| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Neurology, Multiple Sclerosis Research Center, Vanderbilt University Medical Center, Nashville, TN 37212; and
Lawson Research Institute, St. Josephs Care Center, and Department of Medicine, Pharmacology, and Toxicology, University of Western Ontario, London, Ontario, Canada
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, IL-12 p40, and
TNF-
) and NO in response to myelin basic protein. These effects were
associated with a decrease in activation and translocation of NF-
B.
In vivo administration of DHEA significantly reduced the severity and
incidence of acute EAE, along with a decrease in
demyelination/inflammation and expressions of (pro)inflammatory
cytokines in the CNS. These studies suggest that DHEA has potent
anti-inflammatory properties, which at least are in part mediated
by its inhibition of NF-B activation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and TNF-), and NO (free radical NO) play a
critical role in the pathogenesis of the disease
(1, 2, 3).
NF-B, a nuclear transcriptional factor, is activated in response to
inflammatory factors such as microbial products (e.g., LPS, dsRNA, and
DNA) and cytokines (TNF-, IL-1, lymphotoxin, and IL-2; Refs. 4, 5). At least five members of NF-B family have been
identified: p50/p105 (NF-B1), p52/p100 (NF-B2), p65 (RelA), RelB,
and c-rel, which form homo- and/or heterodimers. Of them,
the p50/p65 dimer is the most abundant complex (4, 6).
Activation of NF-B plays a critical role in innate immune responses
by up-regulating expression of cytokines and chemokines (6, 7). Some of these cytokines (IL-1, TNF-, lymphotoxin, IL-2,
IL-12, and IFN-) also directly contribute to the development of
Th1-mediated immune response (8).
Dehydroepiandrosterone (DHEA), a C19 adrenal steroid, and its sulfated form (DHEAS) are synthesized by the adrenal glands, gonadal tissue, and CNS cells (9, 10, 11). In young adult humans, the levels of DHEAS (5–6 µg/ml) and DHEA (2–4 ng/ml) in plasma are considerably higher than any other steroids (9). DHEAS is converted to the bioactive form DHEA by intracellular sulfatases, which are present in a number of cell types, including monocytes and macrophages (12, 13). Although DHEA is an abundant steroid hormone in serum, its biological functions are still unclear.
Although DHEA is widely considered to be a precursor of androgens and/or estrogens (14), increasing evidence indicates that DHEA has, in addition, potent immunoregulatory functions. In animal model systems, administration of DHEA enhances the ability of mice to resist experimental viral and bacterial diseases (15, 16, 17, 18, 19, 20). In humans, many chronic inflammatory diseases are associated with lower serum levels of DHEA or DHEAS (21). Therapy with DHEA has shown benefit in some patients with systemic lupus erythematosus (22) and HIV infection (23). Our previous study has shown that the addition of DHEA to in vitro cultures inhibits the development of Th1 cells (24). In the present study, we examined the effect of DHEA on EAE. We predicted that administration of exogenous DHEA would inhibit Th1-mediated inflammatory responses and would consequently prevent the development of EAE.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Female SJL/J mice (6–8 wk old) were purchased from the National
Institutes of Health (Bethesda, MD) and were maintained in the animal
care facility at Vanderbilt University (Nashville, TN). DHEA was
obtained from Sigma-Aldrich (St. Louis, MO). The DHEA stock solution
(10 mM) was prepared by dissolving it in DMSO (Sigma-Aldrich), and it
was diluted to the experimental concentrations (2–20 µM) in the
culture medium. The monoclonal anti-mIL-12 Abs (C17.5 and C15.6),
gifts from Dr. G. Trinchieri (Wister Institute, Boston, MA), were
purified by QAE Sephadex A-50 and Sephadex G-25 M column
(Pharmacia Biotech, Uppsala, Sweden) from hybridoma ascetic fluid.
C15.6 was biotinylated according to manufacturer’s protocol and was
used as a detecting Ab, whereas C17.5 was used as a capture Ab for
IL-12 p40 ELISA. Abs against p50 and p65 of NF-B were brought from
Santa Cruz Biotechnology (Santa Cruz, CA). rmIL-12 was kindly provided
by Genetics Institute (Cambridge, MA). MBP was purified from guinea pig
spinal cord (Rockland, Gibertsville, PA) by the methods described
previously (25).
