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Department of Immunology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by Staphylococcus
aureus Cowan I-stimulated spleen cells from several mouse
strains such as BALB/c, C3H/HeN, and C57BL/6. When spleen cells were
pretreated with indomethacin (cyclooxygenase (COX)-1 and COX-2
inhibitor) or NS-398 (COX-2-specific inhibitor), S.
aureus Cowan I -induced IFN- production was increased more
markedly in spleen cells from BALB/c mice than from C3H/HeN and C57BL/6
mouse. However, PGE2 production was not significantly
different among spleen cells from three mouse strains. When various
concentrations of PGE2 were exogeneously added to spleen
cells, PGE2 showed a stronger suppressive effect on IFN-
production in spleen cells from BALB/c mice than from other strains of
mice. This suppressive effect of PGE2 in BALB/c mice mainly
depended on IL-12p70 production by APCs. More PGE2 binding
sites were found in BALB/c spleen cells than in C3H/HeN spleen cells,
indicating that the sensitivity difference to the suppressive effect of
PGE2 was due to the difference of the number of
PGE2 receptors. The administration of NS-398 into BALB/c
mice enhanced Ag-specific IFN- production, but not IL-4 production.
This effect is the same as IL-12 administration in vivo. From these
results, we propose that the modulation of PGE2 is
important for Th1 activation via IFN- and IL-12p70 production in
vitro and in vivo and that PGE2 is one of the pivotal
factors in the Th2-dominant immune response in BALB/c
mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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prevents this mouse strain from its
infection by promoting Th1 development. In contrast, B10D2, C3H/He,
C57BL/6, and CBA mice are resistant against L. major
infection, which is related to Th1 activation. Recently, several
reports have shown that in BALB/c mice, CD4+ T
cells bearing Vß4 V
8 TCR were the source of the early burst of
IL-4 and rapidly produced IL-4 down-regulated IL-12R within 48 h
of L. major infection (11, 12). Thus,
modulations of responsive cell types, humoral factors (ILs and IFNs),
and receptors play critical roles in the regulation of Th1 and Th2
subset differentiation in murine models with specific genetic
backgrounds. There are also several reports about the preferential
activation of Th2 in BALB/c mice in other disease models. For example,
in Trypanosoma cruzi infection, BALB/c mice are susceptible
and show higher IL-4 production and parasitemia than resistant C57BL/6
mice (13). In bacillus Calmette-Guérin infection,
spleen cells from infected BALB/c mice produced IFN- 75% lower than
C57BL/6 mice did (14). In Yersinia
enterocolitica infection, resistant C57BL/6 mice produce higher
amount of IFN-, while susceptible BALB/c mice produce
significantly lower amount of IFN- and acquire resistance against
the infection by administration of IL-12 (15, 16, 17). In
murine hepatitis virus infection, susceptible BALB/c mice generate Th2
response and cannot be protected against the infection
(18). On the other hand, BALB/c mice are genetically
resistant to Th1-related experimental autoimmune diseases induced by
the immunization of autoantigen with CFA (19, 20, 21). C57BL/6
mice produce significant level of IFN- by the injection of endotoxin
that causes severe liver injury, but BALB/c mice do not
(22). Cells from BALB/c mice preferentially differentiate
to Th2 not only in vivo, but also in vitro using TCR ß transgenic
mice. These phenomena are due to a selective loss of the expression of
IL-12R (23).
PGE2, an arachidonic acid metabolite produced by
various type of cells, regulates a broad range of physiological
activities in the endocrine, cardiovascular, gastrointestinal, neural,
reproductive, and immune systems, and maintains the local homeostasis
(24). In the immune system, PGE2 is
mainly produced by APCs such as monocytes, macrophages, and dendritic
cells, and the effects are almost suppressive on Th1-related immune
responses. PGE2 suppresses IL-2 and IFN-
production by Th1 clone, but not IL-4 and IL-5 production by Th2 clone
(25). In the differentiation phase of naive T cells,
PGE2 inhibits the differentiation of Th1 and
IL-12R expression via cAMP accumulation (26, 27).
PGE2 also suppresses LPS-induced IL-12 production
by APCs, but enhances IL-10 production (28). In B cell
functions, PGE2 enhances IgE production by IL-4-
and LPS-stimulated B cells in vitro (29). IL-12 and
PGE2 derived from APCs determine IFN- level of
T cells (30). In these reports, PGE2
has a tendency to drive to Th2.
Immune responses are composed of two phases. First, innate immune
responses are activated in response to infectious agents, and then
acquired immune responses are induced. In BALB/c mice, both immune
responses tend to induce Th2 responses. Namely, BALB/c mice produce
lower IFN- in response to bacterial or parasite Ags at an early
phase (innate immunity), and Ag-specific Th2 is preferentially
developed both in vitro and in vivo (acquired immunity).
In this study, first we examined the effect of
PGE2 on IFN- production by
Staphylococcus aureus Cowan I
(SAC)2-stimulated
spleen cells from BALB/c, C3H/He, and C57BL/6 mice in vitro as a model
of innate immune response. Next we examined the effect of a
PGE2 inhibitor on the development of Ag-specific
Th1 and Th2 in vivo as a model of acquired immune response. We show
evidence that PGE2 plays an important role in the
polarization to Th2 in BALB/c mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c, C3H/HeN, and C57BL/6 male mice, 7–8 wk old, were purchased from Seac (Ohita, Japan) and were maintained in our laboratory under a specific pathogen-free condition.
