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RESEARCH ARTICLE | JUNE 15 2008
Peripheral T Cells Are the Therapeutic Targets of Glucocorticoids in
Experimental Autoimmune Encephalomyelitis1 
Simone Wüst; ... et. al

J Immunol (2008) 180 (12): 8434–8443.

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 The Journal of Immunology

Peripheral T Cells Are the Therapeutic Targets of

Glucocorticoids in Experimental Autoimmune
Encephalomyelitis1

Simone Wüst,* Jens van den Brandt,† Denise Tischner,† Anna Kleiman,‡ Jan P. Tuckermann,‡
Ralf Gold,§ Fred Lühder,2,3* and Holger M. Reichardt2,3†
High-dose glucocorticoid (GC) therapy is widely used to treat multiple sclerosis (MS), but the underlying mechanisms remain
debatable. In this study, we investigated the impact of GC administration on experimental autoimmune encephalomyelitis using

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different GC receptor (GR)-deficient mutants. Heterozygous GR knockout mice were less sensitive to dexamethasone therapy,
indicating that the expression level of the receptor determines therapeutic efficacy. Mice reconstituted with homozygous GR
knockout fetal liver cells showed an earlier onset of the disease and were largely refractory to GC treatment, indicating that the
GR in hematopoietic cells is essential for the beneficial effects of endogenous GCs and dexamethasone. Using cell-type specific
GR-deficient mice, we could demonstrate that GCs mainly act on T cells, while modulation of macrophage function was largely
dispensable in this context. The therapeutic effects were achieved through induction of apoptosis and down-regulation of cell adhesion
molecules in peripheral TH17 and bystander T cells, while similar effects were not observed within the spinal cord. In addition,
dexamethasone inhibited T cell migration into the CNS, confirming that peripheral but not CNS-residing T lymphocytes are the essential
targets of GCs. Collectively, our findings reveal a highly selective mechanism of GC action in experimental autoimmune encephalo-
myelitis and presumably multiple sclerosis. The Journal of Immunology, 2008, 180: 8434 – 8443.

G lucocorticoids (GCs)4 are in widespread clinical use for sults in the release of cytokines such as IFN-␥, TNF-␣, and IL-17,
the treatment of multiple sclerosis (MS), the most prev- which initiate and perpetuate an inflammatory response by ac-
alent chronic autoimmune disease of the CNS (1). So tivating microglia and recruiting macrophages (3, 4). As a con-
far, there is no definitive cure available and, despite considerable sequence, the BBB is breached and allows for the influx of more
progress made during the last decade, the clinical management of T lymphocytes, additional immune cells, and the deposition of
MS is still unsatisfactory. Although optic neuritis and other acute humoral components (5). Subsequently, this process leads to
relapses are efficiently treated with high doses of GCs, therapy myelin destruction, induction of oligodendrocyte death, axonal
may still lead to severe complications or incomplete recovery (2). degeneration, and eventually to the development of severe func-
Thus, a better understanding of this commonly used therapeutic tional deficits (6).
regimen is urgently needed. Traditionally, acute relapses of MS patients are treated with high
In the early phases of MS, autoreactive T cells cross the blood- doses of GCs, especially methylprednisolone, for a limited period
brain barrier (BBB) and are restimulated by local APC. This re- of time (2). Under such conditions they exert a variety of proapop-
totic and anti-inflammatory effects and thereby modulate the sur-
*Institute for Multiple Sclerosis Research, University of Göttingen and Gemeinnütz- vival, migration, and the effector functions of multiple cell types,
ige Hertie-Stiftung, Göttingen; †Department of Cellular and Molecular Immunology, including leukocytes, endothelial cells, and neurons (7). Neverthe-
University of Göttingen, Medical School, Göttingen; ‡Tissue Specific Hormone Ac-
tion group, Leibniz Institute for Age Research–Fritz Lipmann Institute, Jena; and less, the relative contribution of these cell-specific effects for ther-
§
Department of Neurology, St. Josef-Hospital, University of Bochum, Bochum, apeutic efficacy remains unknown. Although most GC actions pre-
Germany
sumably require the presence of the glucocorticoid receptor (GR),
Received for publication February 12, 2008. Accepted for publication April 14, 2008. additional nongenomic effects are discussed that may be exerted
The costs of publication of this article were defrayed in part by the payment of page through nonrelated receptors or interactions with lipid membranes
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact. (8, 9). Thus, neither the cellular nor the molecular mechanisms of
1
This work was supported by grants from Gemeinnützige Hertie-Stiftung (1.01.1/06/ GCs have been fully resolved in the context of MS therapy.
010) and Deutsche Forschungsgemeinschaft (Re1631/1-3, Tu-220/3). GC actions can be studied in experimental autoimmune enceph-
2
F.L. and H.M.R. contributed equally to this work and are listed in alphabetical order. alomyelitis (EAE), a widely recognized rodent model of MS (10).
3
Address correspondence and reprint requests to Dr. Fred Lühder, Institute for Mul- Depending on the experimental setup, distinct features of MS are
tiple Sclerosis Research, University of Göttingen and Gemeinnützige Hertie-Stiftung, recapitulated by EAE. In the case of C57BL/6 mice, immunization
Waldweg 33, 37073 Göttingen, Germany. E-mail address: [Link]@[Link]-
[Link] or Dr. Holger Reichardt, Department of Cellular and Molecular Immu- with myelin oligodendrocyte glycoprotein (MOG) leads to a
nology, University of Göttingen, Medical School, Humboldtallee 34, 37073 Göttin- chronic disease, characterized by a fulminant inflammatory re-
gen, Germany. E-mail address: hreichardt@[Link]
sponse, demyelinating lesions, and subsequent axonal damage.
4
Abbreviations used in this paper: GC, glucocorticoid; MS, multiple sclerosis; BBB,
blood-brain barrier; GR, GC receptor; EAE, experimental autoimmune encephalo-
First attempts to treat EAE by administration of GCs were made
myelitis; MOG, myelin oligodendrocyte glycoprotein; Dex, dexamethasone; CHS, almost half a century ago (11, 12). Meanwhile, a series of exper-
contact hypersensitivity; GITR, glucocorticoid-induced tumor necrosis factor receptor iments were conducted to address the mechanism of GCs in EAE,
family related gene; Treg, regulatory T cell; n.s., nonsignificant.
revealing T cells, macrophages, microglia, and endothelial cells as
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 potential targets (7). For instance, GC administration potentiates

