Article

DAB389IL-2 suppresses autoimmune inflammation in the CNS and inhibits T cell-mediated lysis of glial target cells

Department of Radiology, Temple University School of Medicine, Philadelphia, PA 19140.
Experimental and Molecular Pathology (Impact Factor: 2.71). 07/2013; 96(1). DOI: 10.1016/j.yexmp.2013.07.004
Source: PubMed

ABSTRACT

In multiple sclerosis (MS) and its rodent model, experimental autoimmune encephalomyelitis (EAE), activated CD4+ T cells with upregulated IL-2R mediate inflammation and demyelination in the central nervous system (CNS). DAB389IL-2, a chimeric fusion protein of IL-2 and diphtheria toxin, inhibits human and rodent IL-2 activated T cells that express the high affinity interleukin-2 receptor. In the present study, DAB389IL-2 was used to treat rats with EAE. We wanted to investigate the possibility that DAB389IL-2 could prevent tissue destruction within the CNS. We used a suboptimal dose of DAB389IL-2 that allowed substantial transmigration of inflammatory cells across the blood-brain barrier. DAB389IL-2 inhibited infiltration of CD4(+), CD8(+), CD25(+) and TCR αβ(+) associated mononuclear cells and inflammatory macrophages in the spinal cord on day 13 post-immunization, at the peak of disease. Gene expression study showed that DAB389IL-2 treatment suppressed TNF-α and IFN-γ as well as IL-10 cytokine gene expression in the spinal cord of rats with EAE on day 13. DAB389IL-2 in vitro treatment suppressed cytotoxicity of MBP-activated T cells from rats with EAE against oligodendrocytes in culture by 66%. Astrocytes were less targeted by MBP activated T cells in vitro. This study suggests that DAB389IL-2 directly targets CD4(+) and CD25(+) (IL-2R) T cells and effector T cell function and also indirectly suppresses the activation of macrophage CD169(+) (ED3(+)) and microglia CD11b/c (OX42(+)) populations in the CNS.

DAB
389
IL-2 suppresses autoimmune inammation in the CNS and
inhibits T cell-mediated lysis of glial target cells
Mahendra K. Bhopale
a
, Brendan Hilliard
b
, Cris S. Constantinescu
c
, Toshiki Fujioka
d
, Elvira Ventura
e
,
S. Michael Phillips
f
, Abdolmohamad Rostami
g,
a
Department of Radiology, Temple University School of Medicine, Philadelphia, PA 19140, USA
b
Department of Medicine, Temple University School of Medicine, Philadelphia, PA 19140, USA
c
Division of Clinical Neurology, University of Nottingham, England, UK
d
Department of Neurology, Ohashi Hospital, Toho University, Tokyo, Japan
e
Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
f
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
g
Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107, USA
abstractarticle info
Article history:
Received 9 July 2013
Available online xxxx
Keywords:
EAE
DAB
389
IL-2
Oligodendrocyte
T cell cytotoxicity
Cytokine expression
In multiple sclerosis (MS) and its rodent model, experimental autoimmune encephalomyelitis (EAE), activated
CD4
+
T cells with upregulated IL-2R mediate inammation and demyelination in the central nervous system
(CNS). DAB
389
IL-2, a chimeric fusion p rotein of IL-2 and diphtheria toxin, inhib its human and rodent IL-2
activated T cells that express the high afnity interleukin-2 receptor. In the present study, DAB
389
IL-2
was used to treat rats with EAE. We wanted to investigate the possibility that DAB
389
IL-2 could prevent
tissue destruction within the CNS. We u sed a suboptimal dose of DAB
389
IL-2 that allowed s ubstantial trans-
migration of inammatory cells across the bloodbrain ba rrier. D AB
389
IL-2 inhibited inltration of CD4
+
,
CD8
+
,CD25
+
and TCR αβ
+
associated mononuclear cells and inammatory macrophages in the spinal
cord on day 13 po st-immunization, at the peak of disease. Gene expression study showed that DAB
389
IL-2
treatment s uppres sed TNF-α and IFN-γ as well as IL-10 cytokine g ene expression in the spinal cord of
rats with EAE on day 1 3. DAB
389
IL-2 in vitro treatment suppressed cyt otoxici ty of MBP-activated T cells
from rats with EAE against oligodendrocytes in culture by 66%. Astrocytes were less targeted by MBP activated
T cells in vitro. This study suggests that DAB
389
IL-2 directly targets CD4
+
and CD25
+
(IL-2R) T cells and effector
T cell function and also indirectly suppresses the activation of macrophage CD169
+
(ED3
+
) and microglia
CD11b/c (OX42
+
) populations in the CNS.
© 2013 Elsevier Inc. All rights reserved.
Introduction
Multiple sclerosis (MS) is an autoimmune, T-lymphocyte-dependent,
chronic inammatory demyelinating disease of the central nervous sys-
tem (CNS). The inammatory autoimmune response is believed to be
contingent upon activated T cells that produce demyelination through
direct or indirect interactions with myelin and/or oligodendr ocyte s
(Duszczyszyn et al., 2006; Goverman et al., 2005; Haegele et al., 2007;
Ratts et al., 2006; Stüve et al., 2006). The disease is characterized by
the progressive destruction of myelin and axonal loss, with glial scar
formation and frequent motor and sensory dysfunction. Activation of
myelin-specic T cells results in the upregulation of interleukin-2
(IL-2) and IL-2 receptor alpha (IL-2Rα)(CD25)(Lavasani et al., 2007;
Pastor et al., 2006). The effector CD4
+
T cells that mediate EAE, the
experimental model of MS in rodents, are antigen restricted, IL-2 re-
sponsive, and bear the high-afnity IL-2R, i.e. IL-2Rα,IL-2Rβ (CD122)
and IL-2Rγ (CD132) component chains. EAE pathogenesis results from
an induced myelin-specic T cell response when effector CD4
+
T helper
cells cross the bloodbrain barrier and are re-stimulated by antigen pre-
senting cells (APCs) in the target tissue.
The bloodbrain barrier (BBB) disruption is an early and central
event in MS and EAE pathogenesis. Once effector T cells have gained
entry into the CNS, the microenvironment of the CNS becomes very im-
portant in the subsequent interaction between inltrating cells and res-
ident CNS cells. Encephalitogenic T cells initiate the inammatory
process and release cytokines that attract and activate macrophages
that are considered necessary for pathogenesis of EAE (Nguyen et al.,
1994).
Pathological lesions of EAE in the CNS are characterized at disease
onset by the inltration of large numbers of mononuclear cell s, in-
cluding CD4
+
IL-2R
+
T lymphoc ytes, at the time of disease onset.
At this time macrophage/micro glia also become activated and partic-
ipate in the process of inammatory dem yelina tion. Ma crophages
Experimental and Molecular Pathology xxx (2013) xxxxxx
Corresponding author at: Department of Neurology, Thomas Jefferson University,
Health Professions Aca dem ic Building, 901 Walnut Street, Suite 400, Philadelphia,
PA 19107, USA. Fax: +1 215 955 1390.
E-mail address: a.m.rostami@jefferson.edu (A. Rostami).
YEXMP-03486; No of Pages 10
0014-4800/$ see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.yexmp.2013.07.004
Contents lists available at ScienceDirect
Experimental and Molecular Pathology
journal homepage: www.elsevier.com/locate/yexmp
Please cite this article as: Bhopale, M.K., et al., DAB
389
IL-2 suppresses autoimmune inammation in the CNS and inhibits T cell-mediated lysis of
glial target cells, Experimental and Molecular Pathology (2013), http://dx.doi.org/10.1016/j.yexmp.2013.07.004
Page 1
play an important role in autoimmune disease. In rats, CD68
+
(ED1)
and CD169 (ED3) ant igen exp ressing macrophages originate from
perivascular cells (Graeber et al., 1990). Diseased tissues express
the CD169 antigen, which is a receptor for glycoconjugates con-
taining sialic acid. Ia antig ens (class II major histocompatibility
com plex antigens) are found on B-lymp hocytes, ma crophages and
microglia, epidermal cells, macrophages and oligodendrocytes in EAE
(Klyushnenkova and Vanguri, 1997; Papadopoulos et al., 2006). MHC
II expression in the CNS precedes inammatory demyelination in MS
(Ponomarev et al., 2005). Among resident CNS cells, microglial cells
are the most abundant MHC II-expressing populations in Lewis rats
(Miller et al., 2007).