Preparation of cell
Spleen cell suspension was prepared and maintained in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10 mM HEPES, pH 7.2, and 10% FBS (BioWhittaker, Walkersville, MD). For preparation of dish-adherent cells, spleen cell suspension (1 x 107/ml) was incubated in plastic culture dishes (Falcon 3002; Becton Dickinson, Franklin Lakes, NJ) at 37°C for 2 h. After incubation, nonadherent cells were discarded by gentle agitation with PBS. Adherent cells were obtained by scraping them off with a rubber policeman and using them as APCs. The purity of APCs was <10% IgM+ cells and <5% CD90+ cells, as determined by flow cytometry. Purified T cells were prepared by passing them through a nylon wool column (31). The purity of CD90+ cells was >90%. CD4+ T cells were further purified from nylon-purified T cells by magnetic cell sorting, according to the manufacturer’s procedure (Miltenyi Biotech, Bergish-Gladbach, Germany). Depletion of B cells was performed by passing spleen cells through a nylon wool column, and spleen-adherent cells were added back as APCs.
In vitro culture of cells
In the standard culture, spleen cells (3 x
106/ml) were cultured in 24-well culture plates
(Falcon 3047) with or without 1 µM of indomethacin (Sigma, St. Louis,
MO), 100 nM of NS-398 (Cayman Chemicals, Ann Arbor, MI), and indicated
concentrations (0
100 nM) of PGE2 (Sigma) for
12 h, and then they were stimulated with 0.05% SAC, Con A (10
µg/ml), or soluble anti-CD3 Ab (1 µg/ml) for additional 24
h. The culture supernatants were collected and used for ELISA, as
described below. In the experiment to neutralize cytokines or delete
NO, spleen cells were treated with indomethacin and 0 or 10 nM
PGE2 in the presence or absence of 1 µg/ml of
anti-IL-4, anti-IL-10 (PharMingen, San Diego, CA),
anti-TGF-ß Ab (Genzyme, Cambridge, MA), or 250 µM of
L-NG-monomethyl arginine
(L-NMMA; Sigma) for 12 h, and then
stimulated with SAC for additional 24 h. In the experiment using
fractionated cells, purified APCs (5 x
105/ml) or T cells (2 x
106/ml) were treated with indomethacin in the
presence or absence of 100 nM PGE2 for 12 h,
washed twice with PBS, mixed together, and then stimulated with SAC for
additional 24 h. The percentages of IFN- production and the
percentages of suppression of IFN- and IL-12 production were
calculated as follows, and the detailed calculations are described in
figure legends: % IFN- production = [(amounts of IFN- by
agents or Ab-treated cells)/(amounts of IFN- by nontreated cells)]
x 100; and IFN- (IL-12) suppression = {1 - [(amounts
of IFN- (IL-12) by agents and PGE2-treated
cells)/(amounts of IFN- (IL-12) by agent-treated cells)]}
x 100.
ELISA
IL-4, IL-10, IL-12, and IFN- in the culture supernatant were
assayed by ELISA using anti-mouse cytokine mAbs as capture Ab,
biotin-labeled anti-cytokine mAbs as detection Ab, and
streptavidin-conjugated alkaline phosphatase (PharMingen) and
p-nitrophenylphosphate (Zymed Laboratory, San
Francisco, CA) as a substrate. IL-12p70 was measured using BIOTRAK
mouse IL-12p70 ELISA Kit (Amersham Pharmacia Biotech, Aylesbury, U.K.),
according to the manufacturer’s protocol.
PGE2 enzyme immunoassay
PGE2 secreted into the culture supernatant was measured using STAT-PGE2 Enzyme Immunoassay Kit (Cayman Chemicals), according to the manufacturer’s protocol.
PGE2-binding assay
PGE2-binding assay was performed as described by Eriksen et al. (32), with slight modifications. Briefly, spleen cells were suspended at 5 x 107 cells/ml in binding buffer (10 mM phosphate buffer, pH 6, containing 100 mM NaCl and 1 mM EDTA). Two hundred microliters of cell suspension were incubated with [3H]PGE2 (sp. act., 200 Ci/mmol; NEN Life Science, Boston, MA) at the concentrations ranging from 0.25 to 10 nM in the presence or absence of 1000-fold amount of unlabeled PGE2 at 15°C for 45 min. The binding reaction was stopped by the addition of 2 ml of ice-cold binding buffer, and rapidly vacuum filtered through Whatman GF/C glass filters (Whatman, Maidstone, Kent, U.K.). Filters were then washed twice to separate unbound PGE2. Total and nonspecific [3H]PGE2 bounds were measured by a scintillation counter. Specific binding was defined as the difference between the binding in the presence or absence of unlabeled PGE2.