ELISA of cytokines
Levels of cytokines (IL-12 p40, IFN-, IL-4, and TNF-) were
measured by ELISA as described previously (26). The
matched Ab pairs and standards for IL-4, IFN-, and TNF- were
purchased from Endogen (Woburn, MA), and the Abs and standards used for
IL-12 p40 ELISA were described above.
Measurement of NO
NO secreted from cells is rapidly oxidized to nitrite in the culture medium, therefore, determination of nitrite concentrations was used as a measurement of NO production. Fifty microliters of culture supernatant was mixed with 50 µl of Greiss reagent (1% sulfanilamide, 0.1% naphthylethyline diamine dihydrochloride, and 2.5% phosphoric acid) per well in a 96-well plate in triplicate. After incubation at 25°C for 10 min, the absorbance was read at 550 nm. The levels of NO in the culture medium were calculated based on sodium nitrite standards.
Proliferation assay
T cell cultures were grown in a 96-well microtiter plate in triplicate. MBP-primed lymphocytes (2 x 105 per well) were cultured in the presence of MBP for 72 h in RPMI 1640 complete medium (RPMI 1640 medium, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS; Life Technologies, Rockville, MD) under the atmosphere of 5% CO2 and 95% air at 37°C. The cultures were harvested using a Harvester96 (Tomtec, Orange, CT) after incubation with 0.5 µCi/well [3H]thymidine (DuPont Pharmaceuticals, Boston, MA) for the last 18 h. The radioisotope incorporation as index of T cell proliferation was determined using a betaplate liquid scintillation counter (Wallac, Turku, Finland).
Nuclear extraction and EMSA
After treatment, MBP-primed or naive splenocytes (2.5 x 106 cells/ml) were added to 1 mM Na3VO4 at a final concentration and were pelleted by centrifugation at 1,000 x g for 5 min at 4°C. The pellets were washed with 0.5 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 mM Na3VO4, and 5 µg/ml each of aprotinin, leupeptin, pepstatin, chymostatin, and antipain) and were centrifuged again at 1,000 x g for 5 min at 4°C. The cells were lysed in 100 µl of buffer A containing 0.5% Igepal CA-630 (Sigma-Aldrich) for 15 min at 4°C. The resultant nuclei were harvested by centrifugation at 1,500 x g for 15 min at 4°C, resuspended in 30 µl of buffer B (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM DTT, 25% glycerol, 0.5 mM PMSF, and 5 µg/ml each of aprotinin, leupeptin, pepstatin, chymostatin, and antipain), and extracted by vigorous whirling for 15 min at 4°C. The nuclear extracts were cleared by centrifugation at 16,000 x g for 10 min at 4°C. Two microliters of nuclear extract from each sample was taken for measurement of protein content by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA), and the final nuclear extracts were equalized among samples using buffer B, frozen in liquid nitrogen, and kept at -75°C.
The activation or nuclear translocation of NF-B was performed by
using EMSAs. NF-B consensus oligonucleotide
(5'-AGTTGAGGGGACTTTCCCAGGC-3') was purchased from Promega (Madison,
WI), and nuclear factor-Y box binding (NF-Y) oligonucleotide
(5'-GATCTGAGAATTTTCTGATTGGTTCTGGCGAGTTTGG-3') (27) was
synthesized at IDT (Coralville, IA) and purified by polyacrylamide gel.