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The Journal of Immunology 8435

lymphocyte apoptosis in Lewis rats suffering from EAE but it moribund; and 10 ⫽ death. To analyze the effects of GC therapy, dexa-
remains elusive whether this is indeed essential for therapeutic methasone-21-dihydrogen phosphate (Dexa-ratiopharm 100 mg; Mer-
efficacy (13, 14). Furthermore, dexamethasone (Dex) profoundly ckle or Fortecortin Inject 100 mg; Merck) was injected daily i.p. on 3
consecutive days.
suppresses cytokine secretion and cell-cell interactions (15). In
particular, it has been found that GCs target components of the Histology and immunohistochemistry
receptor-ligand pairs LFA-1/ICAM-1, VLA-4/VCAM-1, and
Animals were anesthetized with ketamine/hydrochloride (Inresa) and xy-
CD44/HA that play a crucial role in the extravasation of effector T lazine/hydrochloride (Bayer Vital) in 0.9% NaCl and sacrificed by saline
cells through the BBB (15–17). Finally, additional effects of GCs perfusion through the left ventricle. Following fixation with 4% parafor-
on neuronal survival and the function of the microglia have been maldehyde, the spinal cord was removed, postfixed for 2 h, and embedded
postulated (7). However, despite the large amount of data, the rel- in paraffin. Three-micrometer cross-sections were stained with a rat anti-
evance of these different effects, the identity of the major target humanCD3 Ab (1:200; Serotec) or an anti-mouse Mac-3 Ab (1:200; BD
Biosciences) followed by incubation with a secondary biotinylated rabbit
cells, and the primary site of GC action, i.e., leukocytes in the anti-rat Ab (1:200; Vector Laboratories). Ag unmasking was achieved by
periphery vs inflammatory cells in the lesion, remain largely pretreating the sections in 1 mM EDTA (pH 8.0) for 30 min in a microwave
unclear. oven at 850 W. The peroxidase-based ABC detection system (Dako-
Studies in newborn GR knockout mice (18) as well as mice Cytomation) in combination with diaminobenzidine was used for visual-
ization, and all sections were counterstained with hematoxylin. To detect
reconstituted with hematopoietic stem cells have confirmed that