Immunizations, in vivo treatments, and in vitro culture
Mice were immunized with 50 µg of keyhole limpet hemocyanin (KLH; Sigma) in CFA twice by i.m. injection at a 7-day interval. PBS, IL-12 (250 ng), NS-398 (5 or 50 nmol), or PGE2 (1 nmol) was administered by i.p. injection into mice 3 h before and 1 and 2 days after the first immunization. Fourteen days after the first immunization, spleens were removed and spleen cells (5 x 106/ml) or-CD4+ T cells (1 x 106/ml) mixed with spleen cells (5 x 105/ml) from nonimmunized mice as APCs were cultured with 50 µg/ml of KLH for 48 h. Then, supernatants were collected and used for ELISA.
Statistics
All experiments were repeated at least three times, and some representative results are shown in tables and figures. Statistical analyses were performed between BALB/c mice and C3H/HeN or C57BL/6 mice using the Student’s t test. A confidence level of <0.05 was considered significant (33).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At first, we investigated the effect of indomethacin
(cyclooxygenase (COX)-1 and COX-2 inhibitor) and NS-398 (COX-2-specific
inhibitor) on IFN- production by SAC-stimulated spleen cells from
C3H/HeN, C57BL/6, and BALB/c mice in vitro. Indomethacin pretreatment
increased IFN- production by spleen cells from all mouse strains
when compared with nontreatment, but the degree of increase was
different in the three strains of mouse. As shown in Fig. 1
A, spleen cells from BALB/c
mice showed 95% increase of IFN- production (2727 ± 47 to
5306 ± 106 pg/ml), while spleen cells from C3H/HeN and C57BL/6
mice showed only 30% increase (5445 ± 59 to 6916 ± 88
pg/ml, and 4971 ± 194 to 6654 ± 461, respectively). These
effects were not restricted to indomethacin alone. NS-398 pretreatment
also increased IFN- production, and similar differences between
BALB/c and C3H/HeN mice were observed (Fig. 1A). The amounts
of IFN- produced are variable experiments to experiments, and BALB/c
mice sometimes produce higher amount of IFN- than other strains do.
However, the percent increase of IFN- production by indomethacin or
NS-398 is always higher in BALB/c mice than in other strains of
mouse.
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production
by spleen cells from three mouse strains may be due to differences in
the production of PGE2 in each strain. We then
examined PGE2 production by SAC-stimulated spleen
cells from each mouse strain in vitro. As shown in Table I, however, there was no significant
difference in PGE2 productions among three mouse
strains. Indomethacin-treated spleen cells did not produce detectable
amounts of PGE2 (data not shown).
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production by spleen cells among three mouse strains. Spleen
cells from these mouse strains were preincubated with 0–100 nM of
PGE2. Indomethacin was also added to inhibit the
effect of endogenous PGE2, and then stimulated
with SAC. The percentages of suppression of IFN- production were
calculated and compared with cells pretreated with indomethacin and 0
nM PGE2 and stimulated with SAC. As shown in Fig. 2A, spleen cells from BALB/c
mice were more markedly suppressed by PGE2 than
those of other strains. We also performed the same experiments using
NS-398 instead of indomethacin, and similar results were obtained (data
not shown). These data indicate that spleen cells from BALB/c mice are
highly sensitive to PGE2 as compared with those
from C3H/HeN and C57BL/6 mice, although PGE2 is
similarly produced in three mouse strains. Also, SAC-induced IFN-
production in BALB/c mice seems to be more markedly suppressed by
endogenous PGE2 than in other strains of
mouse.
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in all strains of
mouse, but no significant difference was observed in the degree of
indomethacin-induced increase (Fig. 1
B) and
PGE2-induced suppression of IFN- production
(Fig. 2B) between C3H/HeN and BALB/c spleen cells.
PGE2-mediated suppression of IFN- production in
BALB/c mice is not due to other suppressive cytokines or NO
Some other molecules such as IL-4, IL-10, and TGF-ß and NO are
known to exhibit an inhibitory effect on IFN- production the same as
PGE2 (34, 35, 36). Moreover, since it
has been reported that PGE2 enhances IL-10
production in activated macrophages (28), we examined
whether the higher sensitivity of BALB/c spleen cells to
PGE2 is mediated by other inhibitory cytokines
and NO. Spleen cells from C3H/HeN and BALB/c mice were pretreated with
indomethacin and 10 nM of PGE2 in the presence or
absence of anti-IL-4, anti-IL-10, anti-TGF-ß Ab, or
L-NMMA, an inhibitor of NO synthesis, and then
stimulated with SAC. The percentages of suppression of IFN-
production were calculated and compared with cells pretreated with
indomethacin and 0 nM of PGE2 in the presence or
absence of Ab or L-NMMA and stimulated with SAC. As shown
in Fig. 3, neutralization of inhibitory
cytokines or inhibition of NO production has no effect on reversing the
higher suppression of IFN- production by PGE2
in spleen cells from BALB/c mice. We also examined the amount of IL-4,
IL-10, and NO production by PGE2-treated and
nontreated spleen cells. IL-4 and NO were not detected in culture
supernatants of treated or nontreated spleen cells after stimulation
with SAC (IL-4:ELISA, NO:Griess method, data not shown). SAC-induced
IL-10 production was 20% higher in nontreated cells than in
PGE2-treated cells (data not shown). These
results indicate that the higher sensitivity to the suppressive effect
of PGE2 in BALB/c mice is not due to other
suppressive molecules, and that PGE2 directly
acts on spleen cells from BALB/c mice.