Double-stranded oligonucleotide probe was labeled by
32P in a reaction containing 2 µl of probe (3.5
pmol), 1 µl of T4 polynucleotide kinase buffer (10x), 1 µl of
[-32P]ATP (3000 Ci/mmol; Amersham, Arlington
Heights, IL), 1 µl of T4 polynucleotide kinase (5–10 U; New England
Biolabs, Beverly, MA), and 5 µl of nuclease-free water for 10
min at 37°C. The reaction was stopped by the addition of 1 µl of
0.5 M EDTA (pH 8.0). The labeled probe was purified with a Micro
Bio-Spin 30 Chromatography Column (Bio-Rad). DNA-nuclear protein
binding reactions were done in a final volume of 20 µl for 30 min at
25°C. NF-B-binding reactions contained 5 µg of nuclear protein,
4 µl of binding buffer (5x) (20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM
MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 20% glycerol,
and 0.25 mg/ml fish sperm DNA) and 0.5–1 x
106 cpm 32P-labeled probe;
NF-Y-binding reactions contained 5 µg of nuclear protein, 4 µl of
binding buffer (5x) (20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM
MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 20% glycerol,
and 0.25 mg/ml poly(dI-dC)), and 2–5 x 105
cpm 32P-labeled probe. For supershift assays, 2
µl of anti-NF-B (p50 or p65) Abs was added to NF-B-binding
reactions after binding reaction completed, and they were then
incubated for 1 h. The complex of probe and binding proteins was
electrophoresed in a 4% polyacrylamide gel (16 x 20 cm) in 1x
Tris-borate-EDTA at 100 V, and was autoradiographed at
-75°C.
Induction of EAE
Active EAE was induced by s.c. immunization with 800 µg of mouse spinal cord homogenate (MSCH) in CFA (Sigma-Aldrich) on days 0 and 7, along with i.p. injection of pertussis toxin (200 ng in 0.5 ml of PBS per animal; Sigma-Aldrich) on days 0 and 2. Passive EAE was induced by adoptive transfer of MBP-sensitized T lymphocytes (28). Briefly, SJL/J mice were s.c. immunized with 400 µg of MBP in CFA on days 0 and 7. On day 14, the lymph nodes and spleen cells from the immunized mice were harvested and purified by gradient centrifugation (Histopaque-1077; Sigma-Aldrich), and then incubated in RPMI 1640 complete medium in the presence of 50–100 µg/ml MBP. A proliferation assay of MBP Ag-reactive T cells was performed at the time the lymph nodes and spleen cells were harvested. Cells (2 x 105 per well) were incubated in RPMI 1640 complete medium in the presence of various concentrations of MBP Ag, and the Ag-reactive T cell proliferation assay was conducted as described above. To induce EAE, viable T cell blasts were harvested from MBP-stimulated cultures (a 96-h culture), washed twice with PBS, and injected i.p. into recipient mice (1.0 x 107 cells in 0.5 ml of PBS per animal).
Treatment of animals, and clinical and pathologic evaluation of disease
Mice were treated with DHEA (2 mg/dose) or its carrier, DMSO (Sigma-Aldrich), daily by s.c. injection in a volume of 50 µl. The treatment began from the time of the first immunization (active EAE) or T cell transfer (passive EAE; day 0) until disease recovery. Paralysis was graded as follows: 0, normal; 0.5, still tail; 1, limp tail; 1.5, limp tail with inability to right; 2, paralysis of one limb; 2.5, paralysis of one limb and weakness of one other limb; 3, complete paralysis of both hind limbs; 4, moribund state; and 5, dead.
To assess the degree of inflammation or demyelination, EAE mice with were euthanized on day 25 and were perfused by intracardiac injection of 4% paraformaldehyde and 1% glutaraldehyde in PBS. Transverse sections of the cervical, upper thoracic, lower thoracic, and lumbar regions of the spinal cord were stained with Luxol Fast Blue or H&E. Each spinal cord section was further subdivided into an anterior, posterior, and two lateral columns. Each of them that displayed either lymphocyte infiltration or demyelination was assigned a score of one, thus, each animal had a potential maximum score of 16.
