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Agonist 15-Deoxy-
12,1412,14-Prostaglandin J2 Ameliorates Experimental Autoimmune Encephalomyelitis1
*
Department of Neurology and
Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390; and
Department of Anatomy and Neurobiology, University of Arkansas for Medical Sciences, Little Rock, AR 72205
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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expression in the immune system have been limited. Recently, PPAR-
was identified in monocyte/macrophage cells. In this study we examined
the role of PPAR- in experimental autoimmune encephalomyelitis
(EAE), an animal model for the human disease multiple sclerosis. The
hypothesis we are testing is whether PPAR- plays an important role
in EAE pathogenesis and whether PPAR- ligands can inhibit the
clinical expression of EAE. Initial studies have shown that the
presence of the PPAR- ligand
15-deoxy-
12,14-PGJ2 (15d-PGJ2) inhibits the
proliferation of Ag-specific T cells from the spleen of myelin basic
protein Ac1–11 TCR-transgenic mice. 15d-PGJ2 suppressed
IFN-, IL-10, and IL-4 production by both Con A- and myelin basic
protein Ac1–11 peptide-stimulated lymphocytes as
determined by ELISA and ELISPOT assay. Culture of encephalitogenic T
cells with 15d-PGJ2 in the presence of Ag reduced the ability of these
cells to adoptively transfer EAE. Examination of the target organ, the
CNS, during the course of EAE revealed expression of PPAR- in the
spinal cord inflammatory infiltrate. Administration of 15d-PGJ2 before
and at the onset of clinical signs of EAE significantly reduced the
severity of disease. These results suggest that PPAR- ligands may be
a novel therapeutic agent for diseases such as multiple
sclerosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peroxisome proliferator-activated receptors (PPARs) are members of the
nuclear hormone receptor superfamily of ligand-activated
transcriptional factors that include receptors for steroids, thyroid
hormone, vitamin D, and retinoic acid (4). PPAR binds to
the peroxisome proliferator-responsive element as a heterodimer with
retinoid X receptor (RXR). The transcriptional regulation of target
genes by PPARs is achieved through the binding of these PPAR-RXR
heterodimers to peroxisome proliferator-responsive elements
(5). RXR also forms heterodimers with other members of the
nuclear receptor superfamily, and these interactions influence the
PPAR-regulated transcriptional activation because of the competition
among various RXR heterodimerization partners for RXR (5, 6). To date, three mammalian PPARs subtypes have been isolated
and termed PPAR-
, PPAR-
(also called PPAR-
or NUC-1), and
PPAR- (7, 8). PPAR- is expressed at high levels in
adipose tissue and is a critical regulator of adipocyte differentiation
(9). In addition, the expression of PPAR- has been
detected on macrophages, T cells, endothelial cells, vascular smooth
muscle cells, and colonic tumor cells (10, 11, 12, 13, 14, 15, 16). Recent
data have shown that the natural PG,
15-deoxy-12,14-PGJ2
(15d-PGJ2), and synthetic antidiabetic thiazolidinedione, which are
PPAR- ligands, inhibit phorbol ester-induced NO, TNF-, IL-1, and
IL-6 production by cells of the monocyte/macrophage lineage. These
ligands inhibit gene expression in part by antagonizing the activities
of transcription factors such as AP-1 and NF-
B (10, 11). Moreover, PPAR- ligands have potent tumor modulatory
effects against colorectal, prostate, and breast cancers
(17, 18, 19). They also induce apoptosis in macrophages,
fibroblasts, and endothelial cells (20, 21, 22). Importantly,
PPAR- ligands have been shown to ameliorate a variety of
inflammatory conditions, including arthritis (23),
inflammatory bowel disease (24), atherosclerosis
(25), and a carrageenin-induced pleurisy model in rats
(26).
To explore the role of the PPAR- ligands during the pathogenesis of
EAE, we examined PPAR- expression in the CNS of mice expressing
signs of the disease. We also examined the effect of the PPAR-
ligand 15d-PGJ2 on T cell proliferation and cytokine production in
vitro as well as the effect of administration of exogenous 15d-PGJ2 on
the clinical outcome of EAE induced by either adoptive transfer of
encephalitogenic T cells or active immunization with myelin Ag in
adjuvant. The clinical improvement was accompanied by a significant
decrease in CNS inflammation and decreased cytokine expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transgenic mice bearing the rearranged V2.3, V8.2 gene
encoding the TCR specific for the Ac1–11 peptide
of MBP on the B10.PL background were obtained by crossing transgenic
mice bearing the individual rearranged genes (27). The
V2.3 TCR-transgenic mice and the V8.2 TCR-transgenic were
provided by Dr. J. Goverman (University of Washington, Seattle, WA).