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PGE2 affects various cells such as T cells,
B cells, and APCs. We investigated cell types responsible for the
higher sensitivity to the suppressive effect of
PGE2 in BALB/c spleen cells. B cell depletion
from spleen cells had no effect on IFN- production (2082 ± 119
pg/ml spleen cells to 1875 ± 16.3 pg/ml B cell-depleted spleen
cells in BALB/c mice). Furthermore, B cell-depleted spleen cells show
similar indomethacin-induced enhancement (115.5% increase in spleen
cells to 106.6% increase in B cell-depleted spleen cells in C3H/HeN
mice, and 198.5% increase in spleen cells to 183.6% increase in B
cell-depleted spleen cells in BALB/c mice) and
PGE2-induced suppression of IFN- production
(35% suppression in spleen cells to 33.3% suppression in B
cell-depleted spleen cells in C3H/HeN mice, and 69.8% suppression in
spleen cells to 62.4% suppression in B cell-depleted spleen cells in
BALB/c mice at 10 nM PGE2) the same as in Figs. 1A and 2A. These results suggest that B cells
play no important role in the IFN- production by spleen cells with
SAC stimulation.
It has been reported that PGE2 suppresses IFN-
production of T cells via the down-regulation of IL-12R on T cells and
also suppresses IL-12 production of APCs (27, 28). We
therefore examined whether the higher sensitivity to the suppressive
effect of PGE2 in BALB/c spleen cells was due to
T cells or APCs. As shown in Fig. 4, when
both T cells and APCs were pretreated with 100 nM of
PGE2, IFN- production was more clearly
suppressed in spleen cells from BALB/c mice than those from C3H/HeN
mice. When T cells were pretreated with PGE2 and
mixed with nontreated APCs, there was no difference in the suppression
of IFN- production between two mouse strains. However, when APCs
were pretreated with PGE2, IFN- production by
T cells was suppressed in BALB/c mice, but not in C3H/HeN
mice.
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production were calculated and
compared with spleen cells pretreated with indomethacin, IL-12, and 0
nM of PGE2, and stimulated with SAC. As shown in
Fig. 5, the exogenous addition of IL-12
reversed the higher suppression of IFN- production by
PGE2 in spleen cells from BALB/c mice, and the
percentages of suppression of IFN- production in the presence of
IL-12 were the same in C3H/HeN and BALB/c mice. We also assessed the
capacity of IFN- production by purified T cells stimulated with
plate-coated anti-CD3 Ab and IL-12 in the presence of
PGE2, but no differences were observed in the
suppression of IFN- production by T cells from C3H/HeN and BALB/c
mice (19.6% suppression in C3H/HeN mice and 21.8% suppression in
BALB/c mice). These data indicate that the capacity of IFN-
production by T cells is down-regulated by PGE2,
but the higher suppressive effect of PGE2 on
IFN- production in BALB/c mice is not directed to T cells.
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by the
splenic T and NK cells, because the neutralization of IL-12 by Ab
reduces IFN- production by more than 90% (4064 ± 229 pg/ml by
nontreatment to 213 ± 68 pg/ml by anti-IL-12 Ab treatment).
We investigated IL-12 production by spleen cells from C3H/HeN and
BALB/c mice. Pretreatment of spleen cells with indomethacin increased
IL-12p70 production the same as IFN- production. As shown in Fig. 6A, there is significant
difference between the degree of increase: 8% increase in C3H/HeN mice
(170 ± 5 to 182 ± 3 pg/ml) and 51% increase in BALB/c mice
(89 ± 2 to 135 ± 5 pg/ml). IL-12p40 production was also
increased by the pretreatment with indomethacin in both mouse strains,
but the degree of increase was not significantly different. Spleen
cells from C3H/HeN and BALB/c mice showed about 21% and 19% increase,
respectively (2602 ± 2 to 3158 ± 6 pg/ml in C3H/HeN mice,
and 1870 ± 6 to 2204 ± 6 pg/ml in BALB/c mice).
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(Fig. 6B), IL-12p70 (Fig. 6C), and IL-12p40 (Fig. 6D) production was
calculated and compared with cells pretreated with indomethacin and 0
nM PGE2 and stimulated with SAC. Bioactive
IL-12p70 production by BALB/c spleen cells was highly sensitive to the
suppressive effect of PGE2, which corresponds to
that of IFN-. There was no significant difference in the suppression
of IL-12p40 production between two mouse strains. These data indicate
that the higher sensitivity of IFN- production to the suppressive
effect of PGE2 in BALB/c mice is mediated by
APCs; especially bioactive IL-12 production by APCs plays a key role in
BALB/c mice. BALB/c spleen cells have more PGE2 binding sites than C3H/HeN and C57BL/6 spleen cells
Because spleen cells from BALB/c mice showed a higher sensitivity
to the suppressive effect of PGE2 on IFN- and
IL-12p70 production, we investigated the binding sites of
[3H]PGE2 on spleen cells
from three mouse strains. Fig. 7A showed dose-response curves
of the binding of
[3H]PGE2 on spleen cells
from C3H/HeN and BALB/c mice. From Scatchard plot analysis, the binding
sites of [3H]PGE2 on
spleen cells from C3H/HeN and BALB/c mice were estimated as 131 and 201
molecules per cell, respectively. The
Kd values were estimated as 1.40
x 10-9 M in C3H/HeN mice and 1.56 x
10-9 M in BALB/c mice, respectively (Fig. 7B). The
[3H]PGE2 binding to
spleen cells from C57BL/6 mice was similar to that from C3H/HeN mice,
and the binding sites were 144 molecules per spleen cells and the
Kd value was 1.37 x
10-9 M (Scatchard analysis not shown). We also
assessed the binding capacities of
[3H]PGE2 on purified T
cells and APCs (Table II). The specific
binding of PGE2 to APCs was about 3-fold higher
in BALB/c mice than in C3H/HeN mice. The binding of
PGE2 to T cells was also about 2-fold higher in
BALB/c mice. These data indicate that spleen cells from BALB/c mice,
especially APCs, have more binding sites than ones from other strains,
but the binding affinity is almost similar among three mouse
strains.