Isolation of total RNA and semiquantitative RT-PCR
Mice with EAE were randomly selected by cage numbers for mRNA
isolation and analysis on day 20. Following perfusion with PBS, total
RNA from the spinal cord tissue was extracted using TRI Reagent
(Sigma-Aldrich) in accordance with the manufacturer’s protocol. If
necessary, RNA was further purified by digestion using DNase I. Four
micrograms of total RNA was reverse transcribed (RT) to cDNA
using a GeneAmp RNA PCR kit with oligo d(T)16
primers (PerkinElmer, Branchburg, NJ; RT reaction). PCR amplification
of each cDNA target (CD3, IFN-, IL-4, IL-12 p40, TNF-, and
inducible NO synthase (iNOS)) was performed from the same RT
reaction as an internal control GAPDH. Each PCR contained 5 µl of
cDNA, 2 µl of 10x PCR buffer (PerkinElmer), 1 µl of 25 mM
MgCl2, 0.5 µl of each dNTP (10 mM), 0.5 µl of
sense and antisense target gene-specific primers (50 pmol/µl), 0.25
µl of AmpliTaq DNA polymerase (5 units/µl; PerkinElmer), and 13.75
µl of nuclease-free H2O, and was conducted in
PTC-200 Peltier Thermal Cycler (MJ Research, Watertown, MA). The
specific sense and antisense oligonucleotide PCR primers for each cDNA
target are listed in Table I
.
Amplification conditions including annealing temperatures, number of
cycles, and extension times were optimized for each target. PCR
products were run on a 1.5% agarose gel containing 0.5 µg/ml
ethidium bromide, and were visualized under UV light. The density of
the band was quantitated using a Digital Imaging System (IS-1000
Version 2.0; Alpha Innotech, San Leandro, CA).
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The addition of DHEA to the MBP-primed lymphocyte cultures
resulted in a decrease of MBP-stimulated T cell proliferation, which
was dependent on the doses of both DHEA and MBP (Fig. 1). Following stimulation with 100
µg/ml MBP, the proliferative response of T cells was suppressed at
all concentrations of DHEA. When 50 µg/ml MBP was added, no
inhibition was seen in the cultures with the addition of 5 µM of
DHEA, but significant reduction was observed at 10 or 20 µM of DHEA.
These results are similar to those observed in our earlier studies with
keyhole limpet hemocyanin-primed lymphocytes (24),
indicating that the inhibitory effect of DHEA on the Ag-stimulated T
proliferation depends upon stimulation of Ag. In addition, although the
background of [3H]thymidine incorporation was
low in the cultures without stimulation of MBP, it was reduced by the
presence of DHEA as well.
|
,
IL-12, and TNF-) and NO in MBP-primed splenocyte cultures
We examined whether the inhibition of lymphocytic proliferation by
DHEA was accompanied by a reduction in the levels of Th1-associated
cytokines and NO. As shown in Fig. 2, the
addition of DHEA reduced production of IFN-, IL-12 p40, TNF-, and
NO in MBP-stimulated culture supernatants in a dose-dependent manner.
In cultures treated with 20 µM DHEA, inhibition of IFN- was
complete (100% reduction), IL-12 p40 levels decreased from 3.5 ±
0.12 ng/ml to 1 ± 0.008 ng/ml (72% reduction), TNF- decreased
from 101.4 ± 9.7 pg/ml to 32.3 ± 3.3 pg/ml (68%
reduction), and NO levels were reduced from 9.5 ± 0.5 µM to
2.4 ± 0.002 µM (75% reduction). These observations indicate
that DHEA reduces Th1-mediated inflammatory responses in
MBP-primed T cells following stimulation with Ag. In addition, Th2
cytokine IL-4 production in these cultures following stimulation of MBP
was very low (64 ± 10 pg/ml), and was reduced to undetectable
levels by the presence of DHEA.
|
B
The activation of NF-B is a prerequisite for the expression of
a number of proinflammatory cytokines and mediators, including TNF-,
IL-12 p40, IL-2, and iNOS (4, 29). To determine whether
the molecular mechanism of DHEA action occurred at a site proximal or
distal to NF-B, we examined the effect of DHEA on the induction of
NF-B activation by EMSA in MBP-primed splenocytes. As shown in Fig. 3, nuclear translocation of NF-B was
seen in response to stimulation with MBP in the MBP-primed splenocytes.