These mice were bred and maintained in a federally approved animal
facility at University of Texas Southwestern Medical Center (Dallas,
TX) in accordance with the animal studies committee. All mice were
7–10 wk of age when experiments were performed. B10.PL mice were
purchased from The Jackson Laboratory (Bar Harbor, ME) and then bred in
our animal facility.
Cell culture
Draining lymph nodes and spleens from naive V2.3, V8.2
TCR-transgenic mice were harvested, and single-cell suspensions were
obtained by pressing the tissue through a wire-mesh screen as
previously described (27). The cells were cultured (4
x 106 cell/ml) in complete medium for the time
specified in the text or figures. Con A and MBP peptide
Ac1–11 (CS Bio CO, San Carlo, CA) were used as
stimulating agents.
Lymphocyte proliferation
Proliferative responses of lymph node cells and splenocytes
(4 x 105 cells/well) from V2.3, V8.2
TCR-transgenic mice were determined using different concentrations of
MBP Ac1–11 in the presence of various
concentrations of PPAR- as indicated. Cultures were maintained in
96-well flat-bottom plates for 96 h at 37°C in humidified 5%
CO2/air. The wells were pulsed with 0.5
µCi/well [methyl-3H]thymidine for
the final 16 h of culture. Cells were harvested on glass-fiber
filters, and incorporated
[methyl-3H]thymidine was measured
with a Betaplate counter (Wallac, Gaithersburg, MD). Results were
determined as means from quadruplicate cultures and are shown with
the SEM.
Cytokine ELISA
IFN-, IL-4, and IL-10 were measured in cell culture
supernatants using ELISA plates (Immunol 2; Dynatech, Chantilly, VA)
that were coated with 2 µg/ml (50 µl/well) IFN-, IL-4, or IL-10
mAb (BD PharMingen, San Diego, CA) in 0.1 M carbonate buffer (pH 8.2)
overnight at 4°C. The plates were blocked with 200 µl of 1% BSA in
PBS for 2 h. Tissue culture supernatant (100 µl) were added at
various dilutions titrated to the linear portion of the
absorbance/concentration curve in duplicate and incubated overnight at
4°C. After the plates were washed four times with PBS and 0.05%
Tween 20, 100 µl of biotinylated anti-cytokine-detecting mAb
(directed to a different determinant from the first Ab used to coat
ELISA plates) at 1 µg/ml in PBS and 1% BSA were added for 45 min at
room temperature. Then 100 µl of avidin peroxidase (2.5 µg/ml) was
added, and incubation proceeded for 30 min. Subsequently, the
peroxidase substrate ABTS in 0.1 M citric buffer, pH 4.35, in the
presence of H2O2 was added,
and the absorbance was measured at 405 nm.
ELISPOT assay
The frequencies of IFN--, IL-4-, and IL-10-producing cells
were determined with ELISPOT assays as previously described
(28). Briefly, 96-well nitrocellulose-bottomed microtiter
plates were precoated with 0.4 µg/well of the capture mAb R26A2 for
IFN- (BD PharMingen), 11B11 for IL-4 (BD PharMingen), and mAb
JES5-2A5 for IL-10 (BD PharMingen) and incubated at 4°C overnight.
Each well received 100 µl of 4 µg/ml coating Ab. After three
PBS-Tween 20 (0.05%) and three PBS washes, plates were blocked with
1% BSA for 2 h at room temperature. A single-cell suspension
(4 x 106 cells/100 µl) was added to the
coated plates in duplicate and incubated for 36 h for IFN- and
for 48 h for IL-4 and IL-10. After washing, 2 µg/ml biotinylated
detection mAb XMG1.2 for IFN- (BDPharMingen), BVD6-24G2 for IL-4
(BD PharMingen), or SXC-1 for IL-10 (BD PharMingen) was added to the
wells (100 µl/well). After incubation for 2 h at 37°C, the
plates were washed, streptavidin-alkaline phosphatase (diluted 1/1000
in PBS-Tween 20 containing 1% BSA; DAKO, Carpinteria, CA) was
added to the well, and the plates were incubated for 1 h at
37°C. Color development with 3-amino-9-ethylcarbazole (Sigma-Aldrich,
St. Louis, MO) and H2O2 was
performed. The numbers of red-brown spots in individual well, where
each spot represents a single cytokine-secreting cell, were enumerated
by examining wells under the Alpha Imager 2200 (Alpha Innotech, San
Leandro, CA).