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Because our in vitro experiments indicated that
PGE2 played an important role for IFN- and
IL-12 production in BALB/c spleen cells, we investigated the effect of
NS-398 and PGE2 on IFN- production in vivo.
BALB/c mice were administered with PBS, PGE2, or
NS-398 by i.p. injection during the immunization of KLH. Spleen cells
from immunized mice were then stimulated with KLH in vitro, and
Ag-specific IFN- and IL-4 productions were examined. Spleen cells
from PGE2-administered mice produced IFN-
about 67% lower than those from control mice, while IL-4 production
was the same between PGE2-administered and
PBS-administered mice (data not shown). In contrast with the
administration of PGE2, spleen cells from
NS-398-administered BALB/c mice produced IFN- about 2-fold higher
than control mice did (Fig. 8A). However, as in the case
of PGE2-administered mice, IL-4 production was
the same as compared with control. Administration of IL-12 is a major
and the most effective Th1-activating method. We then compared
NS-398-administered mice with IL-12-administered mice from the
standpoint of the efficiency of Ag-specific IFN- production. As
shown in Fig. 8A, spleen cells from NS-398-administered
BALB/c mice produced higher amounts of Ag-specific IFN- similar to
IL-12 administration. However, NS-398 administration had no effect on
Ag-specific IL-4 production. The enhanced production of Ag-specific
IFN- in NS-398-administered mice was due to activated Th1, because
CD4+ T cell fraction produced increased amount of
IFN- (Fig. 8B). We also tried the same experiment using
indomethacin, but an enhanced development of Th1 was not observed in
indomethacin-administered mice (data not shown). From these data, we
suggest that the modulation of PGE2 plays a
pivotal role in Th1/Th2 balance in vivo, and the administration of
NS-398, like IL-12 administration in BALB/c mice, is useful as an
enhancer of IFN- production.
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production of BALB/c
spleen cells, but keeps balance between production and sensitivity
Because IL-10 is a representative cytokine for suppressing IFN-
and IL-12 production (27, 28, 30), we examined whether
IL-10 had similar effect on IFN- production between C3H/HeN and
BALB/c spleen cells. Anti-IL-10 Ab pretreatment increased IFN-
production, but the degree of increase was similar between C3H/HeN and
BALB/c spleen cells, which is different from the result of indomethacin
treatment (Fig. 9). However, IL-10
production by SAC-stimulated spleen cells from BALB/c mice was about
one-tenth of that from C3H/HeN mice (Table III). From these results, we suggest that
BALB/c spleen cells have higher sensitivity to IL-10, just like
PGE2, but "the balance between production of
and sensitivity to IL-10" is maintained in both mouse strains
concerning the suppression of IFN- production, which are different
from that of PGE2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in BALB/c mice than other mouse strains. This hypothesis is
based on evidence that the immune response in BALB/c mice can be
shifted to Th1 by the neutralization of IL-4 or the administration of
IL-12 at the initial phase of infections or immunizations the same as
C3H/HeN and C57BL/6 mice. There is a report that T cells bearing V8
Vß4 TCR rapidly produce IL-4 by the stimulation with
Leishmania homologue of receptors for activated C kinase of
L. major in BALB/c mice, and this early burst of IL-4 plays
an essential role for instructing the subsequent Th2 differentiation
and consequently becomes susceptible to L. major infection
(11). However, the preferential activation of Th2 and the
failure of Th1 activation in BALB/c mice are induced not only in
L. major infection but also in infections of several
pathogens, and immunizations with CFA as an adjuvant also fail to
induce Th1 responses in BALB/c mice (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). Moreover,
there is a report that in the infection of Nippostrongylus
braziliensis, which generally induces strong Th2 responses, BALB/c
mice show a more excessive Th2 response than C57BL/6 mice do
(37). From these findings, we considered that the
preferential activation of Th2 in BALB/c mice was not only due to the
activation of V8 Vß4 T cells.