Supershift studies showed that the migration of NF-B was retarded
completely by anti-p50 Ab and to a lesser degree by anti-p65
Ab, indicating that the NF-B complex included, at least in part, a
heterodimer of p50/p65. Because the entire panel of anti-NF-B
Abs was not examined, the presence of other Rel proteins and homodimers
of p50/p50 could not be excluded. The addition of DHEA to the cultures
of MBP-primed splenocytes reduced the activation of NF-B in a
dose-dependent manner (Fig. 4). Using a
digital image system to quantity the levels of NF-B, the
translocation of NF-B to the nucleus was inhibited by 43% following
the addition of 20 µM DHEA (Fig. 4B). Furthermore, kinetic
studies showed that in the cultures treated with 10 µM DHEA,
inhibition occurred at as early as 1 h and reached it maximum at
8 h (Fig. 5). These data suggest
that DHEA inhibits T cell proliferation and proinflammatory
cytokine/mediator production at least in part by inhibiting
translocation of NF-B. Because mixed cultures were used in these
investigations, our studies do not identify the phenotype of the cell
in which the effects of DHEA on NF-B activation are maximal.
|
|
|
The development of EAE is closely regulated by the expression of proinflammatory cytokines/mediators (30). Our data has shown that DHEA inhibited immune responses of MBP-specific T cells against MBP by decreasing production of (pro)inflammatory cytokines and NO and/or proliferation. Therefore, we predicted that parental administration of DHEA in vivo would alter the development of autoantigen specific T cells and thereby the incidence and severity of EAE.
When EAE was induced by active immunization in the absence of pertussis
toxin (Fig. 6A), only one of
six mice treated with DHEA was paralyzed (mean maximal clinical score:
0.17), whereas in the vehicle-treated group, five of six mice were
paralyzed (mean maximal clinical score: 1.42; p <
0.01). In the second experiment, EAE was induced with the addition of
pertussis toxin (Fig. 6B). Seven of nine DHEA-treated mice
developed clinical paralysis of EAE. All nine mice treated with vehicle
were paralyzed with high severity, and one died from disease attack.
The severity of EAE was reduced by DHEA treatment from a mean maximal
clinical score of 2.82 in the control group to 1.25
(p < 0.01). In the adoptive transfer model of
EAE (Fig. 6C), none of the nine mice that received DHEA
developed clinical paralysis of EAE, whereas seven of nine mice in the
control group developed clinical signs of EAE with a mean maximal
clinical score of 1.2 (p < 0.001). These data
indicate that administration of DHEA protects animals from the
development of active and adoptively transferred EAE.
|
). In the mice receiving DMSO
(mean clinical score: 1.33), the mean pathologic scores were 3.5 for
demyelination and 4.5 for lymphocyte infiltration. However, in
DHEA-treated mice (mean clinical score: 0), the score of demyelination
or lymphocytes infiltration was reduced to 0.4 or 0.7, respectively
(Fig. 7B). These data indicate that the reduction of EAE
severity in the DHEA-treated mice closely correlates with a decrease of
demyelination and/or lymphocyte infiltration in the CNS.
|
To examine whether the reduction of demyelination/lymphocyte
infiltration following DHEA treatment was accompanied by a decrease in
inflammatory mediators in the CNS, we analyzed the expression of
inflammation/Th1-related genes (CD3, IFN-, IL-12 p40, IL-4,
TNF-, and iNOS) from the spinal cord of mice with EAE. As shown in
Fig. 8, four mice randomly selected from
the DHEA-treated group showed a decrease in levels of all targeted mRNA
(CD3, IFN-, IL-12 p40, TNF-, and iNOS) as compared with four
mice from the vehicle-treated group. The level of each mRNA in each
mouse closely correlated with its clinical score. The digital image
analysis indicated that the levels of expression of CD3, IFN-,
IL-12 p40, TNF-, and iNOS were higher in the vehicle-treated animals
(clinical scores: 3, 2, 3, and 3) than those in the DHEA-treated mice
(clinical scores: 0, 2, 0, and 0; p < 0.05; Fig. 8B). Among the DHEA-treated mice, one animal with paralysis
showed comparable levels of the presence of T cells (marked by CD3)
and (pro)inflammatory cytokines/mediator in the CNS, but in the
remaining animals in which no clinical paralysis was observed, the
levels of all targeted mRNA were either lower (one mouse) or the same
as seen in the naive animal. No expression of IL-4 was seen, and this
would argue against a switch in the immune response from Th1 to Th2
following treatment with DHEA. These data indicate that in vivo
administration of DHEA reduces Th1-mediated autoimmune responses in the
CNS via inhibition of T cell proliferation and/or production of
Th1-related cytokines and inflammatory mediator; as a result, it
prevents EAE.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B in splenocytes
following stimulation with Ag; and in vivo administration of DHEA
inhibits Th1-mediated inflammatory responses in the CNS, and
ameliorates the development of EAE.