Induction and clinical evaluation of EAE
For induction of EAE, naive B10.PL mice were immunized
s.c. with MBP Ac1–11 (200 µg/mouse) in an
emulsion with CFA (Difco, Detroit, MI). Pertussis toxin (200 ng/mouse)
in PBS was injected i.p. at the time of immunization and 48 h
later. For induction of adoptive transfer EAE, lymph nodes and spleens
from TCR-transgenic mice bearing V2.3, V8.2 genes encoding the
TCR specific for Ac1–11 peptide on the B10.PL
background were harvested and pressed through a wire-mesh screen. The
transgenic cells were cultured (4 x 105
cell/well) and combined in medium with MBP peptide
Ac1–11 (5 µg/ml) for 4 days. The activated
cells were suspended in PBS and injected into naive B10.PL mice. Mice
were scored on scale of 0–6 as previously described (29):
0, no clinical disease; 1, limp/flaccid tail; 2, moderate hind limb
weakness; 3, severe hind limb weakness; 4, complete hind limb
paralysis; 5 quadriplegia or premoribund state; and 6, death.
15d-PGJ2 treatment protocol
15d-PGJ2 was purchased from Cayman Chemical (Ann Arbor, MI) as a solution in methyl acetate. To change the solvent, methyl acetate was evaporated under a gentle stream of nitrogen, and the undiluted oil was immediately dissolved in PBS buffer (pH 7.2). For each experiment 15d-PGJ2 was freshly prepared as a 1 mg/ml working stock solution in sterile PBS and diluted further to the desired concentration. Drug or vehicle was administered daily by i.p. route at different doses ranging from 100 µg/kg/day to 1 mg/kg/day in a volume of 100 µl of sterile PBS.
Immunohistochemistry
After various survival times, the mice were deeply
anesthetized with sodium pentobarbitone. The animals were then
transcardially perfused with sterile PBS, followed by 4%
paraformaldehyde in phosphate buffer. Brain and spinal cord were
removed, fixed for 6 h, and cryoprotected in 30% sucrose
overnight at 4°C before being embedded in Tissue-Tek (Ted Pella,
Redding, CA) and quickly frozen in isopentane.
Immunohistochemistry was performed on adjacent sections. PPAR-
immunostaining was conducted using a rabbit polyclonal Ab (Santa Cruz
Biotechnology, Santa Cruz, CA). After pretreatment with 0.3% hydrogen
peroxide in absolute methanol, sections were blocked with 1% BSA for
2 h at room temperature and then incubated with the primary Ab
(1/400 dilution of a 200 µg/ml stock) overnight at 4°C. Other
primary Abs used were MOM2 (1/20) mAb, a marker for macrophages; KT174
(1/20), which recognizes the CD4 Ag of Th cells; CD11b (1/100), which
labels the complement type 3 receptor of the cells of the
macrophage/microglia lineage (BioSource, Camarillo, CA); and rabbit
polyclonal Abs against the glial fibrillary acidic protein (Santa Cruz
Biotechnology).
The binding of the primary Abs was detected using a biotinylated secondary Ab and an avidin-biotin-peroxidase method under humidified conditions (ABC Elite kit; Vector Laboratories, Burlingame, CA). Peroxidase activity was visualized with 3,3'-diaminobenzidine (DAB kit; Vector Laboratories) as a substrate or with methyl green counterstain. Omission of the primary Ab served as a negative control. The specificity of staining was also controlled on sections of peripheral lymphoid organs. The tissue area was measured by a Scion image analysis system (Scion, Frederick, MD). Positive cells were counted by automatic video scanning using a Leica Q500 MC (Zeiss, Oberkochen, Germany), and the numbers of stained cells per 104 square pixels tissue area were calculated.
