE scores (7SD) are depicted. �po0.05, Mann–Whitney U test. (D) Representative H&E, CD11c, CD3 and FoxP3immunohistological stainings of spinal cord transversal sections from Mapk31/1 and Mapk3�/� EAE mice. Camera: JVC KY-F70 (JVC, Yokohama,Japan); acquisition software: DISKUS (Koenigswinter, Germany) and original magnification: � 200.

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T-cell response by controlling the extent of self-antigen presenta-

tion by DC during an autoimmune attack.

In this study we showed that DC deficient of MAPK3 were

unable to present self-antigen to naıve T cells in a tolerogenic

way (Fig. 2). The increased capacity of Mapk3�/� DC to prime

T cells could be the result of the higher constitutive levels of

MHC-II and CD86 on their cell surface (Fig. 1). CD86 that is

constitutively expressed on DC [22] provides the dominant

costimulatory signal during early T-cell activation and acts

predominately within primary lymphoid organs [13]. None-

theless, Mapk3�/� DC were not only superior at priming a T-cell

response towards myelin antigen but also at potentiating the

effector recall response towards the same self-antigen (Fig. 2).

We speculate that the priming of T cells (independent of whether

they are deficient of MAPK3 or not) by endogenous autoantigen-

loaded MAPK3-deficient iDC leads to the differentiation of T cells

that are generally more potent at generating an efficient recall

response.

DC maturation is recognized as a key event in the induction of

an immune response. DC mature in response to LPS (from

pathogens), TNF-a (from endogenous inflammatory signals) or

CD40L (from T-cell feedback signals). Following maturation with

LPS, the phenotypic differences (MHC-II and CD86) between

Mapk3�/� and Mapk31/1 DC were no longer observed. When

confronted with autoantigen, iDC present it weakly to naıve

T cells to maintain tolerance [14]. We believe that at this point

MAPK3 is a key factor that keeps DC in a partial immature state

(MHC-IIlo and CD86lo), thereby preventing an excessive T-cell

response to autoantigen. Following the initial (priming) signal,

T cells feedback activation signals to the DC via CD40–CD40L

interactions. Following DC maturation by exogenous factors such

as LPS or inflammatory mediators, the levels of CD86 achieved

are no longer regulated by MAPK3.

The superiority of Mapk3�/� DC to prime myelin-specific

responses and to potentiate the recall response towards myelin

suggested that absence of MAPK3 during the course of EAE would

result in a failure of Mapk3�/� DC to regulate the autoimmune

response upon encountering myelin antigen. Indeed, we observed

a pronounced role for MAPK3 in the immune system for regu-

lating neuroinflammation (Fig. 3B). Although a deficiency of

MAPK3 in both CNS and immune cell compartments resulted in a

mild but not significant increase in disease severity (Fig. 3A),

chimeric Mapk31/1 mice harboring Mapk3�/� BM (KO-WT)

suffered a severe course of EAE in comparison to control chimeric

mice (WT-WT) (Fig. 3B). On the other hand, Mapk3�/� mice

harboring Mapk31/1 BM (WT-KO) underwent a significantly

milder course of EAE compared with control chimeric mice

(WT-WT) (Fig. 3C). Since in Mapk3�/� mice, MAPK3 is absent

in both CNS and immune system (and there is no distinction

between both systems), it is most likely that the divergent results

in the course of EAE that we observed in the BM chimeras cancel

out each other in the Mapk3�/� mice such that the EAE outcome

is not significant (Fig. 3A). Our results on Mapk31/1 mice

engrafted with Mapk3�/� BM (KO-WT) revealed that MAPK3

obviously plays a beneficial regulatory role in the immune system

by promoting tolerogenic DC (Fig. 3B). On the other hand

MAPK3 exhibited a detrimental role in the CNS considering the

milder EAE course (Fig. 3C) in Mapk3�/� mice harboring

Mapk31/1 BM (WT-KO). In line with the detrimental role of

MAPK3 in the CNS, MAPK3 seems to prevent synaptic plasticity in

the CNS and negatively controls behavioral responses [11].

In this study, we observed a local Th17 response in the CNS of

mice that lack MAPK3 in the immune system (Fig. 4A). It has

been proposed that the DC present in an inflamed CNS activate

transmigrating naıve T cells to initiate epitope spreading [19].

From our study it seems that MAPK3 is an important factor that

regulates the capacity of DC to prime naıve T cells towards an

inflammatory response. The shift towards IL-17-producing T cells

could be the result of a robust priming by the Mapk3�/� DC that

express high levels of surface costimulatory molecule CD86.