Another mechanism of Th2 polarization in BALB/c mice may be the reduced
response of naive Th to IL-12 (23). When naive Th is
exposed to IL-4, they lose IL-12 Rß2 chain and subsequently
differentiate to Th2 (38, 39, 40, 41). However, the
down-regulation of IL-12 Rß2 expression is recovered by the addition
of IL-12 itself or IFN- (12). Thus, IL-12 and IFN-
are important to the maintenance of IL-12 responsiveness and the
suppression of Th2 differentiation. Accordingly, we consider that the
lower production of IL-12 and IFN- is important in Th2 activation in
BALB/c mice in the same way as early and overproduction of IL-4. In
this study, we investigated the immunological functions of BALB/c
mice from the standpoint of the modulation of IL-12 and IFN-
production.
Many effecter molecules have been reported to suppress the production
of IL-12 and IFN-, such as IL-4, IL-10, TGF-ß, NO, PGs, and
corticosteroids (24, 25, 26, 27, 34, 35). Especially,
PGE2 is one of the most important factors for Th2
responses (25, 26, 28, 29). However, to our knowledge,
there has been no report concerning PGE2 and Th2
responses in BALB/c mice, because no significant difference was
observed in the amount of PGE2 produced in BALB/c
and other mouse strains. We also found no significant difference in the
amount of PGE2 produced from spleen cells
stimulated with SAC among these three mouse strains (Table I). However,
we did find that the inhibition of PGE2 synthesis
by indomethacin resulted in a marked production of IL-12p70 and IFN-
in BALB/c mice, but not in C57BL/6 and C3H/HeN mice, suggesting that
BALB/c mice are highly sensitive to the suppressive effect of
PGE2 (Figs. 1A and 2A). The
enhancement of IFN- production in BALB/c mice was also found by the
pretreatment with COX-2-specific inhibitor, NS-398 (Fig. 1A). This result indicates that the COX-2-mediated
PGE2 suppresses IFN- more effectively in
BALB/c mice than the constitutive PGE2 production
mediated by COX-1 does.
The target cells of the suppressive effect of
PGE2 seem to be APCs in BALB/c mice (Fig. 4).
PGE2 also affected IFN- production by T and NK
cells (25, 27). However, in our experimental system, the
pretreatment of T and NK cells with PGE2 did not
show any significant difference in the suppression of IFN-
production between C3H/HeN and BALB/c mice, and an exogenous addition
of IL-12 reversed the suppression of IFN- production by
PGE2 in only BALB/c mice (Fig. 5). Moreover, when
purified T cells were stimulated with plate-coated anti-CD3 Ab and
IL-12 in the presence of PGE2, the degree of
suppression of IFN- production was the same between C3H/HeN and
BALB/c mice. These data suggest that the susceptibility of
IFN--producing T cells to PGE2 is the same
between BALB/c and C3H/HeN mice. On the other hand, IL-12p70 production
by APCs, which corresponds to IFN- production, is more markedly
suppressed by PGE2 in BALB/c mice than in C3H/HeN
mice (Fig. 6). We also found that spleen cells, T cells, and APCs from
BALB/c mice had more PGE2 binding sites than
those of C3H/HeN mice (Fig. 7 and Table II). These data indicate that
the higher sensitivity of IFN- production by T and NK cells to
PGE2 in BALB/c mice is caused by higher
sensitivity of IL-12 production by APCs to
PGE2.
When spleen cells were stimulated with Con A or anti-CD3 Ab instead
of SAC, there was no significant difference in
PGE2-induced suppression of IFN- production
and indomethacin-induced enhancement of IFN- production between
spleen cells from BALB/c and C3H/HeN mice (Figs. 1B and
2B). T cell mitogen also stimulates APCs to produce IL-12
via interaction with T and APCs (42). However, SAC and
other bacterial Ags are known to stimulate directly APCs, and spleen
cells stimulated with Con A produced only 80% lower
PGE2 than SAC stimulation (data not shown). Thus,
we consider that this direct APC stimulation is more sensitive to
PGE2 than indirect APC stimulation in our
experimental system. In BALB/c mice, the failure of Th1 activation is
generally observed in intracellular parasites or bacteria infections
and immunizations with CFA, which contains dead mycobacterium. APCs
seem to be directly stimulated in vivo by these infections and
immunizations. We consider that IL-12 is not fully produced by the
direct stimulation of APCs, and Th2 are preferentially differentiated
in BALB/c mice. A similar result has been reported regarding
bacillus Calmette-Guérin infection, that is, the failure of Th1
activation in BALB/c mice is due to the failure of IL-12 production
by macrophages, while the capacity of IFN- production is the same in
BALB/c and B10D6 mice (43).
Several reports suggest that macrophages and dendritic cells play an
important role for the determination of Th1/Th2 differentiation.
Corticosteroids-treated macrophages preferentially stimulate Th2 by
inhibiting IL-12 production, and human dendritic cells that are
differentiated by GM-CSF and IL-4 in the presence of
PGE2 produce no IL-12 and preferentially
stimulate Th to produce type II cytokines (44, 45, 46). There
is also a report that the amount of IL-12 and
PGE2 derived from APCs determines the IFN-
level of human Th cells (30). In this way, the character
of APC seems to play an important role for the determination of Th1/Th2
responses. Thus, APCs from BALB/c mice have tendency of higher
sensitivity to PGE2 and preferentially activate
Th2 via insufficient production of IL-12.