DHEA is the most abundant steroid in the circulation of humans and many
other warm-blooded animals (9, 31). Plasma levels of DHEA
peak in early adulthood (reaching levels of 2–4 ng/ml) and then
decline through adult life (9). In adult mice, DHEA levels
are 
1 ng/ml and they dramatically increase following parental
administration of DHEA (32). In the present investigation,
the DHEA doses (2–20 µM) we tested in vitro are undoubtedly high
when compared with its levels in sera, but the levels of local and
intracellular DHEA in the steroid target tissue are uncertain. The
average serum DHEAS level in healthy "younger" adults is 7.5
µM, and increases to 20–70 µM following administration of
pharmacological dosages of DHEA without any sign of systemic toxicity
(33). These high levels of recirculating DHEAS can be
converted to DHEA by ubiquitous steroid sulfatase. Therefore, DHEA
levels in plasma may not reflect the real concentrations of DHEA in the
target tissue, which could be much higher than that observed in the
circulation.
The ability of DHEA to regulate proliferation and cytokine production
suggests its potential role in mediating inflammatory response. Prior
studies of DHEA on T cell function have shown conflicting results. It
has been reported that DHEA enhances IL-2 production and, consequently,
T cell proliferation in murine and human T cells (34, 35),
whereas others have noted an inhibition of lymphocyte proliferation and
IL-2 production in the presence of DHEA (36, 37, 38). Our
previous work also has demonstrated that the addition of DHEA in vitro
inhibits T lymphocyte proliferation stimulated by ligation of TCR
complex and favors the induction of a Th2 immune response to Ag
(24). In LPS-stimulated macrophages, DHEA reduces the
production of IL-12 (24), TNF-, IL-1 (39),
and NO (data not shown). In this study, the most dramatic effect of
DHEA was in the suppression of the T cell proliferative response and
the development of EAE.
EAE is a Th1 cell-mediated inflammatory autoimmune disease of the
central nervous system that serves as a prototypic animal model for MS.
The suppression of this disease by DHEA (Fig. 6
) disagrees with the
data presented by Kipper-Galperin et al. (40). This
conflicting result could be due to the differences in the dosage and
the route of administration of DHEA. In this study, we used 2 mg/mouse
every day by s.c. injection, by which way the highest bioavailability
of DHEA in vivo is obtained (41). Although Kipper-Galperin
et al. gave 0.5 mg/mouse every other day by i.p. injection
(40), that may not be enough to suppress the autoimmune
responses in the EAE animals.
The mechanism by which DHEA mediates its effect on T cells in vitro and in vivo is not known. So far, all known steroid receptors belong to a group of nuclear (orphan) receptors, including those for glucocorticoid and progesterone. They undergo structural alternation upon binding to the steroid ligands, conferring upon it the ability to bind DNA and regulate gene transcription (42). The receptors for DHEA/DHEAS are not identified yet, but DHEA binding activity has been detected in murine and human T cells, suggesting that receptors for DHEA may be present in T cells (43, 44). The action of DHEA in vivo is also not fully understood. One of the important questions is whether exogenous DHEA acts directly on T cells and/or other immune cells in vivo as it does in vitro. In many peripheral steroid tissues such as the prostate and mammary glands, DHEA can be converted into androgens and/or estrogens (45), which also have immunomodulatory functions. However, a role independent of its metabolic functions and, in particular, on immune regulation has been previously observed (31, 46). For example, DHEA was shown to have immunomodulatory properties in androgen-unresponsive mice (47). Further studies using an inhibitor of DHEA metabolic conversion or androgen/estrogen receptors double knockout mice are needed to clarify this point.