Recently, a report showed that blockade of CD86, but not CD80,

significantly suppressed IL-17 production [23]. In this study,

Figure 4. MAPK3 deficiency in the immune system promotes a Th17 response in the CNS during neuroinflammation. (A) Frequencies of IL-17-producing CD41 T cells in the brain and LN of EAE mice were determined by FACS using intracellular cytokine staining. (B) Frequencies of CD41

CD251FoxP3-expressing T cells (stained with FoxP3 staining kit, eBioscience) in inflamed CNS from Mapk31/1 and Mapk3�/� EAE mice weremeasured by FACS. Data representative of three experiments are depicted.

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inhibition of CD86 resulted in a greater attenuation in T-cell

accumulation in joints of the arthritis animal model than CD80

inhibition. These results suggest that CD86 plays the predomi-

nant role in local recruitment of pathogenic effector Th17 cells.

IL-17 is associated with many inflammatory diseases, including

MS [24], and is known to be induced by IL-23 that is critical for

the establishment and persistence of inflammatory lesions in EAE

[25, 26]. Therefore, another mechanism by which MAPK3 could

modulate Th17 production during CNS inflammation could be via

an increased secretion of IL-23. However, Agrawal et al. failed to

detect any significant differences in the secretion of IL-23 protein

between WT and Mapk�/� DC [10]. Thus, we think that the shift

towards a Th17 phenotype during CNS inflammation is mediated

by the increased expression of CD86 on Mapk3�/� DC.

Although we observed an enrichment of Th17 cells in the CNS

of mice that lack MAPK3 in the immune system, we did not observe

any differences in the frequency of Treg cells (Fig. 4B) or produc-

tion of immunosuppressive cytokines (unpublished data) in the

CNS of Mapk3�/� mice. This further supports our view that the

immune regulation by MAPK3 seems to be mediated by DC. In

macrophages [27] and DC [10], MAPK3 activation has been

reported to result in the induction of IL-10 and down-regulation of

IL-12. MAPK1/3 activation following TLR-4 triggering is mediated

by the MAPK1/3-specific MAP2-kinase MEK-1/2 that is phos-

phorylated by the upstream MAP3-kinase TPL-2. The TPL-2 MAPK

signaling pathway gives rise to a complex network of cytokine

regulation. While it is crucial for promoting the processing of the

secreted form of the proinflammatory cytokine TNF-a [28], recent

work has shown that it induces the anti-inflammatory cytokine

IL-10 and negatively controls the proinflammatory cytokines IL-12

and IFN-b in macrophages and myeloid DC [29].

MAPK3 has also been reported to phosphorylate and activate

CdGAP, which is a negative regulator of the Rho GTPase Cdc42

[30]. This GTPase is an important regulator of actin dynamics.

Indeed, several lines of evidence suggest a role for MAPK3 in the

regulation of the cell cytoskeleton [3, 30, 31] and we did observe

a more polymerized actin cytoskeleton in Mapk3�/� DC when

compared with WT DC (unpublished data). Cdc42 is important

during the early steps of intracellular protein transport by

controlling retrograde transport of protein from the Golgi

complex to the ER [32]. Thus, the increased levels of MHC-II and

CD86 on the cell surface of Mapk3�/� DC could be the result of

increased trafficking of protein to the membrane.

The mechanism via which MAPK3 acts in a detrimental way in

the CNS (Fig. 3C) during inflammation remains an open ques-

tion. We speculate that MAPK3 negatively regulates the expres-

sion of factors important for the survival or growth of CNS cells,

particularly neurons. Neurofibromatosis that involves cognitive

deficits as a result of neuronal sheath tumors is linked to a defi-

ciency in the neurofibromatosis 1 gene Nf1 that controls activa-

tion of Ras and thus downstream MAPK targets. By

downregulating Ras activity in a mouse model of this disease that

shows learning and memory deficits [33], it is indeed possible to

compensate for the Nf1 mutation since cognitive deficits are

reversed [34]. Thus, activation of MAPK3 pathways may be a

mechanism for neuronal damage during conditions involving

cognitive deficits.

We demonstrate here that a lack of MAPK3 precipitates the

development of DC that are capable of priming autoimmune

reactions beyond normal levels and favor the expansion of

pathogenic effector T cells during acute inflammation. Our study

shows that MAPK3 regulates the capacity of DC to prime naıve

T cells but apparently plays no direct role in T cells. An invol-

vement of MAPK3 in the regulation of other immune cell types

such as macrophages and NK cells still needs to be elucidated. In

this study, we propose MAPK3 as a key molecule that instructs DC

to maintain an immature state and present antigen to T cells in

the context of low MHC-II and CD86 surface expression, thereby

downregulating CNS inflammation.

Materials and methods

Mice

C57BL/6 mice were obtained from Charles River Laboratories.