To identify the cell types in which PPAR- was expressed, a
series of sections from EAE mice was processed for double staining. In
double-Ab immunostaining, tissue specimens were stained with
anti-PPAR- (1/400 dilution in PBS) Ab using the alkaline
phosphatase method and with anti-macrophage Ab using the peroxidase
method. Some tissue specimens were stained with anti-PPAR-
(1/400 dilution in PBS) Ab using the peroxidase method first, followed
by KT174, CD11b, and glial fibrillary acidic protein (GFAP) Abs
using the alkaline phosphatase method. Positive staining was indicated
by a brownish deposit for the peroxidase method, a blue deposit for the
alkaline phosphatase method, and brown-blue deposits for the
double-staining method.
Cell culture of microglia
The N9 microglial cell line is derived from
myc-immortalized mouse microglia (38)
and was provided by P. Ricciardi-Castagnoli (University of Milan,
Milan, Italy). Cells were cultured in MEM containing 10% FBS
(Sigma-Aldrich), 1.4 mM glutamine, and 20 µM 2-ME (Sigma-Aldrich).
Rat HAPI microglial cells were provided by J. Connor (Pennsylvania
State University, State College, PA) (39). HAPI
cells were cultured in DMEM containing 5% FBS, 1.4 mM glutamine, and
100 U/ml penicillin plus 100 µg/ml streptomycin (Life
Technologies/BRL, Grand Island, NY). Where indicated, cells were
treated with the 15d-PGJ2 (Cayman Chemical) or the cytokines IFN-
and TNF- (R&D Systems, Minneapolis, MN).
Flow cytometry
CD40 expression on rodent microglial cells was evaluated
by flow cytometric analysis. Where indicated, cells were pretreated
with 15d-PGJ2 for 1 h before treatment with IFN- and TNF-,
and then CD40 expression on microglial cells was determined 48 h
later. Following treatment, cells were collected and washed thoroughly
with wash buffer (PBS containing 1% goat serum; Sigma-Aldrich). Mouse
N9 microglial cells were then incubated on ice for 45 min with
FITC-labeled rat anti-mouse CD40 Ab or an appropriate
isotype-matched control (IgG2a, isotype; BD PharMingen). Rat HAPI
microglial cells were incubated on ice for 45 min with rabbit
anti-human CD40 Ab, which cross-reacts with rat CD40 protein
(Research Diagnostics, Flanders, NJ). HAPI cells were again washed and
then incubated on ice for 45 min with FITC-labeled goat anti-rabbit
IgG Ab. Following washing, CD40 expression on unfixed mouse N9 and rat
HAPI microglial cells was immediately performed on a FACSCalibur flow
cytometer (BD PharMingen) with WinMDI software (TSRI Cytometry, San
Diego, CA).
Statistical methods
Differences between pairs of groups were tested by Student’s t test. Differences among the three groups were tested by Kruskal-Wallis one-way ANOVA. All significance tests were two-sided.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because EAE is caused by neuroantigen-specific
CD4+ T cells, we sought to determine whether
15d-PGJ2 had any effect on CD4+ T cell
proliferation. Draining lymph nodes and spleen from naive V2.3,
V8.2 TCR-transgenic mice were cultured using different
concentrations of MBPAc1–11 in the presence of
various concentrations of 15d-PGJ2. As shown in Fig. 1
, MBPAc1–11-induced splenocyte proliferation was
strongly suppressed by 2.5 µM 15d-PGJ2. This concentration of
15d-PGJ2 did not affect cell viability as determined by trypan blue
exclusion. However, addition of the same concentration of 15d-PGJ2 (2.5
µM) did not affect lymph node T cell proliferation (Fig. 2). In the unstimulated splenocytes and
lymph node cell cultures, there was no difference in T cell
proliferation.
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. MBPAc1–11-induced IFN- secretion
was significantly suppressed in response to 15d-PGJ2 at 48 h.
Levels of MBP Ac1–11-induced IFN- secretion
were also decreased at 72, 96, and 120 h. Significant levels of
IL-4 and IL-10 were not detected (data not shown).
To enumerate the frequency of cells producing IFN-, IL-4, and IL-10,
splenocytes were cultured in the presence of Con A and
Ac1–11 alone or in the presence of 2.5 µM
15d-PGJ2 for 4 days and replated for the ELISPOT
assays. As shown in Fig. 3, 15d-PGJ2
significantly reduced the levels of Con A- and MBP
Ac1–11-reactive IFN-- and IL-4-secreting
cells. 15d-PGJ2 did not alter the frequency of IL-10-secreting
cells.