NF-B is clearly one of the most important transcriptional factors
regulating expression of many proinflammatory genes, including IL-2,
IL-12 p40, TNF-, IL-1, and iNOS (4, 29). Our gel shift
assay showed that activation of NF-B was reduced in the presence of
DHEA in Ag-primed splenocytes (Figs. 4 and 5), indicating that the
inhibitory action of DHEA on nuclear translocation of NF-B is a key
mechanism mediating the reduction of the proliferative response and
(pro)inflammatory cytokine production. GRs, structurally similar to
DHEA, are known to inhibit the activation of NF-B. Activated GR
directly inhibits activation of NF-B subunits and/or up-regulates
transcriptional activity of IB, resulting in inactivation of
NF-B (48). It is possible that activated DHEA receptor
inhibits NF-B activation in Ag-primed splenocytes in our study
through one of these mechanisms.
It has been demonstrated that the development of the autoimmune disease
EAE requires the activation of inflammation-related genes such as
IL-12, TNF-, and iNOS (2, 49). Virtually all of these
genes have shown the presence of binding sites for NF-B in their
promotor regions (4, 29). In rats with EAE, the activation
of NF-B (p50/p65) is seen in the spinal cord and persists throughout
the disease (50, 51). Disruption of the NF-B gene or
treatment with pyrrodine dithiocarbomate, an inhibitor of NF-B
activation, prevents the development of EAE (51, 52, 53). We
believe that the immunosuppressive effect of DHEA on EAE is closely
associated with the inhibition of activation of NF-B. Because glial
cells synthesize DHEA (10), our findings imply that the
presence of the neurosteriod DHEA may protect the CNS against
autoimmune injury. Additionally, it is possible that genetic
differences in the levels of DHEA in the brain and/or plasma may
predict susceptibility to autoimmune diseases including EAE.
In conclusion, a number of strategies aimed at interrupting the proinflammatory cascade are currently being applied to the treatment of human diseases. Invariably, they are initially tested in experimental model systems with the hope that they will be successful in human disease. Our data show that DHEA has potent anti-inflammatory properties in vitro and in preventing the development of EAE. Furthermore, because the inhibition of EAE was seen in adoptive transfer model system, it is likely that the effect of DHEA is present even after the generation of an immune response. Because DHEA does not possess the undesirable side effects of glucocorticoids, it has the potential to be applied to the treatment of chronic inflammatory diseases in the CNS such as MS.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; DHEA, dehydroepiandrosterone; DHEAS, sulfated form of DHEA; MBP, myelin basic protein; MS, multiple sclerosis; MSCH, mouse spinal cord homogenate; NF-Y, nuclear factor-Y; iNOS, inducible NO synthase.
Received for publication August 14, 2001. Accepted for publication October 11, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B. Annu. Rev. Cell Biol. 10:405.