Treatment with IL-2- diphtheria toxin fusion protein, DAB
389
IL-2,
suppressed EAE in rat and mouse models (Phillips et al., 2007, 2010).
DAB
389
IL-2 is a single chain polypeptide comprised of IL-2 linked to
the membran e association region and th e enzyma tically active do-
main of diphtheria toxin (DT) (Phillips et al., 2007). Three versions,
DAB
389
IL-2, DAB
486
IL-2 and DT
390
IL-2, suppressed IL-2 activated T
lymphocytes in vitro and in vivo (Bacha et al., 1992; Balcer et al.,
1991; Hayashi and Kanagawa, 2006; Kadin and Vonderheid, 2010;
Kawakami et al., 2006; Lansigan et al., 2010; Natarajan et al., 2011;
Pacheco-Silva et al., 1992; Potala and Verma, 2010; Williams et al.,
1987, 1990a,b). Macrophage and T cell activation inuence the CNS
microenvironment by producing pro-inammatory cytokines such
as IL-1β,IL-12,IL-23,TNF-α, IL-17 and GMCSF, which resu lts in
cell-mediated immune pathology in EAE (El-Behi et al., 2011; Sutton
et al., 2006). Activated MBP T cells when directly interacting with oligo-
dendrocytes also cause cytotoxicity (Kawai and Zweiman, 1988).
Our previous studies have shown that DAB
389
IL-2 treatment at later
time points was detrimental (Phillips et al., 2007). In order to examine
whether DAB
389
IL-2 indirectly affects macrophage/microglia activities,
we have conducted experiments using DAB
389
IL-2 at a dose of 1600
kU on days 7 and 9 to avoid detrimental effects in rats and to allow
study of macrophage and microglial activation and the components of
T cell subsets in the CNS. In the present study, we reported the in vivo
effects of the DAB
389
IL-2 fusion protein on the inltration of leukocytes,
particularly T cells and macrophages, the phenotype of microglia, and
the expression of selected CD4
+
T cell-related cytokines during the
development of EAE in rats. We also examined how this fusion protein
affects CD4
+
T cell cytotoxicity against oligodendrocytes and astrocytes
in vitro. The purpose of this study is to examine whether DAB
389
IL-2
indirectly affects macrophage/microglia activities and also the effect of
treatment with this fusion protein on cells downstream from CD4
+
T
effector cells.
Materials and methods
Reagents
DAB IL-2 fusion protein (DAB
389
IL-2) was provided by Seragen, Inc.,
Hopkinton, MA. Gal-C and GFAP antibodies were provided by Dr. David
Pleasure, Department of Neurology, University of California Davis, and
LDH release assay kit was purchased from Boehringer, Inc. (Manheim,
WI); Mycobacterium tuberculosis (Dibco); monoclonal antibodies for
immunohistochemistry were purchased from PharMingen (San Diego,
CA); Luxol fast blue from Sigma; PCR products- PCR buffer II (Perkin-
Elmer, Foster City, CA); dATP, dCPT, dGTP, dTTP (Boeringer, Inc.);
revers e trans cription from Gibco-BRL (Gaithersbur g, MD).
Animals
Lewis rats, female 810 weeks old, were purchased from Charles
River Laboratories (Raleigh, NC). They were kept ve to a cage with an
unrestricted supply of food and water. All studies were performed in
strict accordance with the policies of the University of Pennsylvania
Institute of Animal Care and Use Committee.
Induction of EAE and clinical assessment
Myelin basic protein (MBP) was prepared from guinea pig spinal cord
homogenate (gpSCH-MBP) as previously described (Deibler et al., 1972).
Eight- to 10-week-old Lewis rats were immunized each with 30 μgof
gpSCH-MBP emulsied in CFA suspension of 5 mg/ml of killed and
dried Mycobacterium tuberculos is -H37RA (Difco) in the median footpad
of each hind foot. Each EAE rat received 1600 kU (i.v. injection) of
DAB
389
IL-2 while control rats received an equivalent volume of PBS. We
report here the effect of a suboptimal dosing regimen that allowed us to
study the in situ effects in the CNS of 1600 kU DAB
389
IL-2 on day 7 and
days 7 and 9 for clinical symptoms, and days 7 and 9 to study the transmi-
gration of T cell populations and activation of macrophages and microglia
cellsatasemi-effectivedoseduringthecourseoftheacutephaseofin-
ammatory disease in Lewis rats. Control mutants of DAB
389
IL-2 were
treated in the same way as rats. Mutant DAglu
53
B
389
IL-2 (
M
DAB-IL-2) is
enzymatically inactive but, similar to DAB
389
IL-2, binds to the high afnity
IL-2 receptor. Mutant DAB
389
IL-2
819
(DAB-IL-2
M
) does not bind to
the IL-2 receptor but is enzymatically active, similar to DAB
389
IL-2.
Clinical scores were given as follows: 0 (no abnormality), 1 (limp tail),
2 (hind limb weakness), 3 (partial hind limb paralysis), 4 (total hind
limb paralysis) and 5 (death).
Histological studies
EAE rats from different groups were sacriced at or prior to onset of
peak disease on day 13 for histological study of inammation and de-
myelination at the time when hind limb weakness developed. Animals
under anesthesia were perfused through the left ventricle with
phosphate-buffered saline (PBS) followed by phosphate-bu ffered
10% formaldehyde. Spinal cords were removed and xed in the
same solution. Parafn-embedded 7 μm -thick cross sections of spi-
nal cord tissues were stained with hemato xylin and eosin in order
to conrm inammati on (Deibler et al. 1972). Another set of slides
was s tained with Luxol fast blue (0.5 g of Luxol fast blue (Sigma),
500 ml 95% ethanol, 5 ml 10% acetic acid) for demyelination stud ies.
Slides were incub ated for 48 h at 56 °C and washed in 95% ethanol.
Staine d sectio ns were differentiated in Li
2
CO
3
and counterstained
with hematoxylin for the observation of demyelination (Sadler et
al., 1991). For ultra-t hin se ctions of spinal cord, tiss ues were xed
in 3% glutaral dehyde, post xed in 1% osmium tetroxide for 12h
and embedded in p lastic resin; 1-μm thick cr oss sections were cut
and stained with toluidine blue fo r examination of demyelination
(Calida et al., 2000).
Immunohistochemistry of spinal cord
Immunohistochemistry of cryosections of spinal cord from untreated
and DAB
389
IL-2-treated groups was performed as descr ibed previ-
ously (Phillips et al., 2007). Sp inal cords were snap frozen in liquid
nitrogen. Cryostat sec tions (14 μm) were xed (4 °C) in acetone
and endogenous peroxidase activity was blocked in 0.3 % hydrogen
per oxide in PBS after incubation i n PBS containing 5% normal goat
serum followed by primary antibodies (1:100 or 1:200) against rat
CD4, CD8, CD25 and TCRαβ, CD169 (ED3), CD11b/c (OX42), CD74
(OX6) (PharMingen, San Diego, CA) reaction for 1 h. Antibodies were
detected by biotinylated p olyclonal goat anti-mouse immunoglobulin
(PharMingen) and ABC peroxidase staining kit (Pierce Inc., Rockford,
IL) followin g the manufacturer's instru ctions. Spinal cord lesions
were assessed for the n umber of positively stained cells per mm
2
area at 200x magnication. Positive cells were differentiated using
the avidinbiotin technique (Vec tastain Elite Kit Vector Labs,
Burlingame, CA). Data were quantied by counting positive cells using
the JAVA program.