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the disruption of the BBB, the sections were incubated with an anti-
the GR is essential for the induction of thymocyte apoptosis (19). albumin Ab (1:300; Abcam) that was detected with a biotinylated rabbit
More recently, conditional GR knockout mice were developed that anti-sheep Ab (1:300; Southern Biotechnology Associates).
lack the GR in T cells or myeloid cells (20). In an attempt to
dissect the cell type-specific actions of GCs in contact hypersen- Quantification of histological results
sitivity (CHS), a model of allergic dermatitis, we could show that Enumeration of T cells and macrophages was performed in a double-
macrophages and neutrophils are the only essential targets of GCs blinded manner. Ten visual fields of the cervical, thoracic, and lumbar
in the treatment of this disease while control of T cell function was spinal cord were counted at a 400-fold magnification, two infiltrates
per cross-section and three cross-sections per animal. A median was
dispensable (20). In this study, we have used a similar approach to determined and the results were calculated as number of infiltrating
investigate the mechanisms of GC action in EAE. In contrast to cells per mm2.
CHS, the successful treatment of this neuroinflammatory disease Analysis of BBB disruption was performed by gray scale analysis. Six
by GCs depends on the presence of the GR in T lymphocytes but photographs were made of comparable histological areas of cross-sections
not myeloid cells, involves induction of apoptosis and down-reg- of the cervical, thoracic, and lumbar spinal cord at a 200-fold magnification
(three cross-sections per animal). The photographs were analyzed by using
ulation of cell adhesion molecules in peripheral lymphoid organs, Scion Image software and the results of the pixel densities are depicted as
and interferes with T cell migration to the CNS. Thus, we describe arbitrary units.
for the first time the underlying mode of GC action at the cellular
and molecular level in the treatment of EAE and presumably its Isolation of spinal cord infiltrates
human counterpart MS. Lymphocytes were isolated from the spinal cord by density centrifugation
following perfusion of the mice with NaCl. To this end, the dissected tissue
Materials and Methods was passed through a metal mash and homogenized in PBS containing
Mice 0.1% BSA, 1% glucose, and 100 ␮g/ml DNase I (Roche). After centrifu-
gation, the spinal cord homogenate was resuspended in 6 ml of 30% Per-
C57BL/6 mice used for EAE induction were purchased from Charles coll, overlaid on a Percoll gradient containing 4 ml of 45% and 2 ml of 70%
River. GRN⫹/⫺ mice were reported elsewhere (21); GRflox mice (22) were Percoll, and spun for 20 min (2300 rpm, 4°C). Finally, the lymphocytes
bred to mice expressing Cre either as transgene under the control of the were harvested at the interfaces between the layers, washed with PBS, and
proximal lck promoter (lckCre) (23) or having it knocked into the LysM analyzed by flow cytometry.
locus (LysMCre) (24). The different mouse lines were backcrossed to
C57BL/6 mice for at least six generations. All animal experiments were Flow cytometry
approved by the responsible authorities in Lower Saxonia and Bavaria.
All Abs and reagents were obtained from BD Biosciences unless otherwise
Generation of HSC chimeric mice indicated: anti-CD3␧ (145-2C11), anti-CD4 (RM4-5), anti-CD44 (IM7),
anti-CD11a/LFA-1 (2D7), anti-CD49d/VLA-4 (R1-2), anti-GITR (DTA-
GRN⫹/⫺ mice were intercrossed and pregnant females were sacrificed on
1), anti-IL-17 (TC11-18H10.1), anti-active caspase 3 (C92-605), annexin
embryonic day 14.5. Fetuses were dissected and stored in ice-cold PBS
V, and anti-Foxp3 (FJK-16s; eBioscience). The Abs were directly labeled
while genotyping by PCR as previously described (21). The livers of fe-
with FITC, PE, PerCP, PE-Cy7, Cy5, allophycocyanin, or allophycocya-
tuses were removed, passed through a nylon mash, washed, and the cell
nin-Cy7. Extracellular staining was performed as previously described
number counted. In parallel, 6-wk-old female CD45.1- congenic C57BL/6
(26); for the intracellular staining of Foxp3 and caspase 3, we followed the
mice (The Jackson laboratory) were gamma-irradiated at 11.5 Gy (6.5 and
manufacturer’s protocols. To allow for intracellular staining of IL-17, cells
5 Gy with a 4-h intercept). Subsequently, 2 ⫻ 106 fetal liver cells in 500
were stimulated with PMA (5 ng/ml) and ionomycin (500 ng/ml) for 2 h
␮l of PBS were injected i.v. and the reconstituted mice were kept in in-
followed by a treatment with GolgiPlug (BD Biosciences). All analyses
dividually ventilated cages for 3 wk with water supplemented with peni-
were performed on a FACSCanto II device allowing for the detection of six
cillin (Sigma-Aldrich). At an age of 12 wk, the mice were subjected to the
fluorescent dyes (BD Biosciences).
EAE experiments.
EAE induction and therapy Tracking experiments
Mice were immunized with 50 ␮g of MOG35–55 peptide in PBS, emulsified Splenic T cells were isolated from diseased mice by magnetic cell sorting
in an equal volume of CFA containing Mycobacterium tuberculosis using an AUTOMACS machine according to the manufacturer’s instruc-
H37RA (Difco) at a final concentration of 1 mg/ml, and given s.c. into the tions (Miltenyi Biotec). One ⫻ 106 cells/ml were labeled with 50 nM CFSE
flanks as previously described (25). Two injections of pertussis toxin (List for 10 min at 37°C and the reaction was stopped with 2% FCS followed by
Biological Laboratories; 400 ng/mouse in total) were given, one immedi- extensive washings. Cells (5 ⫻ 106) were injected into each recipient
ately after immunization and the second 2 days after immunization. Ani- mouse also suffering from EAE (grades 3 and 4), followed by daily treat-
mals were weighed and scored daily for clinical signs of disease on a scale ment with Dex or PBS for three times, starting 1 h after adoptive transfer.
from 0 to 10 depending on severity; scores were as followed: 0 ⫽ normal; Fifty-eight hours later, the leukocytes were isolated from spinal cord and
1 ⫽ reduced tone of tail; 2 ⫽ limp tail, impaired righting; 3 ⫽ absent spleen and the frequency of the CFSE⫹ cells among the CD3⫹CD4⫹ T
righting; 4 ⫽ gait ataxia; 5 ⫽ mild paraparesis of hind limbs; 6 ⫽ moderate lymphocytes was determined. The ratio of their relative abundance in spi-
paraparesis; 7 ⫽ severe paraparesis or paraplegia; 8 ⫽ tetraparesis; 9 ⫽ nal cord vs spleen was taken as a measure of specific migration.
8436 GCs TARGET PERIPHERAL T CELLS IN EAE

Statistical analysis
Analyses were routinely performed using the Mann-Whitney U test. When
comparing more than two experimental groups, the Kruskal-Wallis test
followed by the Dunn multiple comparison test was used (Microsoft Excel
and GraphPad Prism version 4). Data are depicted as mean values ⫾ SEM;
p values above 0.05 were considered as nonsignificant (n.s.); ⴱ, p ⬍ 0.05;
ⴱⴱ, p ⬍ 0.01; and ⴱⴱⴱ, p ⬍ 0.001. To determine differences referring to the
disease course, the whole curves rather than individual time points were
compared between experimental groups. Strictly speaking, statistical anal-
ysis was performed from the onset of the disease (preventive setting) or the
day after the first Dex treatment (therapeutic setting) until the end of the
observation period.