The major inhibitory molecules, such as IL-4, IL-10, and TGF-ß and
NO, inhibit IFN- production the same as PGE2
(34, 35, 36). Moreover, it has been reported that
PGE2 enhances IL-10 production in activated
macrophages (28, 48). IL-10 itself is not a direct Th2
inducer, but is known as a strong suppressor of IFN- production,
while IL-4 and NO are suppressors for Th1 differentiation (35, 36, 47). We assessed the role of other inhibitory molecules in
this system, but anti-IL-4, anti-IL-10, and anti-TGF-ß
Abs and L-NMMA did not recover the suppressed IFN-
production by PGE2 in BALB/c and C3H/HeN mice
(Fig. 3). These results suggest that the suppressive activity of
PGE2 is not mediated by other inhibitory
molecules.
We obtained interesting findings regarding IL-10. BALB/c mice showed
high sensitivity to IL-10 in the same way as
PGE2. However, different from
PGE2, the amount of IL-10 was 90% lower in
BALB/c mice than in C3H/HeN mice, and the enhancement of IFN-
production by the neutralization of IL-10 was similar between two mouse
strains. From these results, we suggest that "the balance between
production of and sensitivity to IL-10" is maintained in both mouse
strains, but not of PGE2 (Fig. 9 and Table III).
We did not investigate whether these balances were maintained or
impaired in other suppressive molecules (IL-4, TGF-ß, and NO) and
type I cytokines (IL-12 and IFN-), and there is a possibility that
"the balance between production of and sensitivity to" is impaired
in some cytokines in the same way as PGE2 in some
immunological diseases. One suggestion that we want to emphasize in
this study is that "the balance between production of and sensitivity
to," rather than the amount of production, is one of the most
important factors in determining the level of immune responses.
We obtained marvelous results that the administrations of NS-398 in
vivo induced the activation of Th1 the same as the administration of
IL-12. These results further indicate that PGE2
works as a Th1 suppressor in BALB/c mice (Fig. 8). However, the
administration of indomethacin instead of NS-398 did not enhance
Ag-specific IFN- production (data not shown). We suspect that
indomethacin inhibits not only COX-2, but also COX-1, and a
constitutive production of PGE2 by COX-1 may need
to maintain the activation of several cell types, because
PGE2 produced by COX-1 serves to maintain the
homeostasis and is produced by a wide variety of cell types to regulate
a broad range of physiological activities. However, in in vitro system,
we handled only immune systems that are easily affected by
indomethacin, and different from in vivo system. We also investigated
the effect of PGE2 in vivo. When
PGE2 is administered at the initial phase of
immunization, Ag-specific IFN- production was about 67% lower than
that of the control (data not shown). These results indicate that
Th1 development can be regulated by the modulation of
PGE2 production.
Other arachidonic acid derivatives, such as leukotriene (LT), also
may play some roles in the regulation of IFN- production.
Cyclooxygenase and lipoxygenase catalyze arachidonic acid to synthesize
PGs or LTs, respectively. There is a possibility that COX inhibitors
increase LT synthesis because COX inhibitors shut off the stream of
arachidonic acid to PGs and shift to the lipoxygenase stream. It is
reported that LTB4 up-regulates IL-1, IL-2, and
IFN- production and enhances NK cell activity (49).
These effects of LTB4 are just opposite of that
of PGE2. We did not examine the
LTB4 production by spleen cells treated with COX
inhibitor or PGE2. However, it is interesting to
investigate the relationship between Th1/Th2 development and
LTB4, and cross-regulation of
PGE2 and LTB4 by
APCs.
In conclusion, we did find that IFN- and IL-12 productions of BALB/c
mice were highly sensitive to the suppressive effect of
PGE2 as compared with C3H/He and C57BL/6 mice,
while the production of PGE2 was the same among
three strains of mouse. The main target of
PGE2-induced suppression seems to be on APCs in
BALB/c mice. BALB/c spleen cells have more PGE2
binding sites than those of other mouse strains. These results suggest
that PGE2 plays an important role to polarize Th2
type response in BALB/c mice. As discussed above, we want to emphasize
that "the balance between production of and sensitivity to" is an
important factor for the determination of the level of immune
responses. The disturbance of "the balance between production of and
sensitivity to" in some cytokines should be investigated in several
immunological diseases.
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Footnotes |
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2 Abbreviations used in this paper: SAC, Staphylococcus aureus Cowan I; COX, cyclooxygenase; KLH, keyhole limpet hemocyanin; L-NMMA, L-NG monomethyl arginine; LT, leukotriene.