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Interaction of costimulatory molecules on APC with their cognate
receptors on T cells influences the differentiation of
CD4+ T cells. Two well-studied costimulatory
pathways are the B7/CD28 and CD40/CD40L pathways (30, 31).
We determined by FACS analysis that rat HAPI (Fig. 4A) and mouse N9 (Fig. 4B) microglial cells expressed low constitutive levels of
CD40. Culture with IFN- and TNF- induced the expression of CD40
on these cells, and addition of 15d-PGJ2 partially inhibited this
induction (Fig. 4).
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Because 15d-PGJ2 inhibited the proliferation and IFN-
production of MBP Ac1–11-specific T cells in
vitro, we next wished to address whether this also inhibited the
ability of these cells to adoptively transfer EAE. When TCR Tg T cells
specific for MBP Ac1–11 were activated in vitro
with Ag for 4 days in the presence or the absence of 15d-PGJ2 and
subsequently transferred into naive B10.PL recipients, the presence of
15d-PGJ2 inhibited the encephalitogenicity and resulted in a delay in
the onset and a decrease in the severity of disease (Fig. 5). These data all indicate that 15d-PGJ2
has a significant effect on T cell responses in vitro.
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Because PPAR- agonists had been shown to inhibit a number of
inflammatory conditions (23, 24, 25, 26), we determined whether
administration of 15d-PGJ2 could affect the pathogenesis of EAE.
Administration of 15d-PGJ2 i.p. to B10.PL mice induced to develop EAE
was initiated on day 1 postimmunization and continued daily for 10
days. 15d-PGJ2 administration delayed the appearance of clinical
signs of EAE induced by MBP Ac1–11 peptide and
reduced the severity of the disease. As shown in Fig. 6A, mice treated with 15d-PGJ2
at 1 mg/kg/day and 100 µg/kg/day developed a less severe course of
disease. Whereas seven of eight mice receiving PBS developed clinical
EAE, only five of seven animals receiving 15d-PGJ2 at 100
µg/kg/day and three of seven mice receiving 15d-PGJ2 at 1 mg/kg/day
developed EAE. Mice treated with higher 15d-PGJ2 doses had a mild delay
in the onset of clinical signs (Fig. 6A). However, the
higher dose exhibited signs of toxicity to some animals. On the basis
of these results, 15d-PGJ2 at doses of 500 and 200 µg/kg/day was used
in another experiment. Three groups of seven animals were given
15d-PGJ2 at 500 µg/kg/day, 200 µg/kg/day in 0.1 ml of PBS, or 0.1
ml of PBS. Mice were treated daily with i.p. injections for 28 days
beginning on day 1 postimmunization. Of the seven mice receiving PBS,
six developed EAE and one died of severe EAE, while of seven mice
receiving 15d-PGJ2 at 500 µg/kg/day, five developed EAE and two died
of EAE. In the 15d-PGJ2-treated mice at 200 µg/kg/day, three of seven
developed EAE and no mortality was observed. As shown in Fig. 6B, animals that received 15d-PGJ2 at 500 and 200
µg/kg/day had a significantly improved disease course compared with
those that received PBS alone (PBS vs 500 µg/kg/day,
p < 0.001; PBS vs 200 µg/kg/day, p
< 0.001). The onset of disease was delayed in 15d-PGJ2-treated mice
given 200 µg/kg/day (Fig. 6B).
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Having ascertained that 15d-PGJ2 suppressed EAE, we then
tested the effect of drug treatment on the later course of EAE. EAE was
induced as before, and the animals were monitored for clinical signs.
We examined the effect of 15d-PGJ2 administration on disease course at
the onset of EAE on day 10 postimmunization. Three groups of mice were
treated with 15d-PGJ2 at 500 µg/kg/day, 200 µg/kg/day 15d-PGJ2 in
0.1 ml of PBS, or 0.1 ml of PBS. Mice were treated daily with i.p.
injections for 21 days beginning on day 10. Mice treated with 15d-PGJ2
at the two different doses had lower maximum clinical scores of EAE
than control mice treated with PBS. However, both doses had a
significant effect (PBS vs 500 µg/kg/day, p < 0.001;
PBS vs 200 µg/kg/day, p < 0.001). Animals treated
with 200 µg/kg/day had a later onset of clinical signs and less
severe maximum clinical scores (Fig. 7).
Thus, administration of 15d-PGJ2 before disease onset or at the time of
onset of clinical disease had beneficial clinical effects in this EAE
model.