2 M.K. Bhopale et al. / Experimental and Molecular Pathology xxx (2013) xxxxxx
Please cite this article as: Bhopale, M.K., et al., DAB
389
IL-2 suppresses autoimmune inammation in the CNS and inhibits T cell-mediated lysis of
glial target cells, Experimental and Molecular Pathology (2013), http://dx.doi.org/10.1016/j.yexmp.2013.07.004
Page 2
Oligodendrocyte and astrocyte culture
Lewis rat oligodendrocyte- and astrocyte-enriched cultures were
derived from the brain of a 1-day old pup (Kawai and Zweiman, 1988,
1990). Briey, the primary brain culture was established in a T75 ask
at 37 °C in 5% CO
2
and 95% humidity in Eagle's minimum medium
supplemented with 10% fetal calf serum, 2 mM glutamine, 2 mg/ml
glucose, 5 μg/ml penicillin, and 50 μg/ml streptomycin. After 13 days'
in vitro culture, oligodendrocytes were isolated by shaking in an orbital
shaker and passing through nylon meshes of 70 μ,40μ and 20 μ,then
plated on poly-
L-lysine coated rounded cover slips (8 mm diameter
size) placed in 24 well plates at a density of 2 × 10
4
cells per cover
glass. Astrocytes were obtained from primary cell cultures after gentle
shaking, then plated in T75 asks for 2 hrs and oating glial cells were
removed. These cells were grown in asks for one week. Astrocytes
were then collected after trypsinization and washing in DMEM 10%
FCS medium and plated on poly-L-lysine coated cover slips placed in
24 well plates.
DAB
389
IL-2 treatment of restimulated MBP-reactive T cell culture in vitro
Primary cultures of lymph node cells obtained from PBS a nd
DAB
389
IL-2 treated EAE rats, on day 13 post-immunization separately,
were stimulated with 5 μg/ml puried MBP in RPMI-1640 medium con-
taining 2% rat normal serum at 37 °C in 5% CO
2
and 95% humidity. After
4 days of incubation, MBP reactive T cells were restimulated with MBP
for 3 days in RPMI-1640 medium containing 5% FCS and used for cyto-
toxic assay to study the effect of in vivo treated DAB
389
IL-2 on effector T
cells against target oligodendrocyte cells for 20 h incubation.
Ex vivo DAB
389
IL-2 treatment of MBP-reactive T cells
MBP reactive T cells obtained from the lymph nodes of MBP im-
munized rats on day 13 post-immunization. MBP reactive T cells
were treated, without restimulating cells in vitro, with either PBS
or DAB
389
IL-2 (0.2 μg/ml) for 4 h in RPMI-1640 growth medium con-
taining 10% FCS culture medium and were then washed wi th RPMI
growth medium three times followed by RPMI-1640 1% serum medium
to study the ex-vivo effect of DAB
389
IL-2 treated effector T cells against
the target oligodendrocyte cells for 4 h incubation.
Cytotoxicity assay
Cytotoxicity assays of MBP-reactive T cells against oligodendro-
cytes or astrocytes were performed using an LDH release assay
(Boehringer, Inc.) according to the manufacturer's instructions. Target
cells (oligodendrocytes [97% Gal-C positive] or astrocytes [93% GFAP
positive]) were cultivated on individual cover slips in the wells of sterile
24 well tissue culture plates, Target cells were suspended separately at
three different densities 1250, 5000, and 20000 and incubated over-
night at 37 °C in 5% CO
2
and 95% humidity. MBP reactive T cells
(2 × 10
5
) were added to corresponding wells containing the adherent
brain cells of either oligodendocytes or astrocytes for Effector Target ra-
tios of 160, 40 and 10 respectively. Non-specic T cells were used from a
group of female Lewis rats, weighing 140150 g. These rats were im-
munized with 150 μg of synthetic peptide SP-26 corresponding to the
5378 amino acid sequence of bovine myelin P2 protein as reported
previously (Rostami et al., 1990). Briey, 150 μg of high-performance
liquid chromatography (HPLC)-puried SP26 was injected into the
hind foot pads subcutaneously with 100 μl of phosphate buffered saline
and 100 μl of complete Freund's adjuvant (CFA, Sigma, St. Louis, MO)
per rat.
Both effector MBP reactive T cells and target glial cells (oligodendro-
cytes or astrocytes) were suspended in DMEM 1% FBS medium and
were allowed to incubate for a period of 4 h or 20 h as per the
experiment. M aximum LDH release was determined by adding 2%
triton X-100 in a total volume of 100 μl containing oligodendrocytes
or astrocytes in a culture medium. Supernatant of 100 μl/well was
removed from each well and transferred to a 96 well plate. The absor-
bance of the samples at 490 nm (reference 600 nm wavelength) was
measured by using an ELISA plate reader.
Cytotoxicity percentage was calculated as follows
% Cytotoxicity ¼ Experimental OD
490
ðÞSpontaneous OD
490
ðÞ100
Maximum LDH release OD
490
ðÞ
RT-PCR
Total RNA was prepared from 10 mm
3
tissue samples of lumbar
spinal cords from PBS-treated and DAB
389
IL-2 treated EAE rats following
the method described previously (Siegling et al., 1994). PCR primer
pairs for the 5 and 3 region of β-actin, tumor necrosis factor (TNF)-α,
Interferon (IFN)-γ, IL-4 and IL-10 sequences were selected by using
the following OLIGO program:β-actin sense primer 5-CTA TCG GCAA
TG AGC GGTTC-3 and antisense primer 5-CTT AGG AGT TGG GGG
TGG CT-3;IFN-γ sense primer 5-CCC TCT CTGG CTGTTACTGC-3 and
antisense primer 5-CTCCTT TTCC GCTTCCTTAG-3;TNF-α specic
sense primer 5-CGAGTGACAA GCCCGTAGCC-3 and antisense primer
5-GGATGAACAC GCCAGTCGCC-3; IL-4 specic sense primer 5-ATG
CACCGAG ATGTTTGTACC-3 and antisense primer 5-TTTCAGTGTT CT
GAGCGTGGA-3; IL-10-specic sense primer 5-TGCCTTCAG T CA AGT
GAAGACT-3 and antisense primer 5-AAA CTCATTC ATGGC CTTGTA-
3 amplied to 35 cycles. The PCR product was electrophoresed on 1.6
% agarose gel for separation. Analysis of the amplied product was
performed by an image analyzer (BIO-RAD Gel Doc 1000) in a semi-
quantitative PCR for cytokine IFN-γ,TNF-α, IL-4 and IL-10 gene expres-
sion. Cytokine mRNA amounts were expressed as a ratio of β-actin
mRNA amounts in order to compensate for the variable efciency of
PCR amplication of each cytokine.
Statistical analysis
The statistical signi
cance of differences between groups was
rst evaluated by utilizing the Student's t-test (analysis of variance).
Statistical signica nce of differences was determined by the analysis
of P values using the StudentNewmanKuels and Dunnett tests.
Differences between pairs were considered signicant at P b 0.05.
Experiments were replic ated at least three times, giving similar re-
sults. The gures show the mean value ± SEMs of a representative
experiment with four or more animals. Student's t test was applied
for comparing histological scores, body weights a nd clinical s cores
and cytotoxicity by using the StateWork program. Values of P b 0.05
were considered signicant.
Results
In order t o examine whether DAB
389
IL-2 directly affects th e com -
ponents of T cell s ubsets and indirectly affects macrophage/microglia
in the CNS, we conducted experiments using DAB
389
IL-2 at a dose of
1600 kU on days 7 and 9 to avoid detrimental effects in rats. The im-
mune mechanisms by which the IL-2 fusion protein downregulates
monocyte/macrophage acti vity within the CNS is a reductio n in
number of C D4
+
effector cells along with a reduced number of mac-
rop hages migrating into the CNS, causing inhibi tion of cytotoxicity
against oligodendrocytes. The cause of IL-10 suppression is unknown.
At this timepoint IL-10 producing cells were drastically affected due to
the toxic effect of DAB
389
IL-2 as this cytokine is considered to be an
essential molecule in reducing EAE-mediated inammation.