Results
Administration of dexamethasone ameliorates MOG-induced
EAE in mice
The beneficial effect of GCs is well established in the treatment of

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human MS and monophasic EAE in the Lewis rat, but has not yet
been systematically applied to the chronic MOG-induced EAE
model in C57BL/6 mice. Therefore, we first investigated whether
GC application around the time of immunization impacts on the
disease course. Dex was chosen instead of methylprednisolone
because it is most commonly used for the treatment of EAE in
rodents (7) and has a higher potency in reducing clinical symp-
toms in C57BL/6 mice (our unpublished data). EAE was in-
duced by immunization with MOG and at days ⫺1, 0, and 1 we
administered 100 or 20 mg/kg Dex i.p. In this preventive set-
ting, the onset and the severity of the disease were significantly
reduced, an effect that lasted until the end of the observation
period (Fig. 1A).
Next, we turned to the clinically more relevant therapeutic set-
ting. Dex administration was started when more than half of the
mice showed first signs of disease, resulting in an average score of
2–3 at the 10-point scale. When Dex was applied at an ultrahigh
dose of 100 mg/kg for 3 days, therapy led to an immediate ame-
lioration of the disease (Fig. 1B). However, a few days later, EAE
exaggerated again, although its severity remained below the one of
untreated mice (Fig. 1B). To establish a dose-efficiency relation-
ship, we tested a series of different hormone concentrations (Fig.
1C). The therapeutic effect of Dex gradually decreased at lower
doses but remained efficient even at 0.8 mg/kg (Fig. 1C). Taken
together, GCs are an effective means to treat EAE in C57BL/6
mice, both in a preventive and a therapeutic setting, and act in a
dose-dependent manner.
FIGURE 1. Dex administration interferes with EAE. A, In a preventive
GCs impact on leukocyte infiltration and the BBB setting, EAE was induced by immunization with MOG and the mice were
To investigate whether the clinical efficacy of GCs in EAE therapy treated with the indicated doses of Dex for 3 days, starting 1 day before
was reflected by a reduction in leukocyte infiltration, we performed immunization (arrows); n ⫽ 8 for each group, statistical analysis: days
an immunohistochemical analysis of spinal cord sections from 6 –31. B, In a therapeutic setting, Dex was applied three times after the first
appearance of clinical signs, starting at an average score of 2–3 (arrows);
mice that had been treated with 100 mg/kg Dex either in a pre-
n ⫽ 11 for each group, statistical analysis: days 11–30. C, In a therapeutic
ventive or a therapeutic setting. T cells and macrophages that com- setting, different doses of Dex were applied after the first appearance of
prise the majority of the CNS infiltrate were almost absent from clinical signs (arrows); n ⫽ 5 for each group, statistical analysis: days
the spinal cord of mice that had undergone preventive Dex admin- 10 –27.
istration. In therapeutically treated mice, the infiltrate was less but
still significantly reduced (Fig. 2, A and B). Another hallmark of
EAE and MS is the disruption of the BBB, which can be studied marginally affected their relative abundances (Fig. 2C). The frequency
by staining spinal cord sections for the presence of albumin. Both, of LFA⫹ and CD44⫹ T cells, which represent characteristic cell pop-
in the preventive and the therapeutic setting, the BBB was partially ulations present in CNS lesions of diseased mice, also remained un-
protected by Dex administration, indicating that GCs impact on the altered (Fig. 2C). This suggests that GC administration reduces infil-
integrity of the BBB (Fig. 2, A and B). tration of the spinal cord largely independent of the cell type.
To determine the influence of Dex administration on the cellular
composition of the spinal cord infiltrate, we isolated leukocytes by Therapeutic GC effects in EAE require the presence of the GR
density centrifugation using a Percoll gradient and analyzed them and are receptor dosage dependent
by flow cytometry. GC treatment significantly reduced the absolute There is a long-standing debate as to whether GC therapy of MS and
numbers of TH17 cells as well as other T lymphocytes, but only EAE is mediated by the nuclear GC receptor (GR). Alternatively, it
The Journal of Immunology 8437

A T cells Macrophages Albumin

Control

FIGURE 2. Dex suppresses leuko-

cyte infiltration and preserves BBB
integrity. A, Immunohistochemical
stainings for infiltrating T cells, mac-
rophages, and the integrity of the BBB
(albumin) in the cervical spinal cord of Dex
one control and one therapeutically therap.
Dex- treated mouse sacrificed on day
12. Original magnification, ⫻200. B,
Quantification of immunohistochemi-

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cal stainings of spinal cord in control
and Dex-treated mice for T cells, mac-
B T cells Macrophages Albumin
rophages, and albumin as in A. Each
symbol represents one animal. n ⫽ *** *** *
5–7. C, Quantification of T cell subsets
* * **

Mac3+ cells / mm2

CD3+ cells / mm2

arbitrary units
in the spinal cord of control and Dex-
treated mice by flow cytometry follow-
ing isolation by density centrifugation.
IL-17 (intracellular), LFA-1, and CD44
(surface) expression was determined
after gating on live CD3⫹CD4⫹ T
cells. Total cell numbers refer to all
l

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.
tro

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ap

ev

ap

ev

ap

ev
cells isolated from the spinal cord of a
on

on

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pr

pr
er

er

er
th

th

th
C

C
ex

ex

ex
mouse and percentages refer to the rel-
ex

ex

ex
D

D
D

D
ative frequency of the respective cell
population among the CD4⫹ T cells.
n ⫽ 8. Control ⫽ PBS-treated mice; C IL-17+ IL-17- IL-17 + LFA-1 + CD44 +
Dex prev. ⫽ preventive application of n.s. n.s.
100 mg/kg Dex on days ⫺1, 0, and 1; * *
Dex therap. ⫽ therapeutic application
percentage
total cells

total cells

of 100 mg/kg Dex on days 9 –11.

*
.