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Having determined that administration of 15d-PGJ2 protected
against EAE, we next examined the effects of 15d-PGJ2 on the pathology
of EAE. Four groups of three B10.PL mice induced to develop EAE were
given 15d-PGJ2 at 500 or 200 µg/kg/day, PBS control, or no treatment.
Mice were treated daily with i.p. injections for 21 days beginning on
day 10 postimmunization. Mice were sacrificed and the spinal cords were
removed for histologic analysis. Infiltrating macrophages and
CD4+ T cells were detected by
immunohistochemistry. The difference in the number of infiltrating
cells between the control groups and the experimental treatment groups
were quantified by a Scion image analysis system. For the parameters we
measured, the number of CD4+ T cells and
macrophages infiltrating the CNS, there was no significant difference
between the mice that were untreated and those that received a vehicle
injection (data not shown). The number of CD4+ T
cells recruited into the lesions was significantly reduced (Fig. 8A), independently of whether
15d-PGJ2 was given at the high dose of 500 µg/kg/day (40% reduction,
p < 0.01) or a lower dose of 200 µg/kg/day (49%
reduction, p < 0.001). Also, both doses of 15d-PGJ2
significantly reduced the total number of macrophages in the lesions by
58 and 72%, respectively (p < 0.001; Fig. 8B). These results suggest that administration of the
PPAR- agonist, 15d-PGJ2, reduces the inflammation in actively
induced EAE.
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in the CNS during
the course of EAE. Spinal cord and brain tissues obtained from EAE and
CFA-inoculated mice 2–3 wk postimmunization were analyzed by
immunohistochemistry for PPAR- expression. No PPAR-
immunoreactivity was detected in the CNS of CFA control mice. PPAR-
in macrophage-like cells was prominently expressed in the perivascular
cuffs, associated with large mononuclear cells in the perinuclear and
cytoplasmic regions (Fig. 9). There was
pronounced astrogliosis noted in the CNS lesions; PPAR-
immunoreactivity was associated with GFAP-staining astrocytes.
Immunostaining with normal rabbit serum was completely negative in all
animals (data not shown).
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in the macrophages, we
immunostained the same sections from the CNS tissue of mice by the
double-Ab staining method with anti-macrophage or anti-GFAP and
anti-PPAR- Ab. Intense brown-blue deposits (double-positive
cells) were detected in macrophages and astrocytes (Fig. 10). Control staining with normal
rabbit serum was uniformly negative (data not shown). These
observations suggest that PPAR- is localized in infiltrating
macrophages and astrocytes during the inflammatory response in
EAE.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is functionally
relevant in freshly isolated T cells. These authors demonstrated that
ligands for PPAR- mediated inhibition of IL-2 secretion by the T
cell clones while not inhibiting IL-2-induced proliferation of such
clones, suggesting that PPAR- ligation may affect signaling pathways
that are activated after TCR stimulation but not activated after IL-2R
ligation. These results are consistent with our data, which show that
15d-PGJ2 can inhibit T cell proliferation and the ability of those T
cells to transfer EAE when T cells are cultured with 15d-PGJ2 in vitro
(Fig. 5
).
PPAR- activators have previously been shown to effectively inhibit
manifestations of inflammatory disorders. This statement is supported
by in vivo animal studies indicating that PPAR- agonists induce
synoviocyte apoptosis and suppress adjuvant-induced arthritis in rats
(23), inhibit the inflammatory response in atherosclerosis
and a carrageenin-induced pleurisy model in rats (25, 26),
and ameliorate inflammatory bowel disease in mice (24).
Various mechanisms have been proposed to explain this therapeutic
efficacy. Mechanistically, PPAR- acts at least in part by inhibiting
the activity of transcription factors AP-1, STAT-1, and NF-B. In the
case of NF-B, 15d-PGJ2 inhibits the activity of IB kinase, which
normally phosphorylates the NF-B inhibitor, IB, resulting in
trans-activation of NF-B target genes (32, 33).
Recently, administration of the PPAR- agonist troglitazone was shown
to inhibit signs of MOG35–55-induced EAE in
C57BL/6 mice (34). In this study it was demonstrated that
administration of troglitazone could reduce the clinical signs of
disease and reduce the size of lesions in the lumbar cord. However, it
was also noted that there was little difference in T cell-mediated
proliferation or cytokine production from mice receiving troglitazone
vs placebo, leading the authors to conclude that troglitazone had
little effect on T cell differentiation or T cell expansion
(34). Our data suggest that that the presence of the
PPAR- agonist 15d-PGJ2 can affect T cell proliferation and cytokine
production, similar to the observation made by Clark et al.