Received for publication August 9, 1999. Accepted for publication December 20, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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8 CD4+ T cells in BALB/c mice infected with Leishmania major instructs Th2 cell development and susceptibility to infection. Immunity 6:541.[Medline]
interferon. Infect. Immun. 62:2590. production in NK cells and CD4+ T cells. J. Immunol. 156:1458.[Abstract]
-inducing factor) regulates early cytokine production in, and promotes resolution of, bacterial infection in mice. J. Immunol. 160:299. and IL-2, but not IL-4 and IL-5. J. Immunol. 155:4604.[Abstract]
level of activated human CD4+ T cells. J. Immunol. 156:1722.[Abstract]
and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression. Eur. J. Immunol. 27:647.[Medline]
ß T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065.This article has been cited by other articles:
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|
|
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 |
|
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|
|
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 |
|
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 G. Donofrio, S. Herath, C. Sartori, S. Cavirani, C. F. Flammini, and I. M. Sheldon Bovine herpesvirus 4 is tropic for bovine endometrial cells and modulates endocrine function Reproduction, July 1, 2007; 134(1): 183 - 197. [Abstract] [Full Text] [PDF]
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|
C. H. Serezani, J. H. Perrela, M. Russo, M. Peters-Golden, and S. Jancar Leukotrienes Are Essential for the Control of Leishmania amazonensis Infection and Contribute to Strain Variation in Susceptibility. J. Immunol., September 1, 2006; 177(5): 3201 - 3208. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
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|
|
|
|
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|
J. Noguchi, E. Kuroda, and U. Yamashita Strain difference of murine bone marrow-derived mast cell functions J. Leukoc. Biol., September 1, 2005; 78(3): 605 - 611. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
 N. Ray, M. E. Bisher, and L. W. Enquist Cyclooxygenase-1 and -2 Are Required for Production of Infectious Pseudorabies Virus J. Virol., December 1, 2004; 78(23): 12964 - 12974. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Newton, T. Lu, S. J. Nazian, I. Perkins, H. Friedman, and T. W. Klein The THC-induced suppression of Th1 polarization in response to Legionella pneumophila infection is not mediated by increases in corticosterone and PGE2 J. Leukoc. Biol., October 1, 2004; 76(4): 854 - 861. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Harizi, C. Grosset, and N. Gualde Prostaglandin E2 modulates dendritic cell function via EP2 and EP4 receptor subtypes J. Leukoc. Biol., June 1, 2003; 73(6): 756 - 763. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Kuroda and U. Yamashita Mechanisms of Enhanced Macrophage-Mediated Prostaglandin E2 Production and Its Suppressive Role in Th1 Activation in Th2-Dominant BALB/c Mice J. Immunol., January 15, 2003; 170(2): 757 - 764. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Liu, T. Matsuguchi, N. Tsuboi, T. Yajima, and Y. Yoshikai Differences in Expression of Toll-Like Receptors and Their Reactivities in Dendritic Cells in BALB/c and C57BL/6 Mice Infect. Immun., December 1, 2002; 70(12): 6638 - 6645. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. Martin, M. Suzuki, K. Maghni, R. Pantano, D. Ramos-Barbon, D. Ihaku, F. Nantel, D. Denis, Q. Hamid, and W. S. Powell The Immunomodulatory Actions of Prostaglandin E2 on Allergic Airway Responses in the Rat J. Immunol., October 1, 2002; 169(7): 3963 - 3969. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Kuroda, T. Kito, and U. Yamashita Reduced Expression of STAT4 and IFN-{gamma} in Macrophages from BALB/c Mice J. Immunol., June 1, 2002; 168(11): 5477 - 5482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Harizi, M. Juzan, V. Pitard, J.-F. Moreau, and N. Gualde Cyclooxygenase-2-Issued Prostaglandin E2 Enhances the Production of Endogenous IL-10, Which Down-Regulates Dendritic Cell Functions J. Immunol., March 1, 2002; 168(5): 2255 - 2263. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Schwacha, C.-S. Chung, A. Ayala, K. I. Bland, and I. H. Chaudry Cyclooxygenase 2-mediated suppression of macrophage interleukin-12 production after thermal injury Am J Physiol Cell Physiol, February 1, 2002; 282(2): C263 - C270. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Lipscomb and B. J. Masten Dendritic Cells: Immune Regulators in Health and Disease Physiol Rev, January 1, 2002; 82(1): 97 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. O. Yarovinsky and G. W. Hunninghake Lung fibroblasts inhibit activation-induced death of T cells through PGE2-dependent mechanisms Am J Physiol Lung Cell Mol Physiol, November 1, 2001; 281(5): L1248 - L1256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-H. Su, H.-i. Chen, and C. J. Jen C57BL/6 and BALB/c Bronchoalveolar Macrophages Respond Differently to Exercise J. Immunol., November 1, 2001; 167(9): 5084 - 5091. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. THURNHER, C. ZELLE-RIESER, R. RAMONER, G. BARTSCH, and L. HOLTL The disabled dendritic cell FASEB J, April 1, 2001; 15(6): 1054 - 1061. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Ikegami, Y. Sugimoto, E. Segi, M. Katsuyama, H. Karahashi, F. Amano, T. Maruyama, H. Yamane, S. Tsuchiya, and A. Ichikawa The Expression of Prostaglandin E Receptors EP2 and EP4 and Their Different Regulation by Lipopolysaccharide in C3H/HeN Peritoneal Macrophages J. Immunol., April 1, 2001; 166(7): 4689 - 4696. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I-C. Ho, J. P. Arm, C. O. Bingham III, A. Choi, K. F. Austen, and L. H. Glimcher A Novel Group of Phospholipase A2s Preferentially Expressed in Type 2 Helper T Cells J. Biol. Chem., May 18, 2001; 276(21): 18321 - 18326. [Abstract] [Full Text] [PDF]
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