3M.K. Bhopale et al. / Experimental and Molecular Pathology xxx (2013) xxxxxx
Please cite this article as: Bhopale, M.K., et al., DAB
389
IL-2 suppresses autoimmune inammation in the CNS and inhibits T cell-mediated lysis of
glial target cells, Experimental and Molecular Pathology (2013), http://dx.doi.org/10.1016/j.yexmp.2013.07.004
Page 3
DAB
389
IL-2 suppresses clinical and histological features of EAE
All rats inoculated with gpSCH-MBP and CFA developed neurological
signs between days 9 and 13 post-immunization; peaking on day 13 at
clinical scores of 3.04.0 (Fig. 1A). Rats treated with DAB
389
IL-2 on day 7
or days 7 and 9 developed lower grade neurological clinical score
(mean ± SD = 1.79 ± 0.84) than PBS-treated EAE animals (3.0 ±
1.26) (P N 0.055) Histological examination of all rats sacriced on
day 13 post-immunization at the peak of clinical disease showed in-
ammation and demyelination. Staining was performed on spinal
cord sections from animals on days 8 and 13 after immunization.
On day 8, rats immunized with CFA and CFA + gpSCH-MBP animals
had identical H&E staining showing no pathology (Fi g. 1 B). All an-
imals that received CFA + gpSCH-MBP had i nammation with
perivascular cufng consi stent with EAE on day 13 (Fig. 1C). On
day 8 CFA + gpSCH-MBP-immunized animals showed normal
myelin staining, while on day 13 they showed slight demy elin-
ation when stained with Luxol fast blue (Fig. 1D). Demyeli nation
was more evident at day 13 in untreated EAE animals when
assessed by to luidine b lue on Epon embedded 1 μ thick sections
(Fig. 1E).
DAB
389
IL-2 suppresses mononuclear T cells and macrophage/microglial
cells in EAE
All immunized rats, with and without DAB
389
IL-2 treatment, scored
grade 0 as normal at days 7 and 8 post-immunization with no inltra-
tion of any of the cell types examined histologically at day 8. However,
rats that had manifested clinical disease on day 13 showed large num-
bers of mononuclear cells (MNC) and macrophages in the histology of
spinal cords. Immunohistochemistry for CD4
+
,CD8
+
,CD25
+
and TCR
αβ
+
cells showed a decrease in MNC cell population in the spinal
cords of DAB
389
IL-2 treated rats (Table 1; Fig. 1F). CD169
+
,CD11b/c
+
,
and CD74
+
cell inltrates in the spinal cord were observed considerably
les s frequently in DAB
389
IL-2 treated EAE rats on day 13 (Table 1).
Immunohistochemical assessment of spinal cords from PBS and
Fig. 1. A. Clinical scores of rats treated on days 7 or 7 and 9 with 1600 kU DAB
389
IL-2 or with phosphate-buffered saline (PBS). Clinical score values are mean ± SEM for four animals. B. EAE
rat spinal cord parafnsectionsof7μm stained with H&E showing no inammation on day 8. C. EAE rat spinal cord parafnsectionsof7μm stained with H&E showing inammation on
day 13. D. EAE rat spinal cord parafnsectionsof7 μm stained with Luxol Fast blue, counterstained with hematoxylin, showing inammation and slight demyelination on day 13. E. EAE rat
spinal cord section embedded in Epon, cut at 1 μ; section stained with toluidine blue. In this white matter section there are several demyelinated large axons of which the axon cylinder is
preserved (magnication of ×1000). F. EAE rat spinal cord cryosections (14 μm) stained with CD4
+
,CD8
+
,CD25
+
and TCR-αβ
+
. Treatment with DAB
389
IL-2 (1600 kU/rat i.v.) was given
on days 7 and 9 before onset of clinical signs.Rats were sacriced on day 13. DAB
389
IL-2affects inhibition of T cells in the spinal cord. CD4, CD8, CD25 and TCRαβ positive cells present in the
spinal cord lesions were assessed by number of positive stained cells per mm2 area at 200× magnication. There was a drastic reduction in marked stained cell populations of CD4
+
(P b 0.005); CD8
+
(P b 0.002); CD25
+
(P b 0.005) and TCR-αβ
+
(P b 0.035).
4 M.K. Bhopale et al. / Experimental and Molecular Pathology xxx (2013) xxxxxx
Please cite this article as: Bhopale, M.K., et al., DAB
389
IL-2 suppresses autoimmune inammation in the CNS and inhibits T cell-mediated lysis of
glial target cells, Experimental and Molecular Pathology (2013), http://dx.doi.org/10.1016/j.yexmp.2013.07.004
Page 4
DAB
389
IL-2 treated EAE rats showed a decrease in C D169
+
cells
(53.94 ± 30.2 vs. DAB
389
IL-2 4.13 ± 1.51 P b 0.002) (Fig. 2A); CD11b/c
+
cells (83.07 ± 25.2 vs. DAB
389
IL-2 29.59 ± 14.18 P b 0.001) (Fig. 2B)
and CD74
+
cells (73.75 ± 25.13 vs. DAB
389
IL-2 36.5 ± 15.71 P b 0.012)
(Fig. 2C) with their respective total MNC. Thus, DAB
389
IL-2 indirectly
or directly suppresses the activation of macrophage/microglia cells in
the spinal cord. Our study suggests that the fusion protein DAB
389
IL-2
inhibited activation of macrophages/microglia.
Table 1
Inhibition of immune mononuclear cells inltrated into CNS by the treatment of DAB
389
IL-2. A comparative illustration of mononuclear cells specic stained lymphocytes in spinal cord
sections from rats treated with PBS or DAB
389
IL-2 (1600 kU) on days 7 and 9.
Cell type Number of cells/mm
2
area
Mononuclear cells (average) Speciccell
PBS DAB
389
IL-2 % cell inhibition % cells inhibition
231.06 101.20 56.20 CD4
+
69.85
184.40 77.41 58.02 CD8
+
63.06
229.69 91.26 60.26 CD25
+
54.93
206.33 88.63 57.04 TCRαβ
+
62.09
159.03 36.43 79.59 CD169
+
92.34
133.96 78.40 41.47 CD11b/c
+
64.37
124.65 85.23 31.66 CD74
+
50.50
Four rats in each group, treated with either PBS or DAB
389
IL-2, were sacriced on day 13. Spinal cords from PBS or DAB
389
IL-2 treated rats were sectioned and stained and counted in mm
2
area. Cells were quantied by percent cell inhibition. Data represent mean of six serial sections of four animals in each group.
Fig. 2. Quantitative immunohistological studies of mononuclear cell inltration in spinal cord section (14 μm) i n mm
2
area of EAE and DAB
389
IL-2 treated rats with respect t o
A. CD169
+
,B.CD11b/c
+
and C. CD74
+
markers. DAB
389
IL-2treatment(1600 kU/rati.v.)givenondays7and9beforeonsetofclinicalsigns.Ratsfromeachgroupweresacriced
on day 13 for histological studies. DAB
389
IL-2 inhibits mononuclear cells and macrophage/microglia cell population in the spinal cord sections. P hotographs showing reduced
number of macrophag e (CD169
+
) and microglia (CD11b/c
+
) stained cells, respectively, in the spinal cords of DAB
389
IL-2 treated rats (magnication of × 200).
5M.K. Bhopale et al. / Experimental and Molecular Pathology xxx (2013) xxxxxx
Please cite this article as: Bhopale, M.K., et al., DAB
389
IL-2 suppresses autoimmune inammation in the CNS and inhibits T cell-mediated lysis of
glial target cells, Experimental and Molecular Pathology (2013), http://dx.doi.org/10.1016/j.yexmp.2013.07.004
Page 5
DAB
389
IL-2 suppresses the cytotoxicity of MBP-activated T cells
against oligodendrocytes
Primary cultures of gpSCMBP reactive T cells derived from lymph
node cells of untreated or DAB
389
IL-2 treated EAE were restimulated
with gpSCMBP in RPMI 10% FCS medium followed by 1% serum medium
to use for cytotoxic assay. Cytotoxicity levels of MBP reactive T cells from
EAE rats against oligodendrocytes showed a signicantly higher value
than against astrocytes ( 71.4% vs. 41.5% respectively) (P b 0.001).