C57BL/6-Tg(Tcra2D2,Tcrb2D2)1Kuch/J (2d2) mice (express a

transgenic TCR recognizing the MOG35–55 epitope) and

B6.129S7/Rag1tm1Mom/J (Rag1�/�) mice were from Jackson

Laboratories. Mapk3�/� mice were kindly provided by Dr. Gilles

Pages, CDTA CNRS, France. Mapk3�/� OT-2 double transgenic

mice were generated by cross-breeding OT-2 mice (C57BL/6-

Tg(TcraTcrb)425Cbn) with Mapk3�/� mice. Animals were bred under

specific pathogen-free conditions at the central animal facility of

the Charite - Universitaetsmedizin Berlin (FEM) and kept in-

house for experiments in individually ventilated cages. Animal

experiments were approved by the state committee for animal

welfare.

Active EAE

Female Mapk31/1 or Mapk3�/� mice, 8–10 wk old, were

immunized s.c. with 200mg MOG35–55 peptide (Pepceuticals)

together with CFA and heat-killed Mycobacterium tuberculosis

(H37Ra, Difco). Bordetella pertussis toxin (350 ng; List Biological

Laboratories) was administered i.p. at days 0 and 2. Active EAE in

BM chimeras was similarly induced using 150mg of MOG35–55

and 200 ng pertussis toxin. Mice were assigned a clinical score

daily: 0, no disease; 1, tail weakness; 2, paraparesis; 3,

paraplegia; 4, paraplegia with forelimb weakness; 5, moribund

or dead animals.

Generation of BM chimeras

Conventional BM chimeras were generated as described

previously [35]. In brief, congenic B6.SJL mice (expressing the

CD45.1 B cell antigen) recipient animals were sublethally

Eur. J. Immunol. 2010. 40: 1–10 Molecular immunology 7

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

irradiated and reconstituted with 12� 106 CD90-depleted (CD90

microbeads, Miltenyi Biotec) donor CD45.2 BM cells isolated

from Mapk31/1 or Mapk3�/� animals. Alternatively, we gener-

ated BM chimera from Mapk31/1 or Mapk3�/� recipient mice

that were reconstituted with B6.SJL BM. Successful engraftment

was determined by FACS after 8 wk.

Isolation of CNS mononuclear cells and intracellularFACS analysis

Isolation of CNS mononuclear cells was performed by tissue

digestion with clostridiopeptidase A (Sigma) and 200 U/mL DNase I

(Roche) for 30 min at 371C. The homogenate was resuspended in

23% Percoll (Amersham Pharmacia) and layered over 73% Percoll.

The gradients were centrifuged for 30 min and the interphase was

collected. The obtained fractions of mononuclear cells were stained

intracellularly with anti-IL-17 and anti-IFN-g (both BD Pharmingen)

and anti-FoxP3 (eBiocience) antibodies according to the manufac-

turers’ protocols and analyzed by FACS using a FACSCanto II flow

cytometer and the FACSDiva software.

Immunohistochemistry

For immunostaining, frozen sections were air-dried, fixed in

acetone and incubated with primary antibodies against CD3

(Dako), FoxP3 (eBioscience) and CD11c (eBioscience) for 30 min.

For detection, secondary biotinylated goat anti-rabbit, rabbit anti-

rat or goat anti-armenian hamster (Dianova) antibodies were used,

followed by the streptavidin alkaline phosphatase kit (Dako) or the

EnVision peroxidase kit (Dako). Alkaline phosphatase was revealed

by Fast Red as chromogen and peroxidase was developed with a

highly sensitive diaminobenzidine (DAB) chromogenic substrate for

approximately 10 min. Negative controls were performed by

omitting the primary antibody.

Generation of mouse BM-derived DC

BM suspensions from femurs of C57BL/6 Mapk31/1 or Mapk3�/�

mice were grown in 100 mm Petri dishes in RPMI-1640 medium

containing 10% FCS (Biochrom) supplemented with 10 ng/mL

GM-CSF (from supernatants of 293FT HEK cells transfected with

pGMCSF, a kind gift from Dr. Alex Scheffold, DRFZ, Berlin). Cells

were replenished with new GM-CSF medium every 3 days. On

day 10, the fully differentiated iDC were harvested.