.
th l

th l

th r o l

th rol

th rol
ex n t r o

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could act through a yet unknown alternative receptor or by inter- topathology, we analyzed spinal cord sections of Dex-treated EAE
action with lipid membranes (9, 27). To address this question, we mice (Fig. 3B). Importantly, we found higher numbers of infiltrat-
first studied heterozygous GR knockout mice (GRN⫹/⫺) be- ing T cells and macrophages and a reduced integrity of the BBB in
cause homozygous mutants die around birth due to lung atel- GRN⫹/⫺ mice. This suggests that the GR is essential for GC ther-
ectasis (18). Importantly, GR expression in those animals is apy in EAE.
reduced by half and T cells show an increased resistance to
GC-induced apoptosis (21).
We first compared the clinical course of EAE between GRN⫹/⫹ The GR in hematopoietic cells is central to the therapeutic
and GRN⫹/⫺ mice and found a minor but statistically significant efficacy of GCs in EAE
difference in the disease course (Fig. 3A). This indicates that en- A plethora of potential GC target cells is discussed in the context
dogenous GCs impact on EAE via the GR. Next, we tested the of EAE therapy but their individual relevance is unknown. Among
therapeutic protocol on these mice. EAE was induced by MOG them are hematopoietic cells, mainly T cells and macrophages, and
immunization and, once the mice showed the first symptoms, they nonhematopoietic cells such as endothelial and neural cells. To
were injected with Dex for 3 consecutive days. To reveal a poten- discriminate between these two major tissue compartments, we
tial gene dosage effect, we used a concentration of only 4 mg/kg, generated chimeric mice in which GR expression was exclusively
which was still effective in wild-type mice (Fig. 1C). Dex admin- abrogated in the hematopoietic lineage. CD45.1-congenic mice
istration to GRN⫹/⫹ control mice led to a halt of disease progres- were lethally irradiated and reconstituted by transplantation of
sion, while the same dose was not effective in heterozygous CD45.2⫹ fetal liver cells from embryonic 14.5-day-old GRN⫹/⫹
GRN⫹/⫺ mice (Fig. 3A). To answer whether the impact of GC control or homozygous GRN⫺/⫺ embryos. As confirmed by flow
treatment on the disease course was also reflected by immunohis- cytometry, all nonhematopoietic cell types in the resulting
8438 GCs TARGET PERIPHERAL T CELLS IN EAE

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FIGURE 3. Haploinsufficiency of the GR attenuates the therapeutic ef-
fects of GC treatment. A, GRN⫹/⫹ and GRN⫹/⫺ mice were therapeutically
treated with 4 mg/kg Dex or PBS (control), starting at an average disease
score of 2–3 (arrows). n ⫽ 7; statistical analysis: days 14 –23. B, Immu-
nohistochemical analyses of T cells, macrophages, and albumin in the spi-
nal cord of GRN⫹/⫺ and GRN⫹/⫹ mice treated with Dex as in A and
sacrificed 6 h after the last injection. Each symbol represents one animal.
n ⫽ 8 –10.

GRNHSC⫹/⫹ and GRNHSC⫺/⫺ mice were wild type while the leu-
kocytes either lacked or expressed the GR. The number of repop-
ulating lymphocytes as well as their subset distribution was similar
after reconstitution in both groups (data not shown), which is in
accordance with previously published results (19). A functional
test further showed that T cells from GRNHSC⫺/⫺ mice were com-
pletely refractory to Dex- induced apoptosis, confirming the suc-
cessful generation of chimeric mice (data not shown).
MOG-EAE was induced in the two types of chimeric mice fol-
lowed by administration of 100 mg/kg Dex to half of each group
at time points when an average disease score of 2–3 was reached.
Notably, EAE was more severe in untreated GRNHSC⫺/⫺ mice as FIGURE 4. The GR in the hematopoietic system is required for the
compared with GRNHSC⫹/⫹ controls, manifesting in a premature anti-inflammatory effects of Dex in EAE. A, MOG-EAE was induced in
disease onset and rapid morbidity (Fig. 4A). This was confirmed by HSC chimeric mice followed by therapy with 100 mg/kg Dex, starting at
an average score of 2–3 (GRNHSC⫺/⫺ mice ⫽ dotted arrows, GRNHSC⫹/⫹
statistical analysis based on three individual experiments, reveal-
mice ⫽ full arrows); n ⫽ 6, statistical analysis: days 13–23, one represen-
ing that GRNHSC⫺/⫺ mutants showed clinical symptoms on aver- tative experiment of three is shown. The cross indicates that all animals
age 4 days earlier than control mice (Fig. 4B). This suggests that were prematurely sacrificed for ethical reasons. B, Kaplan-Meier curve
endogenous GCs exert a suppressive effect on hematopoietic cells illustrating the day of the disease onset in GRNHSC⫹/⫹ (n ⫽ 22; dotted line)
and thereby reduce the susceptibility to EAE in wild-type mice. compared with GRNHSC⫺/⫺ mice (n ⫽ 26; full line). C and D, Immuno-
Next, we studied the effect of therapeutic GCs in the chimeric histochemical analyses of T cells (C), macrophages (C), and albumin (D)
mice. As expected, GRNHSC⫹/⫹ mice could be efficiently treated in the spinal cord of GRNHSC⫹/⫹ and GRNHSC⫺/⫺ mice that had been
by Dex, resulting in an immediate and long-term amelioration of treated with Dex or PBS (control (Con)) as in A and sacrificed 6 h after the
the disease, while GRNHSC⫺/⫺ mice hardly responded to high- last injection. Each symbol represents one animal. n ⫽ 3– 6.
dose GC therapy (Fig. 4A). Although we observed a mild and
transient therapeutic effect in mutants on the first day, the disease
rapidly aggravated and eventually all GRNHSC⫺/⫺ mice became Thus, the presence of the GR in hematopoietic cells is essential for
moribund within a short period of time irrespective of Dex treat- the therapeutic success of Dex administration.
ment (Fig. 4A). Immunohistochemical analysis of the spinal cord GC treatment partially restores the integrity of the BBB (Fig. 2,
confirmed that Dex failed to diminish the number of T cells and A and B), but it is unclear whether this is achieved through direct
macrophages in GRNHSC⫺/⫺ mice while the infiltrate in GC actions on endothelial cells or whether it is rather due to a
GRNHSC⫹/⫹ control mice was significantly reduced (Fig. 4C). diminished production of proinflammatory cytokines by leukocytes in
The Journal of Immunology 8439

the lesion. Interestingly, GCs did not impact on the permeability of the
BBB in GRNHSC⫺/⫺ mice despite the presence of the GR in endo-
thelial cells (Fig. 4D). This indicates that Dex primarily reduces lym-
phocyte infiltration of the CNS and that the protective effect on the
BBB is therefore mainly indirect.