At the diseas e recovery pha se on day 30, activa ted T cells showed
an insignicant difference in cytotoxicity effect between oligoden-
drocytes (50.1%) and astrocytes (42.4%). MBP reactive T cells did
not make any di fferenc e at the beginning of disease on day 9 in
PBS -treated (51.3%) an d DAB
389
IL-2 treated T cells (53.2%) against
oligodendrocyte cytotoxicity. T cells obt ained from rats after recov-
ery from disease (day 30 post-immunization) had reduced oligoden-
dro cyte cytoto xic ity (54.1%) compared to the acute phase of disease,
and DAB
389
IL-2 treatment of these T cells further reduc ed cytotoxic-
ity (42.13%); however, this d ifference did not reach statistical signif-
icance. There was a signicant decrease in cytotoxicity level of
DAB
389
IL-2 treated MBP reactive T cells against oligodendrocytes
(27.9%) when compared with the same numbe r of PBS -treated MBP
activated T cells (71.4%) (P b 0.001). T cells obtained from non-
imm unized rats treated with DAB
389
IL-2 (31.7%) showed insigni-
cant change (P N 0.050). Nonspecic MBP-activated T cells from
lymph nodes of rats with EAN showed an insi gnicant cy totoxicit y
effect on oligodendrocytes and astrocytes (Table 2, Fig. 3A, B, C, D, E).
DAB
389
IL-2 ex-vivo treatment suppresses MBP reactive T cells
MBP reactive T cells derived from the lymph nodes of EAE rats ,
without re-stimulat ion in vitro, were treated either with PBS or
DAB
389
IL-2 for 4 h and then washed with RPMI gro wth medium
three times followed by RPMI-1640 1% serum medium. MBP reactive
Tcells,treatedoruntreatedwithDAB
389
IL-2, showed a dose-dependent
cytotoxicity against oligodendrocytes with increasing effector:target ra-
tios (Table 3; Fig. 3F). Spontaneous LDH release by oligodendrocytes at
4 h incubation was 1.082 ± 0.533 and 2.723 ± 2.32 at 20 h (n =3).
MBP reacti ve T cells irradiated with γ-radiation to make antigen pre-
senting cells (APC) did not make a ny difference in PBS treated
(51.303%) and DAB
389
IL-2 treated T cells (53.24%) a gainst oligoden-
dro cyte cytotoxicity.
DAB
389
IL-2 treatment suppresses TNF-α,IFN-γ and IL-10 but not IL-4 gene
expression by RT-PCR
Analysis of cytokine expression of spinal cords showed signicantly
decreased IFN-γ and TNF-α gene expression in DAB
389
IL-2 treated EAE
rats as compared to control PBS-treated EAE rats on day 13 at the peak
of disease. IFN-γ and IL-10 gene expression was also signicantly
decreased in spinal cord tissue samples of DAB
389
IL-2 treated EAE rats
(Fig. 4A, B). These results favor the downregulation of Th1 cytokines
and up-regulation of Th2 cytokines by DAB
389
IL-2 treatment on day
13. Characteristically high expression of IFN-γ wasseeninthespinal
cord tissue of gpSCH-MBP-immunized rats treated with PBS and also
the mutant chimeric proteins DAB
389
IL-2 glu- and DAB
389
IL-2 del-
treated samples (Fig. 4C).
Discussion
Because DAB
389
IL-2 suppresses immunoreactivity in vitro and
in vivo by inactivation of antigen or IL-2 activated lymphocytes
(Bacha et al., 1988, 1991; Williams et a l., 1990a,b), we examined
the effect of tre atment with this fusion protein on cells downstrea m
from the CD4
+
T effector cells in experimental autoimmune disease.
We conducted our study with two doses of DAB
389
IL-2 (1600 kU) on
days 7 and 9 such that treatment did not completely inhibit the inltra-
tion of mononuclear cells. Otherwise, as has been shown, repeated
treatment at a later period kills rats, probably due to IL-2 suppression
of Treg cells (Golovina and Vonderheide, 2010; Hobeika et al., 2011;
Litzinger et al., 2007; Matsushita et al., 2008; Petro, 2011). DAB
389
IL-2
fusion protein either down reg ulates or blocks migration of mononu-
clear cells into the CNS, and monocyte activation is reduced. By re-
duc ing the numbers of inltrating inammatory cells, the fusion
pro tein has a direct inhibitory effect on EAE.
The pre sent study indicates that DAB
389
IL-2 suppresses clinical
demyelinating di sease in a n acute rat m odel of EAE b y suppressing
accumulation of a variety of cell populations in CNS lesions, including
macrophages that are important for the pathogenesis of inammation
and production of pro-inammatory cytokines. The accumulation of
these cells is contingent upon pr ior activation or migration of Th1
IL-2R
+
cells (Aktas et al., 2003; Elliott et al., 1996; Gao et al., 2001;
Juedes a nd Ruddle, 2001; McMah on et al., 2005; Ramadan et al.,
1995). CD169
+
and CD11b/c
+
monocyte/macrophages trafcinto
sites of ina mmation. These cells are implicated in microglia or
macrophage-mediated myelin phagocytosis. Our studies demon-
strate that DAB
389
IL-2 treatment reduced the number of cells express-
ing these antigens in the CNS of rats. We have also previously shown
that DAB
389
IL-2 treated mice have a sharply reduced number of
CD11b
+
and TCRαβ cells in spinal cord lesions (Graeber et al. 1990),
which possibly affects the TCRγδ inammatory process (Bauer et al.,
1994, 1995; Bullard et al., 2007; Murzenok et al., 2002; Ponomarev
et al., 2004; Rinner et al., 1995). Phagocytic activity of macrophages
and microglia cells during the course of acute and chronic relapsing
EAE was observed in blood-borne macrophages inltrating into the
brain, which, together with activated lymphocytes and microglia cells,
started a new demyelination process. In the present study, the suppres-
sive effect of DAB
389
IL-2 is associated with a striking reduction in
CD169
+
, CD11b/c
+
and CD74
+
cells in the spinal cord. DAB
389
IL-2
therapy inhibits CD4
+
,CD8
+
,CD25
+
,andTCRαβ
+
cells from cross-
ing the bloodbrain barrier (BB B). DAB
389
IL-2 acts indirec tly a s a po-
tent deactivating factor f or macrophage s and resident mi croglia,
thu s ultimately suppressing Th1 response and ameliorating inam-
matory disease in EAE rats. At the beginning of disease, lymp hocytes
do not appear in spinal cord tissue until day 9 post immunization,
coincident with the onset of clinical signs. The integrity of the BBB
fails within a short period of time (1218 h) before the onset of clinical
signs, and inltration of inammatory cells does not occur until this fail-
ure takes place. Microglia cells also become activated early in EAE dis-
ease, and then differentiate into macrophages and dendritic-like cells
(Ponomarev et al., 2005). CD45
high
brain macrophages (Ia-positive)
represent non-parenchymal microglial cells which are stimulated even
in a normal T cell response (Remington et al., 2007). Macrophages/
microglia, which have the capacity for antigen ingestion, contribute to
either protective or damaging effects in the brain of MS (Zhang et al.,
2011).
Table 2
Comparison of cytotoxicity produced by the interaction of effector T cells and target
oligodendrocytes or astrocytes.
%cytotoxicityLDH
Source of effector T cells Target glial cells
Oligodendrocytes Astrocytes
EAE* Day 13 71.468 ± 4.74* 41.595 ± 4.619
EAE* Day 30 50.104 ± 6.152 42.464 ± 0.194
Normal Day 13 10.926 ± 4.95 10.895 ± 6.767
EAN** (Non-specic) Day 13 32.645 ± 8.724 30.518 ± 13.112
Cytotoxicity produced when effector T cells (2 × 10
5
/well) interact directly with target
oligodendrocytes or astrocytes (2 × 10
4
/well) cells in vitro. It shows a highly signicant
LDH values when targeted oligodendrocytes compared to astrocytes (P b 0.001) for 20 h
incubation. Each value represents the mean ± SD of individual percent LDH (n =3).
EAE* = Experimental autoimmune encephalomyelitis induced rats.
EAN** = Experimental autoimmune neuritis induced rats.
6 M.K. Bhopale et al. / Experimental and Molecular Pathology xxx (2013) xxxxxx
Please cite this article as: Bhopale, M.K., et al., DAB
389
IL-2 suppresses autoimmune inammation in the CNS and inhibits T cell-mediated lysis of
glial target cells, Experimental and Molecular Pathology (2013), http://dx.doi.org/10.1016/j.yexmp.2013.07.004
Page 6
It has been reported that CNS damage apparently results from direct
or indirect effects of chemokines and pro-inammatory cytokines such
as IFN-γ,LT/TNF(Hochman et al., 19951996), IL-17 (Jadidi-Niaragh
and Mirshaey, 2011)andGMCSF(El-Behi et al., 2011), produced by
pathogenic CD4
+
T cells, residential glial cells or non-specic immune
cells recruited from blood. Expression of INF-γ and TNF-α in spinal
cord tissu e of DA B
389
IL-2 treated EAE rats decreased in our study.