(14). Our data also suggest that T cells that home to the
spleen are more sensitive to low doses of 15d-PGJ2 with regard to the
inhibition of proliferation then T cells that home to lymph nodes. In
addition, our data demonstrate that another PPAR- agonist
demonstrates a positive therapeutic effect in another EAE model,
suggesting that these observations are more general and are not due to
the specific model examined.
There are a number of other possibilities for how 15d-PGJ2 mediated its
therapeutic effect in vivo. For example, we have recently shown that
15d-PGJ2 can inhibit the production of TNF- and NO by microglial
cells (35). In that study 15d-PGJ2 also inhibited the
production of IL-12, which is important for the differentiation of T
cells to a Th1 phenotype and plays a pivotal role in EAE
pathogenesis. Administration of 15d-PGJ2 to mice developing EAE could
result in protection by inhibition of the secretion of these
inflammatory mediators known to be toxic to oligodendrocytes.
Future studies will attempt to determine whether the main
mechanism for EAE suppression is due to its effect on T cells as
opposed to its effect on cells that are more likely to mediate the
pathogenesis of EAE at the level of the effector response. Thus,
15d-PGJ2 may modulate EAE by inhibiting the activation of
microglia/macrophages as well as by inhibiting the differentiation of T
cells into a Th1 phenotype that have been recruited into the inflamed
CNS. It is important to note that our observation that PPAR- is
expressed in the CNS of mice with EAE suggests that this molecule plays
a role in the remissions that are normally observed in EAE, although it
is likely that there are several pathways that contribute to the
remissions in EAE.
It has recently been shown that PPAR- ligands can inhibit the
IFN--induced expression of the CXC chemokines inducing protein-10,
and monokine induced by IFN-/IFN-inducible T cell
chemoattractant by endothelial cells (36). These
chemokines have been shown to play an important role in T cell
recruitment to sites of inflammation. Thus, another possibility for
inhibition of clinical signs of EAE by 15d-PGJ2 is its ability to
inhibit the molecules that contribute to the development of
inflammatory cell infiltrates into the CNS.
Finally, it was also interesting that administration of 15d-PGJ2 in
vivo did not follow a strict dose response with regard to inhibition of
EAE (Figs. 6 and 7), with a dose of 200 µg/kg/day being more
effective than a dose of 500 µg/kg/day. This observation may be due
to the fact that PPAR- binds to DNA as a heterodimer with RXR and
that PPAR--RXR can be activated by ligands specific for either
receptor (37). PPAR- and RXR interact with distinct
multisubunit coactivator complexes; thus, trans-activation
of target genes may also be regulated at the level of selective
coactivator recruitment and affect the dynamics of the responses
observed.
Overall, our results have shown that 15d-PGJ2 can inhibit the
clinical expression of EAE. It probably can accomplish this through
inhibition of expansion of encephalitogenic T cells as well as through
inhibiting a number of pathways that play a significant role in the
final effector pathways in EAE. These results suggest that PPAR-
activators such as 15d-PGJ2 may have the potential to modulate human
CNS inflammatory demyelinating diseases such as MS.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael K. Racke, Department of Neurology and Center for Immunology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9036. E-mail address: michael.racke{at}utsouthwestern.edu
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; 15d-PGJ2, 15-deoxy-12,14-PGJ2; MBP, myelin basic protein; MS, multiple sclerosis; PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; GFAP, glial fibrillary acidic protein.
Received for publication November 7, 2001. Accepted for publication December 21, 2001.
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) in 0.1 ml of PBS
(n = 7), 15d-PGJ2 at 100 µg/kg/day (
) in 0.1
ml of PBS (n = 7), or 0.1 ml of PBS (
;
n = 8). Mice were treated daily with i.p.
injections for 10 days beginning on day 1 as indicated by the bar. Mice
were monitored daily for clinical disease. B, Male
wild-type B10.PL mice were immunized with MBP Ac1–11 (200
µg) in CFA on day 0. Pertussis toxin (200 ng) was injected i.p. on
days 0 and 2. Three groups of animals were given 15d-PGJ2 at 500
µg/kg/day (