T cells but not macrophages are the major targets of GCs

To further dissect the therapeutic Dex effect on hematopoietic
cells, we studied the individual contribution of T cells vs macro-
phages, which are the main CNS-infiltrating cell types in MOG-
EAE. To this end, we analyzed cell type-specific GR knockout
mice lacking the receptor in either of the two cellular compart-
ments (20, 28). First, we investigated GRlysMCre mice in which the
GR is absent from myeloid cells. Recombination of the floxed GR
allele in these animals is almost complete in macrophages and

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neutrophils, whereas almost no depletion is observed in T and B
lymphocytes (20). The onset of the disease in GRflox control and
mutant GRlysMCre mice was comparable. Furthermore, EAE in
mice of both strains could be efficiently treated with Dex, each
resulting in a three-point lower disease score as compared with
untreated mice (Fig. 5A). Thus, modulation of macrophage func-
tions does not significantly contribute to the efficacy of GCs in
EAE therapy.
To reveal a potential role of the GR in T lymphocytes, we used
GRlckCre mice (28), which are capable of mounting normal T cell-
dependent immune responses, e.g., in CHS (20). First, the onset of
EAE after immunization with MOG was significantly earlier in
GRlckCre mice as compared with GRflox controls (Fig. 5B). This is
in line with the premature development of clinical signs in
GRNHSC⫺/⫺ mice (see Fig. 4B) and demonstrates that endogenous
GCs exert an inhibitory effect on the susceptibility to EAE via their
action on T cells. Second, there was a clear attenuation of the
therapeutic GC effect (Fig. 5B). Three daily injections of Dex, FIGURE 5. T cells but not myeloid cells are essential targets of GCs
starting at an average disease score of 2–3 (day 8 for GRlckCre and in the treatment of EAE. A, Mice lacking the GR in macrophages
day 12 for GRflox), resulted in an immediate halt of the disease (GRlysMcre) and GRflox control mice were therapeutically treated with
progression and a long-term reduced disease severity in GRflox 100 mg/kg Dex (arrows); n ⫽ 5, one representative experiment of two
is shown; statistical analysis: days 13–17. B, Mice lacking the GR in T
control mice (Fig. 5B). In contrast, GRlckCre mice only transiently
cells (GRlckCre) and GRflox control mice were therapeutically treated
responded to the administration of Dex and already on the second with 100 mg/kg Dex (GRlckCre mice ⫽ marked by full arrows; GRflox
day of the treatment the disease aggravated again. During the next control mice ⫽ marked by dotted arrows); n ⫽ 5, one representative
few days, the disease worsened substantially in both PBS- and experiment of two is shown; statistical analysis: days 9 –17 and 13–17,
Dex- treated mice, leading to very high clinical scores, for which respectively. The cross indicates that all animals were prematurely sac-
reason the majority of mice had to be sacrificed for ethical reasons rificed for ethical reasons; in the GRlckCre group treated with PBS half
(Fig. 5B). In conclusion, our findings demonstrate that T cells are of the mice had to be sacrificed.
the hematopoietic cell population that is most relevant for the ther-
apeutic efficacy of GCs in the treatment of EAE.

GCs induce T cell apoptosis in peripheral lymphoid organs but Next, we studied splenic T cells from GRflox and GRlckCre mice.
not in the CNS Interestingly, the level of apoptosis in CD4⫹ T cells from GRflox
Having established T lymphocytes as the major targets of GCs control animals was significantly increased by Dex at 58 h. This
during EAE, we investigated the biological processes that were was also true for LFA-1⫹ T lymphocytes known to home to the
affected by this treatment. At first, we studied induction of T cell site of inflammation, as well as for T cells expressing the activation
apoptosis in the spinal cord of wild-type mice that had been treated marker and cell adhesion molecule CD44 (Fig. 6C and data not
three times with Dex after the onset of EAE by annexin V staining shown). To assess the level of apoptosis in TH17 cells, we had to
and flow cytometry. Detailed analysis of the disease course re- study caspase 3 activation ex vivo due to the incompatibility of
vealed that GC administration gradually reduced the clinical symp- intracellular cytokine staining with annexin V binding. Indeed, the
toms (Fig. 6A). Concomitantly, the number of apoptotic T cells in number of apoptotic IL-17⫹ cells was significantly increased fol-
the CNS increased in both experimental groups between 10 and lowing Dex treatment as compared with controls, indicating that
58 h after the beginning of treatment, presumably as a consequence GC treatment also targets bona fide encephalitogenic effector T
of activation-induced cell death. Unexpectedly, however, induc- cells in the spleen (Fig. 6D). Most importantly, the level of apo-
tion of apoptosis in infiltrating T cells in the spinal cord was com- ptosis was unaltered in all cell populations of GRlckCre mice after
pletely unaltered by Dex at both time points (Fig. 6B). This sug- Dex administration, confirming that induction of cell death re-
gests that therapeutic efficiency of GCs in EAE is not a quires the presence of the GR in T cells (Fig. 6, C and D). In
consequence of enhanced cell death in the CNS lesion itself. summary, peripheral but not infiltrating T cells undergo enhanced
8440 GCs TARGET PERIPHERAL T CELLS IN EAE