There was a total down-regulation of IL-10 gene expression in
DAB
389
IL-2 treated rats, while it was elevated in the recovery phases
of the disease (Almeras et al., 2002).
Myelin basic protein reactive T cells treated with DAB
389
IL-2 had
reduced cytotoxicity against oligodendrocytes as measured by LDH
release compared to untreated T cells. IL-2 enhances proliferation of
MBP-reactive T cells whereas DAB
389
IL-2 decreases its effect. Several in-
vestigators have shown indirect evidence of in vitro damage to oligo-
dendrocytes by T cells. Lyman et al. (1986) found that MBP-reactive
lymphocytes induced an increased release from CNS organotypic cul-
ture of CNPase (cyclic nucleotide phosphatase), a myelin-specicen-
zyme. Röyttä et al. (1985) have reported damage to oligodendrocytes
in organotypic cultures when they interact with T cells derived from
lymphoid cells of rodents with EAE. To explore the effector mechanisms
underlying the effect of DAB
389
IL-2 on clinical EAE manifestations,
we used an in vitro assay of the cytotoxic effect of MBP-reactive
lymphocytes on syngeneic oligodendrocytes. We report here a reduced
cytotoxic effect of DAB
389
IL-2 treated MBP reactive T cells with and
without restimulation against oligodendrocytes.
In conclusion, we have demonstrated a reduction in the number of
IL-2Rbearing cells during the sensitization process using the fusion
protein DAB
389
IL-2. Our results indicate that DAB
389
IL-2, in addition to
its suppressive effect on myelin specic T cells, mediates its suppressive
effect on EAE by preventing inltration/activation of mononuclear cells
and inammat ory c el ls including macr ophag es and/or activat ed
Fig. 3. A. Oligodendrocytes and B. astrocytes were cultured on the cover slip at the bottom of each well in 24 well plates as described in Materials and methods. C. Interaction of MBP
reactive T cells (effector cells) treated with PBS and oligodendrocytes (target cells) during 4 h incubation. D. MBP reactive T cells treated with DAB
389
IL-2 and oligodendrocyte for 4 h
incubation. E. LDH value of target oligodendrocyte upon interaction with MBP reactive T cells derived from the lymph node of EAE rat on days 9 and 13 separately. DAB
389
IL-2 treated
MBP activated T cells at a concentration of 0.5 mg/ml (50 U/ml) showing signicantly decreased cytotoxicity (P b 0.001) at 20 h incubation. F. Cytotoxicity of target oligodendrocytes
is dependent on increased target: effector ratio for 20 h incubation.
7M.K. Bhopale et al. / Experimental and Molecular Pathology xxx (2013) xxxxxx
Please cite this article as: Bhopale, M.K., et al., DAB
389
IL-2 suppresses autoimmune inammation in the CNS and inhibits T cell-mediated lysis of
glial target cells, Experimental and Molecular Pathology (2013), http://dx.doi.org/10.1016/j.yexmp.2013.07.004
Page 7
microglia. DAB
389
IL-2 reduces cell-mediated immunity in EAE rats by
targeting IL -2R
+
activated T cells and may affect TNF-α and IFN-γ
activation, which contributes to the breakdown of the BB B and the
inltration of leuk ocytes . I n addition, DAB
389
IL-2 inhibits the activa-
tion of other target cells, such as macrophages, which are important
for the path ogenesis of autoimmune dem yelinating disease.
Conict of interest statement
There are no nancial conicts of interest.
Acknowledgments
This study was supported by a grant from the National Institutes
of Health (AMR). We thank Seragen, Inc. for the gift of DAB
389
IL-2 and
mutants for this research. We are thankful to Dr. David Pleasure,
Department of Neurology, University of California, Davis, CA, for
Table 3
Cytotoxicity production of effector MBP-reactive T cells against target oligodendrocytes
with and without DAB
389
IL-2 treatment ex-vivo.
Target:Effector ratio
1:160 1:40 1:10
MBP + T cell 91.00 ± 6.422
1
28.276 ± 8.549 6.042 ± 2.03
MBP + T cell
+DAB
389
IL-2
76.454 ± 11.855 37.827 ± 7.305 8.353 ± 5.49
Normal T Cell 20.896 ± 5.194 19.870 ± 6.292 2.503 ± 2.15
Normal T cell
+DAB
389
IL-2
44.696 ± 3.593 22.939 ± 7.946 2.536 ± 1.77
High control 100.528 ± 9.567 100.0 ± 12.157 100.031 ± 11.233
Low control 7.849 ± 4.849 2.340 ± 2.031 4.426 ± 5.845
Cytotoxic effects of effector MBP reactive T cells (2 × 10
5
cells/well) on oligodendrocyte
target cells at different cell ratios by LDH method after 4 h incubation. Each value
represents mean percentage ± standard deviation (SD) of LDH concentration of triplicate
cultures compared to the Triton X-100 treatment. Differences between values are non-
signicant except MBP T cells vs. normal T cells at the 1:160 ratio by the Student's t-test
*P b 0.05.
Fig. 4. Cytokine gene expression in spinal cords of EAE and DAB
389
IL-2-treated EAE rats showing (A) IL-4 upregulation, IL-10 downregulation gene expression compared to β-actin images;
(B) β-actin: cytokine ratio of IFN-γ,TNF-α, IL-4 and IL-10 with PBS-treated or DAB
389
IL-2-treated EAE; (C) β-actin:INF-γ from spinal cord samples of animals treated with DAB
389
IL-2 and
mutants: DAglu
53
B
389
(
M
DAB-IL-2) and DAB
389
IL-2
819
(DAB-IL-2
M
). DAB
389
IL-2 decreases TNF-α,INF-γ whereas IL-4 shows trend toward up-regulation in gene expression.
8 M.K. Bhopale et al. / Experimental and Molecular Pathology xxx (2013) xxxxxx
Please cite this article as: Bhopale, M.K., et al., DAB
389
IL-2 suppresses autoimmune inammation in the CNS and inhibits T cell-mediated lysis of
glial target cells, Experimental and Molecular Pathology (2013), http://dx.doi.org/10.1016/j.yexmp.2013.07.004
Page 8
providing us with rat Gal-C and GFAP antibodies. We also thank
Katherine Regan for editorial assistance.
References
Aktas, O., Waiczies, S., Smorodchenko, A., Dorr, J., Seeger, B., Prozorovski, T., Sallach, S.,
Endres, M., Brocke, S., Nitsch, R., Zipp, F., 2003. Treatment of relapsing paralysis in
experimental encephalomyelitis by targeting Th1 cells through atorvastatin. The
Journal of Experimental Medicine 197, 725733.
Almeras, L., Meresse, B., Seze, J., De Lefranc, D., Dubucquoi, S., Fajardy, I., Vermersch, P.,
Prin, L., 2002. Interleukin-10 promoter polymorphism in multiple sclerosis: associa-
tion with disease progression. European Cytokine Network 13, 200206.
Bacha, P., W illi ams, D.P., Waters, C., Williams, J.M., Murphy, J.R., Strom, T.B., 1988.
Interleukin 2 receptor-targe ted cytotoxicity. Interleukin 2 receptor-mediated
action of a diphtheria toxin-related interleukin 2 fusi on protein. The Journal
of Experimental Medicine 167, 612622.
Bacha, P.A., Forte, S.E., McCarthy, D.M., Estis, L., Yamada, G., Nichols, J.C., 1991. Impact of
interleukin-2-receptor-targeted cytotoxins on a unique model of murine interleukin-
2-receptor-expressing malignancy. International Journal of Cancer 49, 96101.
Bacha, P., Forte, S.E., Perper, S.J., Trentham, D.E., Nichols, J.C., 1992. Anti-arthritic effects
demonstrated by an interleukin-2 receptor-targeted cytotoxin (DAB486IL-2) in rat
adjuvant arthritis. European Journal of Immunology 22, 16731679.