In vivo and in vitro T-cell priming

iDC derived from BM of Mapk31/1 and Mapk3�/� mice were

incubated for 3h with MOG35–55 peptide (25mg/mL). For in vivo

priming, 4� 106 cells were injected i.c. into T-cell-deficient Rag1�/�

mice. Ten million CFSE-labeled naıve CD412d2 T cells (isolated by

magnetic cell sorting using Naıve T-cell isolation kit, Miltenyi Biotec)

were injected i.v. 24 h later. On day 5, the animals were sacrificed

and single-cell suspensions were prepared from draining popliteal, as

well as inguinal, axillary and mesenterial LN. Cells were stained with

anti-Vb11- and anti-CD4- antibodies and PI was added to exclude

dead cells. In vivo proliferation of living 2d2 T cells (Vb11 CD4-double

positive, PI-negative cells) was measured by assessing the decrease of

CFSE fluorescence on a FACSCanto flow cytometer using the

FACSDiva software (BD Pharmingen). For experiments determining

the direct impact of MAPK3 on T cells, we incubated WT iDC for 3 h

with OVA323–339 peptide (0.3mM) and injected them i.c. into T-cell

deficient Rag1�/� mice. After 24 h, CFSE-labeled naıve CD41 T cells

from OT-2 Mapk3�/� or OT-2 Mapk31/1 double transgenic mice

were injected in the Rag1�/� mice. On day 5, cells from inguinal,

axillary and mesenteric LN were stained with anti-Va2-, anti-CD4-

antibodies and PI. In vivo proliferation of living OT-2 T cells was

measured as above. For in vitro priming, MOG-loaded iDC were co-

incubated at different ratios with CFSE-labeled naıve CD41 2d2

T cells isolated from spleen and LN cells of 2d2 animals by magnetic

cell sorting (as for the in vivo priming experiment above). On day 4,

cells were harvested, stained and measured similarly as for the in

vivo priming assay. Culture supernatants were collected and stored

at –801C for later quantification of soluble cytokines (see Detection of

soluble cytokines below).

Surface staining of costimulatory molecules andMHC-II

BM-derived DC, LN and spleen cells were washed with FACS

buffer and stained with anti-CD80, anti-CD86 and MHC-II (IAb)

antibodies and analyzed by FACS, using a FACSCanto II flow

cytometer and the FACSDiva software (BD Pharmingen).

Detection of soluble cytokines

Culture supernatants of iDC/T-cell co-cultures were analyzed for

soluble cytokines using xMAP (Luminex) technology that combines

principles of flow cytometry and plate-based immunoassays.

Detection of IL-17 and IFN-g were performed with the Milliplex

Map Mouse Cytokine/Chemokine Panel kit in a 96-well microplate

according to the manufacturer’s protocol (Millipore). Briefly, 25mL

of culture supernatant were incubated with the capturing bead

mixture, washed, incubated with biotinylated detection antibodies

and labeled with streptavidin-phycoerythrin. After the final

washing steps, sample plates were run on a Luminex 100 IS

analyzer to measure fluorescent intensities, and cytokine concen-

trations were calculated by comparison with a standard curve.

Statistical analysis

Statistical analysis was performed with SPSS 12 (SPSS, Germany)

and graphical presentation with SigmaPlot 10 (Systat Software,

Eur. J. Immunol. 2010. 40: 1–10Ivo Bendix et al.8

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Germany). For EAE course comparisons, the Mann–Whitney U

test was applied. To compare groups, the Bonferroni correction

for mutiple comparison or the t-test was used. �po0.05,��po0.01, ���po0.001.

Acknowledgements: We thank Dr. Gilles Pages and Professor

Jacques Pouyssegur for providing us with the Mapk3-/- mice;

Nancy Nowakowski, Janet Lips, Eva Katrin Wirth and Simone

Spieckermann for excellent technical assistance and Lena Mann

for carefully proof-reading this manuscript. This work was

supported by a university grant to S.W. (Charite Rahel Hirsch

Scholarship), the Deutsche Forschungsgemeinschaft (DFG) to

F.Z. (SFB 650, SFB-TRR 43), C.F.P. (Exc 257) and C.L. (SFB 650).

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: iDC: immature DC � MOG: myelin oligodendrocyte

glycoprotein

Full correspondence: Dr. Sonia Waiczies, Charite–University Hospital

Berlin, Room 1.12, H.88, Robert-Roessle-Str. 10, 13125 Berlin, Germany

e-mail: sonia.waiczies@charite.de

Additional correspondence: Professor Frauke Zipp, Department of

Neurology, University Medicine Mainz, Johannes Gutenberg University,

Mainz, Germany

e-mail: zipp@uni-mainz.de

Current addresses: Ivo Bendix, Department of Pediatrics I/ Neonatology,

University Hospital Essen, Hufelandstr. 55, 45122 Essen, Germany;

Timour Prozorovski, Department of Neurology, Life-Science-Center 1a,

Heinrich-Heine-University, 40225 Dusseldorf, Germany

Received: 28/8/2009

Revised: 13/1/2010

Accepted: 8/2/2010

Accepted article online: 23/2/2010

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2010. 40: 1–10Ivo Bendix et al.10