B as a frequent target for immunosuppressive and anti-inflammatory molecules. Adv. Immunol. 65:111.[Medline]
B and Rel proteins: evolutionarily conserved mediators of immune response. Annu. Rev. Immunol. 16:225.[Medline]
B and rel proteins in innate immunity. Adv. Immunol. 58:1.[Medline]
-amyloid peptide. J. Neurochem. 74:847.[Medline]
in the trigeminal ganglion correlate with protection against HSV-1-induced encephalitis following subcutaneous administration with androstenediol. J. Neuroimmunol. 89:160.[Medline]
. J. Immunol. 147:1792.[Abstract]
in ANA-1 cells. J. Leukocyte Biol. 64:92.[Abstract]
B half-site. Mol. Cell. Biol. 15:5258.[Abstract]
production in vitro. Cell. Immunol. 184:58.[Medline]
and interlukin-6 in astrocytes. Int. J. Dev. Neurosci. 17:765.[Medline]
B and IB proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
B is activated in microglia during experimental autoimmune encephalomyelitis. J. Neuroimmunol. 55:99.[Medline]
B in the spinal cord of experimental allergic encephalomyelitis. Neurosci. Lett. 287:17.[Medline]
B-deficient mice: roles of NF-B in the activation and differentiation of autoreactive T cells. J. Immunol. 163:2937.This article has been cited by other articles:
|
|
 |
|
  A. Vojdani, F. Hebroni, Y. Raphael, J. Erde, and B. Raxlen Novel Diagnosis of Lyme Disease: Potential for CAM Intervention Evid. Based Complement. Altern. Med., September 1, 2009; 6(3): 283 - 295. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mondal, A. Roy, and K. Pahan Functional Blocking Monoclonal Antibodies against IL-12p40 Homodimer Inhibit Adoptive Transfer of Experimental Allergic Encephalomyelitis J. Immunol., April 15, 2009; 182(8): 5013 - 5023. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Dasgupta, A. Roy, M. Jana, D. M. Hartley, and K. Pahan Gemfibrozil Ameliorates Relapsing-Remitting Experimental Autoimmune Encephalomyelitis Independent of Peroxisome Proliferator-Activated Receptor-{alpha} Mol. Pharmacol., October 1, 2007; 72(4): 934 - 946. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Roy and D. C. Hooper Lethal Silver-Haired Bat Rabies Virus Infection Can Be Prevented by Opening the Blood-Brain Barrier J. Virol., August 1, 2007; 81(15): 7993 - 7998. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Brahmachari and K. Pahan Sodium Benzoate, a Food Additive and a Metabolite of Cinnamon, Modifies T Cells at Multiple Steps and Inhibits Adoptive Transfer of Experimental Allergic Encephalomyelitis J. Immunol., July 1, 2007; 179(1): 275 - 283. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. H. van den Broek, J. G. Damoiseaux, M. H De Baets, and R. M. Hupperts The influence of sex hormones on cytokines in multiple sclerosis and experimental autoimmune encephalomyelitis: a review Multiple Sclerosis, June 1, 2005; 11(3): 349 - 359. [Abstract] [PDF]
|
|
|
|
 |
|
 D. R. Jeffery Use of combination therapy with immunomodulators and immunosuppressants in treating multiple sclerosis Neurology, December 28, 2004; 63(12_suppl_6): S41 - S46. [Abstract] [Full Text]
|
|
|
|
 |
|
 R. Bergamaschi, C. Livieri, E. Candeloro, C. Uggetti, D. Franciotta, and V. Cosi Congenital Adrenal Hyperplasia and Multiple Sclerosis: Is There an Increased Risk of Multiple Sclerosis in Individuals With Congenital Adrenal Hyperplasia? Arch Neurol, December 1, 2004; 61(12): 1953 - 1955. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Dasgupta, M. Jana, Y. Zhou, Y. K. Fung, S. Ghosh, and K. Pahan Antineuroinflammatory Effect of NF-{kappa}B Essential Modifier-Binding Domain Peptides in the Adoptive Transfer Model of Experimental Allergic Encephalomyelitis J. Immunol., July 15, 2004; 173(2): 1344 - 1354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Charalampopoulos, C. Tsatsanis, E. Dermitzaki, V.-I. Alexaki, E. Castanas, A. N. Margioris, and A. Gravanis Dehydroepiandrosterone and allopregnanolone protect sympathoadrenal medulla cells against apoptosis via antiapoptotic Bcl-2 proteins PNAS, May 25, 2004; 101(21): 8209 - 8214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Dasgupta, Y. Zhou, M. Jana, N. L. Banik, and K. Pahan Sodium Phenylacetate Inhibits Adoptive Transfer of Experimental Allergic Encephalomyelitis in SJL/J Mice at Multiple Steps J. Immunol., April 1, 2003; 170(7): 3874 - 3882. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Du, S.-Y. Yao, A. Ljunggren-Rose, and S. Sriram Chlamydia pneumoniae Infection of the Central Nervous System Worsens Experimental Allergic Encephalitis J. Exp. Med., December 16, 2002; 196(12): 1639 - 1644. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