Balcer, L.J., McIntosh, K.R., Nichols, J.C., Drachman, D.B., 1991. Suppression of immune
responses to acetylcholine receptor by interleukin 2-fusion toxin: in vivo and
in vitro studies. Journal of Neuroimmunology 31, 115122.
Bauer,J.,Sminia,T.,Wouterlood,F.G.,Dijkstra,C.D.,1994.Phagocytic activity of
macrophages and microglial cells during the course of acute and chronic relapsing
experimental autoimmune encephalomyelitis. Journal of Neuroscience Research 38,
365375.
Bauer, J., Huitinga, I., Zhao, W., Lassmann, H., Hickey, W.F., Dijkstra, C.D., 1995. The role of
macrophages, perivascular cells, and microglial cells in the pathogenesis of experi-
mental autoimmune encephalomyelitis. Glia 15, 437446.
Bullard, D.C., Hu, X., Adams, J.E., Schoeb, T.R., Barnum, S.R., 2007. p150/95 (CD11c/CD18)
expression is required for the development of experimental autoimmune encephalo-
myelitis. American Journal of Pathology 170, 20012008.
Calida, D.M., Kremlev, S.G., Fujioka, T., Hilliard, B., Ventura, E., Constantinescu, C.S., Lavi, E.,
Rostami, A., 2000. Experimental allergic neuritis in the SJL/J mouse: induction of
severe and reproducible disease with bovine peripheral nerve myelin and pertussis
toxin with or without interleukin-12. Journal of Neuroimmunology 107, 17.
Deibler, G.E., Martenson, R.E., Kies, M.W., 1972. Large scale preparation of myelin basic
protein from central nervous tissue of several mammalian species. Preparative Bio-
chemistry 2 (2), 139165.
Duszczyszyn, D.A., Beck, J.D., Antel, J., Bar-Or, A., Lapierre, Y., Gadag, V., Haegert, D.G., 2006.
Altered naive CD4 and CD8 T cell homeostasis in patients with relapsingremitting
multiple sclerosis: thymic versus peripheral (non-thymic) mechanisms. Clinical and
Experimental Immunology 143, 305313.
El-Behi, M., Ciric, B., Dai, H., Yan, Y., Cullimore, M., Safavi, F., Zhang, G.X., Dittel, B.N.,
Rostami, A., 2011. The encephalitogenicity of T(H)17 cells is dependent on IL-1- and
IL-23-induced production of the cytokine GM-CSF. Nature Immunology 12, 568575.
Elliott, J.I., Douek, D.C., Altmann, D.M., 1996. Mice lacking alpha beta + T cells are resis-
tant to the induction of experimental autoimmune encephalomyelitis. Journal of
Neuroimmunology 70, 139144.
Gao, Y.L., Rajan, A.J., Raine, C.S., Brosnan, C.F., 2001. gammadelta T cells express activation
markers in the central nervous system of mice with chronic-relapsing experimental
autoimmune encephalomyelitis. Journal of Autoimmunity 17, 261271.
Golovina, T.N., Vonderheide, R.H., 2010. Regulatory T cells: ov ercoming su ppression
of T-cell immunity. Cancer Journal 16 (4), 342347 (Review).
Goverman, J., Perchelle t, A., Huseby, E.S., 2005. TheroleofCD8(+)Tcellsinmultiple
sclerosis and its animal models. Current D rug Targets. In
ammation and Allergy
4, 239245 (Review).
Graeber, M.B., Streit, W.J., Kiefer, R., Schoen, S.W., Kreutzberg, G.W., 1990. New expression
of myelomonocytic antigens by microglia and perivascular cells following lethal
motor neuron injury. Journal of Neuroimmunology 27, 121132.
Haegele, K.F., Stueckle, C. A., Mal in, J.P ., Sinde rn, E., 20 07. Increase of CD8+ T-
eff ector memory cells in periph eral blo od of patients with relapsing-remitting
multiple sclerosis compared to healthy controls. Journal of Neuroimmunology 183,
168174.
Hayashi, R.J., Kanagawa, O., 2006. Requirement of high-afnity IL-2-IL-2R interaction for T
cell anergy induction. International Immunology 18 (5), 645651 (Epub 2006 Mar
30).
Hobeika, A.C., Morse, M.A., Osada, T., Peplinski, S., Lyerly, H.K., Clay, T.M., 2011. Depletion
of human regulatory T cells. Methods in Molecular Biology 707, 219231.
Hochman, P.S., Majeau, G.R., Mackay, F., Browning, J.L., 19951996. Proinammatory re-
sponses are efciently induced by homotrimeric but not heterotrimeric lymphotoxin
ligands. Journal of Inammation 46 (4), 220234.
Jadidi-Niaragh, F., Mirshaey, A., 2011. Th17 cell, the new player of neuroinammatory
process in multiple sclerosis. Scandinavian Journal of Immunology 74, 113.
Juedes, A.E., Ruddle, N.H., 2001. Resident and i nltrating central nervous system APCs
regulate the emergence and resolution of experimental autoimmune encephalo-
myelitis. Journal of Immunology 166, 51685175.
Kadin, M.E., Vonderheid, E.C., 2010. Targeted therapies: Denileukin diftitox-a step towards
a 'magic bullet' for CTCL. Nature Reviews. Clinical Oncology 7, 430432.
Kawai, K., Zweiman, B., 1988. Cytotoxic effect of myelin basic protein-reactive T cells on
cultured oligodendrocytes. Journal of Neuroimmunology 19, 159165.
Kawai, K., Zweiman, B., 1990. Characteri stics of in vitro cytotoxic effects of myelin
basic protein-react ive T ce ll lines on syngeneic oligodendrocytes. Journ al of
Neuroimmunology 26, 5767.
Kawakami,K.,Nakajima,O.,Morishita,R.,Nagai,R.,2006.Targ eted anticancer
immunotoxins and cytotox ic agents wit h direct kil ling moiet ies. The Scie ntic
World Journal 6, 781790 (Review).
Klyushnenkova, E.N., Vanguri, P., 1997. Ia expression and antigen presentation by glia:
strain and cell type-specic differences among rat astrocytes and microglia. Journal
of Neuroimmunology 79, 190201.
Lansigan, F., Stearns, D.M., Foss, F., 2010. Role of denileukin diftitox in the treatment
of persistent or recurrent cutaneous T-cell lymphoma. Cancer Management and
Research 2, 5359.
Lavasani, S., Dzhambazov, B., Andersson, M., 2007.
Monoclonal antibody against T-cell
receptor alphabeta induces self-tolerance in chronic experimental autoimmune
encephalomyelitis. Scandinavian Journal of Immunology 65, 3947.
Litzinger, M.T., Fernando, R., Curiel, T.J., Grosenbach, D.W., Schlom, J., Palena, C., 2007. IL-2
immunotoxin denileukin diftitox reduces regulatory T cells and enhances vaccine-
mediated T-cell immunity. Blood 110, 31923201.
Lyman, W.D., Roth, G.A., Chiu, F.C., Brosnan, C.F., Bornstein, M.B., Raine, C.S., 1986. Antigen-
specic T cells can mediate demyelination in organotypic central nervous system
cultures. Cellular Immunology 102, 217226.
Matsushita, N., Pilon-Thomas, S.A., Martin, L.M., Riker, A.I., 2008. Comparative meth-
odologies of regulatory T cell de pletion in a murine melanoma model. Journal of
Immunological Methods 333, 167 179.
McMahon, E.J., Bailey, S.L., Castenada, C.V., Waldner, H., Miller, S.D., 2005. Epitope spread-
ing initiates in the CNS in two mouse models of multiple sclerosis. Nature Medicine
11, 335339.
Miller, S.D., McMahon, E.J., Schreiner, B., Bailey, S.L., 2007. Antigen presentation in the CNS
by myeloid dendritic cells drives progression of relapsing experimental autoimmune
encephalomyelitis. Annals of the New York Academy of Sciences 1103, 179191.