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FIGURE 7. Impact of GC treatment on Treg, expression of adhesion
FIGURE 6. GCs induce apoptosis in peripheral but not CNS-infiltrating molecules, and T cell migration. A, GRflox and GRlckCre mice were thera-
T cells. A, C57BL/6 mice were daily treated with Dex (dashed line) or PBS peutically treated daily for three times with Dex or PBS, starting at an
(solid line) three times after the disease onset at an average score of 4 average score of 2–3 and after 58 h the splenocytes were analyzed by flow
(arrows); the clinical score was determined at the time of injection and 10, cytometry. Treg were identified among the CD3⫹CD4⫹ cells by the con-
34, and 58 h later; n ⫽ 14. B, Leukocytes were isolated at 10 and 58 h from comitant expression of GITR and Foxp3 and their relative frequency is
the spinal cord of C57BL/6 mice treated with Dex or PBS as in A and indicated in each dot blot (left panel). One representative analysis is de-
apoptosis was determined in CD3⫹CD4⫹ cells by flow cytometry based on picted; n ⫽ 3–11. The relative expression level of Foxp3 on Treg was
the binding of annexin V. n ⫽ 6 –13. C, Splenocytes were isolated from GRflox determined as the mean fluorescence intensity (MFI; right panel). B, Using
and GRlckCre mice at 58 h after the treatment with Dex or PBS as in A and the same mice as in A, surface expression of LFA-1, VLA-4, and CD44 on
apoptosis was determined in CD3⫹CD4⫹ cells (left panel) or CD3⫹CD4⫹ splenocytes was determined by flow cytometry. One repre-
CD3⫹CD4⫹LFA-1⫹ cells (right panel) by flow cytometry. n ⫽ 5. D, Spleno- sentative analysis is depicted; n ⫽ 3–11. C, Splenic T cells were isolated
cytes from PBS-treated mice were isolated as in C and cultured ex vivo in the from diseased mice, labeled with CFSE, and retransferred into recipient
absence (control (Con)) or presence of 10⫺7 M Dex for 20 h. Subsequently, the mice also suffering from EAE. The mice were daily treated with Dex or
degree of apoptosis was determined by intracellular staining for active caspase PBS three times, starting 1 h after adoptive transfer. Fifty-eight hours later,
3 in combination with an anti-IL-17 Ab. n ⫽ 2. the leukocytes were isolated from spinal cord and spleen and the percent-
ages of CFSE⫹ cells among the CD3⫹CD4⫹ T lymphocytes were deter-
mined in both organs. The ratio of their relative frequencies in spinal cord
apoptosis after GC treatment, suggesting that an impaired recruit- and spleen is depicted as a measure of specific migration. n ⫽ 3.
ment of T cells to the site of inflammation must be central to
therapeutic efficiency.
crease the relative number of Treg, which was postulated to con-
Regulatory T cells (Treg) do not account for the beneficial effect tribute to their anti-inflammatory activity (30, 31). Therefore, we
of GCs on EAE wondered whether Dex administration might have an effect on Treg
Naturally occurring CD4⫹CD25⫹Foxp3⫹ Treg play a crucial role in our model. To address this question, we induced EAE in GRflox
in the control of pathogenic immune responses such as EAE and and GRlckCre mice and treated them three times with Dex similar
MS (29). Experimental evidence further suggested that GCs in- to the previous experiments. Subsequently, Treg were identified
The Journal of Immunology 8441

among the CD4⫹ T cells in spleen (Fig. 7A) and spinal cord (data anism underlying GC actions in neuroinflammatory disorders is
not shown) based on the concomitant expression of FoxP3 and still missing.
GITR. Against all expectations, Dex administration rather dimin- Traditionally, GCs are thought to act through their cognate re-
ished than increased the relative frequency of Treg in the periphery ceptor, the GR (8). However, the observation of rapid responses
as well as in the lesions of GRflox mice. Noteworthy, this effect was challenged the model that all GC actions were transcriptionally
much less pronounced in GRlckCre mice. Moreover, Dex also re- mediated through the GR and supported the hypothesis that so-
duced the expression level of FoxP3 in splenic Treg of GRflox called nongenomic effects exist (27). These could be mediated by
mice while it remained unaltered in GRlckCre mice (Fig. 7A). the GR itself (33) through an unrelated protein as recently ob-
Taken together, these data essentially rule out that Treg contrib- served for the estrogen receptor (34) or even by unspecific phys-
ute to the anti-inflammatory activity of GCs in MOG-induced icochemical interactions with lipid membranes. From this it is de-
EAE in C57BL/6 mice. batable whether the GR is necessary for the therapeutic effects of
GCs or not. Our experiments now unequivocally demonstrate that
Cell adhesion molecules on T cells are down-regulated the GR is responsible at least for most GC actions in the treatment
by GCs of EAE and presumably MS. There are three lines of evidence in
Because Dex administration reduces T cell infiltration in the spinal favor of this notion. 1) Heterozygous GRN⫹/⫺ mice are signifi-
cord without directly inducing cell death in situ, one could assume cantly less susceptible to Dex treatment as compared with wild-

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that GCs presumably block the recruitment of new leukocytes to type littermates. Thus, the level of the GR determines therapeutic
the site of inflammation. Although induction of peripheral T cell success, indicating that GCs impact on the disease via the GR
apoptosis is one mechanism, down-regulation of cell adhesion itself. 2) Mice reconstituted with fetal liver cells from homozygous
molecules could be another. To test this hypothesis, we determined GRN⫺/⫺ mice are almost fully refractory to GC therapy. The fact
surface expression of CD44, LFA-1, and VLA-4 on CD3⫹CD4⫹ that the GR is completely absent from all hematopoietic cells in
splenocytes, which all represent molecules that are involved in T these animals confirms that GC effects on leukocytes in EAE
cell extravasation and homing to the inflamed CNS during EAE intervention require the presence of the receptor itself. 3) Simi-
(16, 17). Importantly, all three surface receptors were down-reg- larly, T cell-specific GR knockout mice hardly respond to Dex,
ulated on splenic T cells from Dex-treated GRflox mice while their underscoring the relevance of GR actions. We believe that this
expression remained unaltered in GRlckCre mice (Fig. 7B). It is proves that the majority of GC actions in EAE depend on the
noteworthy that expression of CD44, LFA-1, and VLA-4 was only presence of the GR. Consequently, the contribution of other mech-
diminished on peripheral T cells after Dex administration while anisms of GC action is, at best, of minor importance.
CNS-residing lymphocytes were completely unaffected (data not In the context of EAE therapy, GCs act on a plethora of cell
shown). Thus, GCs not only reduce the number of peripheral T types. T lymphocytes, macrophages, microglia, endothelial cells,
cells through induction of apoptosis but also compromise their and neurons are only the most important to mention (7). Therefore,
ability to replenish the pool of infiltrating T cells in the CNS by we wondered whether GC actions on all these cell types were
repressing cell adhesion molecules needed for homing to the site of equally crucial for the efficacy of GC therapy or whether this treat-
inflammation. ment primarily impacts on a specific subset. Our studies with HSC
chimeric mice and conditional knockout mouse strains now show
Dex interferes with T cell migration to the CNS that T cells are the most important targets of Dex in the treatment
of EAE. Mice that lack the GR in all hematopoietic cells while
The previous experiments suggested that GCs might interfere with retaining expression in endothelial and neural cells were almost
T cell migration to the spinal cord. To test this hypothesis, we fully refractory to Dex treatment and did not restore the integrity
performed a tracking experiment in the context of an ongoing of the BBB. The same was true for T cell-specific GR mutant mice,
EAE. Splenic T cells were isolated from strongly diseased wild- whereas myeloid cell-specific GR mutants respond normally to GC
type mice and labeled with CFSE. Subsequently, 5 ⫻ 106 cells treatment. In contrast, a small but reproducible effect of Dex on the
were adoptively transferred i.v. into syngeneic mice also suffering disease course was observed in GRNHSC⫺/⫺ and GRlckCre mice,
from EAE (grades 3– 4) and allowed to equilibrate for 1 h. The indicating that nonhematopoietic cell types may, to a minor extent,
mice were then injected with Dex or PBS daily for three times and also contribute to the therapeutic efficacy of GCs. In the first place,
58 h after the cell transfer they were sacrificed, and the frequency this applies to endothelial cells because the adhesion molecules
of the CFSE⫹ cells among the CD3⫹CD4⫹ T cells was determined ICAM-1 and VCAM-1 (35) and the chemokine CXCL10/IFN-␥-
by flow cytometry. As a measure of specific cell migration to the inducible protein 10 (36) are known to be down-regulated after GC
CNS, we analyzed the abundance of CFSE-labeled cells in the administration. However, the integrity of the BBB was not restored
spinal cord relative to their abundance in the spleen. Most impor- by Dex in GRNHSC⫺/⫺ mice, suggesting that a direct impact on
tantly, our results indicate that the frequency of CFSE⫹ infiltrating endothelial cells is at best transient. An alternative explanation for
T cells in the spinal cord was significantly reduced by GC therapy the limited effects of GC treatment in GRNHSC⫺/⫺ and GRlckCre
(Fig. 7C). This suggests that Dex interferes with T cell migration mice would be nongenomic effects mediated through a GR-inde-
and thereby diminishes the fresh supply of peripheral T cells to the pendent mechanism. Thus, additional experiments are required to
CNS lesion. distinguish between these two possibilities. In summary, our data
suggest that GC effects on T cells are most crucial for the efficacy
Discussion of Dex in the treatment of EAE.
High-dose GC therapy is the measure of choice to treat acute re- Several mechanisms are currently discussed on how GCs impact
lapses such as optic neuritis in MS patients (2). Currently, the on T cells in the treatment of MS and EAE. One is elimination of
standard regimen is a daily injection of 0.5–2.0 g of methylpred- T lymphocytes by apoptosis, which was previously demonstrated
nisolone i.v. for 5 days followed by a rapid taper (32). However, for the monophasic EAE model in Lewis rats (13, 14, 37). We
adverse effects such as aseptic bone necrosis or psychosis may could now show that GCs indeed induce apoptosis in TH17 effector
sometimes complicate the treatment. Although this highlights the as well as other T cells. However, in contrast to previous reports
need for improved compounds, a deep understanding of the mech- (38), increased apoptosis after Dex administration was restricted to
8442 GCs TARGET PERIPHERAL T CELLS IN EAE

peripheral T lymphocytes and not seen in infiltrating T lympho- Acknowledgments

cytes. Our observation that the level of cell death in the CNS We thank Niklas Beyersdorf for help with the generation of the HSC chi-
increases over the 3-day observation period (see Fig. 6B) offers a meric mice and Alexandra Bohl, Martina Weig, Amina Bassibas, and
possible explanation for this discrepancy. Apparently, due to the Nicole Tasch for technical assistance.
high local levels of proinflammatory cytokines, pronounced acti-
vation-induced cell death occurs during chronic EAE in the spinal Disclosures
The authors have no financial conflict of interest.
cord of C57BL/6 mice, possibly mediated through Fas or galec-
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