Murzenok, P.P., Matusevicius, D., Freedman, M.S., 2002. gamma/delta T cells in multiple
sclerosis: chemokine and chemokine receptor expression. Clinical Immunology 103,
309316.
Natarajan, N., Telang, S., Miller, D., Chesney, J., 2011. Novel immunotherapeutic agents
and small molecule antagonists of signalling kinases for the treatment of metastatic
melanoma. Drugs 71, 12331250.
Nguyen, K.B., McCombe, P.A., Pender, M.P., 1994. Macrophage apoptosis in the central
nervous system in experimental autoimmune encephalomyelitis. Journal of
Autoimmunity 7, 145152.
Pacheco-Silva, A., Bastos, M.G., Muggia, R.A., Pankewycz, O., Nichols, J., Murphy, J.R.,
Strom, T.B., Rubin-Kelley, V.E., 1992. Interleukin 2 receptor targeted fusion toxin
(DAB486-IL-2) treatment blocks diabetogenic autoimmunity in non-obese diabetic
mice. European Journal of Immunology 22, 697702.
Papadopoulos, D., Pham-Dinh, D., Reynolds, R., 2006. Axon loss is responsible for chronic
neurological decit following inammatory demyelination in the rat. Experimental
Neurology 197, 373385.
Pastor, S., Vaccaro, C.G., Minguela, A., Ober, R.J., Ward, E.S., 2006. Analyses of
TCR clustering at the T cell-antigen-presenting cell interface and its impact on the
activation of naive CD4+ T cells. International Immunology 18, 16151625.
Petro, T.M., 2011. Reg ulatory role of resveratrol on Th17 in autoimmune di sease.
International Immunopharmacology 11 (3), 310318 (Review).
Phillips, S.M., Bhopale, M.K., Constantinescu, C.S., Ciric, B., Hilliard, B., Ventura, E., Lavi, E.,
Rostami, A., 2007. Effect of DAB(389)IL-2 immunotoxin on the course of experimen-
tal autoimmune encephalomyelitis in Lewis rats. Journal of the Neurological Sciences
263, 5969.
Phillips, S.M., Bhopale, M.K., Hilliard, B., Zekavat, S.A., Ali, M.A., Rostami, A ., 2010.
Suppression of murine experimental autoimmune encephalomyelitis by interleukin-2
receptor targeted fusion toxin, DAB(389)IL-2. Cellular Immunology 261, 144152.
Ponomarev, E.D., Novikova, M., Yassai, M., Szczepanik, M., Gorski, J., Dittel, B.N., 2004.
Gamma delta T cell regulation of IFN-gamma production by central nervous system-
inltrating encephalitogenic T cells: correlation with recovery from experimental
autoimmune encephalomyelitis. Journal of Immunology 173, 1587
1595.
Ponomarev, E.D., Shriver, L.P., Maresz, K., Dittel, B.N., 2005. Microglial cell activation and
proliferation precedes the onset of CNS autoimmunity. Journal of Neuroscience Re-
search 81 (3), 374389.
Potala, S., Verma, R.S., 2010. Modi ed DT-IL2 fusion toxin targeting uniquely
IL2Ralpha expressing leukemia cell lines construction and characterization.
Journal of B iot ech no l ogy 148, 147155.
Ramadan, M.A., Gabr, N.S., Bacha, P., Günzler, V., Phillips, S.M., 1995. Suppression
of immunopathology in schistosomiasis by interleukin-2-targeted fusion toxin,
DAB389IL-2. I. Studies of in vitro and in vivo efcacy. Cellular Immunology 166,
217226.
Ratts, R.B., Karandikar, N.J., Hussain, R.Z., Choy, J., Northrop, S.C., Lovett-Racke, A.E., Racke,
M.K., 2006. Phenotypic characterization of autoreactive T cells in multiple sclerosis.
Journal of Neuroimmunology 178, 100110.
Remington, L.T., Babcock, A.A., Zehntner, S.P., Owens, T., 2007. Microglial recruitment, ac-
tivation, and proliferation in response to primary demyelination. American Journal of
Pathology 170 (5), 17131724.
Rinner, W.A., Bauer, J., Schmidts, M., Lassma nn, H., Hickey, W.F., 1995. Resident mi-
croglia and hematogenous macrophages as phagocytes in adoptively transferred
experimental autoimmune encephalomyelitis: an inve stigat ion using rat rad ia-
tion bone marrow chimeras. Glia 14, 257266.
Rostami, A., Gregorian, S.K., Brown, M.J., Pleasure, D.E., 1990. Induction of severe experi-
mental autoimmune neuritis with a synthetic peptide corresponding to the 5378
amino acid sequence of the myelin P2 protein. Journal of Neuroimmunology 23,
145151.
9M.K. Bhopale et al. / Experimental and Molecular Pathology xxx (2013) xxxxxx
Please cite this article as: Bhopale, M.K., et al., DAB
389
IL-2 suppresses autoimmune inammation in the CNS and inhibits T cell-mediated lysis of
glial target cells, Experimental and Molecular Pathology (2013), http://dx.doi.org/10.1016/j.yexmp.2013.07.004
Page 9
Röyttä, M., Lyman, W.D., Roth, G.A., Bornstein, M.B., Raine, C.S., 1985. Preliminary analysis
of cell and serum-induced demyelination in vitro using a syngeneic system. Acta
Neurologica Scandinavica 71, 226236.
Sadler, R.H., Sommer, M.A., Forno, L.S. , Smith, M.E. , 1991. In duct ion o f anti-myelin an-
tibodies in EAE and their possible role i n demyelination. Journal of Neuroscience
Research 30 (4), 616624.
Siegling, A., Lehmann, M., Platzer, C., Emmrich, F., Volk, H.D., 1994. A novel multispecic
competitor fragment for quantitative PCR analysis of cytokine gene expression in
rats. Journal of Immunological Methods 177, 2328.
Stüve,O.,Marra,C.M.,Bar-Or,A.,Niino,M.,Cravens, P.D., Cepok, S., Frohman, E.M., Phillips, J.T.,
Arendt, G., Jerome, K.R., Cook, L., Grand'Maison, F., Hemmer, B., Monson, N.L., Racke, M.K.,
2006. Altered CD4+/CD8+ T-cell ratios in cerebrospinal uid of natalizumab-treated
patients with multiple sclerosis. Archives of Neurology 63, 13831397.
Sutton, C., Brereton, C., Keogh, B., Mills, K.H., Lavelle, E.C., 2006. Acrucialroleforinterleukin
(IL)-1 in the induction of IL-17-prod ucing T cells that mediate autoimmune
encephalomyelitis. The Journal of Experimental Medicine 203, 16851691.
Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., Genbauffe, F.,
Strom , T . B., Mu rphy, J.R., 1987. Diphtheria toxin receptor binding domain
substitution with interleukin-2: genetic con structio n and properties of a diph-
theria toxin-related interleukin-2 fusion protein. Pr otein Engineering 1 (6),
493498.
Williams, D.P., Snider, C.E., Strom, T.B., Murphy, J.R., 1990a. Structure/function analysis of
interleukin-2-toxin (DAB486-IL-2). Fragment B sequences required for the delivery
of fragment A to the cytosol of target cells. Journal of Biological Chemistry 265,
1188511889.
Williams, D.P., Wen, Z., Watson, R.S., Boyd, J., Strom, T.B., Murphy, J.R., 1990b. Cellular pro-
cessing of the interleukin-2 fusion toxin DAB486-IL-2 and efcient delivery of diph-
theria fragment A to the cytosol of target cells requires Arg194. Journal of Biological
Chemistry 265, 2067320677.
Zhang, Z., Zhang, Z.Y., Schittenhelm, J., Wu, Y., Meyermann, R., Schluesener, H.J., 2011.
Parenchymal accumulation of CD163(+) macrophages/microglia in multiple sclero-
sis brains. Journal of Neuroimmunology 237, 7379.
10 M.K. Bhopale et al. / Experimental and Molecular Pathology xxx (2013) xxxxxx
Please cite this article as: Bhopale, M.K., et al., DAB
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IL-2 suppresses autoimmune inammation in the CNS and inhibits T cell-mediated lysis of
glial target cells, Experimental and Molecular Pathology (2013), http://dx.doi.org/10.1016/j.yexmp.2013.07.004
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