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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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, or the neutralization of IFN-
or IL-12 had
no effect on MDC production. B cells, macrophages, and dendritic cells
were main producers of MDC, while T cells produced only a small amount
of MDC. MDC production by B cells was equally stimulated by LPS and
anti-CD40 Ab, while that by macrophages and dendritic cells was
more markedly stimulated by anti-CD40 Ab, and PGE2
further enhanced MDC production by these stimulated cells. These
results indicate that PGE2 regulates Th1/Th2-related
chemokine production by B cells, macrophages, and dendritic cells, and
that this is a new function of PGE2 for the regulation of
Th2 immune responses at the induction and activation
stages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production and is
related with the elimination of intracellular pathogens, while the
activation of Th2 enhances humoral immune responses via IL-4 and IL-5
and is related with allergic diseases. Th1 and Th2 are differentiated
from naive T cells after the Ag recognition, which are influenced by
surface molecules of APCs, Ag doses, and cytokines (1, 2, 3, 4, 5, 6).
The selective recruitment of Th subset to an inflammatory site is also
important to determine the character of immune responses. Although the
mechanism of this selective recruitment still remained unclear, recent
studies suggest that the patterns of chemokines and their receptors
determine the selective recruitment of Th1 and Th2
(7, 8, 9, 10).
Th1 and Th2 express different chemokine receptors and respond to
distinct types of chemokines. CXC chemokine receptor 3
(CXCR3)3 and CCR5 are
highly expressed on Th1, and IFN-inducible protein-10 (IP-10, new
nomenclature: CXCL10 (7)), which binds to CXCR3, induces
chemotaxis of Th1, while CCR3 and CCR4 are preferentially expressed on
Th2, and ligands for CCR4, macrophage-derived chemokine (MDC, CCL21
(7)), and thymus and activation-regulated chemokine (TARC,
CCL17 (7)) promote the migration of Th2
(7, 8, 9, 10, 11, 12, 13). The relationship between chemokines and Th1/Th2
responses in vivo has been also investigated in several human diseases
and mouse models. In multiple sclerosis and rheumatoid arthritis, which
are Th1-related diseases, CCR5+ and
CXCR3+ T cells are accumulated, and their
ligands, macrophage-inflammatory protein (MIP)-1
(CCL3
(7)) and IP-10, are detected at inflammatory sites
(14, 15). On the other hand, a higher expression of TARC
is detected on Reed-Sternberg cells of Hodgkin’s lymphoma that have
the characteristic feature of infiltration of Th2 (16). In
murine models of allergic airway inflammation and atopic dermatitis,
NC/Nga mouse, and endotoxin-induced liver injury, the accumulation of
CCR4+ Th2 and the production of MDC and TARC play
an important role in the induction of tissue injuries
(17, 18, 19, 20).
IP-10 and MDC are mainly produced by APCs such as dendritic cells,
monocytes, macrophages, and B cells, and highly regulated by Th1- and
Th2-derived cytokines. In general, IFN- up-regulates IP-10, but
suppresses MDC production, while IL-4 and IL-13 have opposite effects
(9, 12, 13, 21, 22, 23). Thus, Th1 and Th2 cytokines regulate
Th1- and Th2-related chemokine (IP-10 and MDC) productions, and
reversely these chemokines also regulate Th1/Th2 responses. However,
there is no report about the role of PGE2 in
Th1/Th2-related chemokine productions.
PGE2, an arachidonic acid metabolite, produced in
various types of cells, regulates broad range of physiological
functions (24). In the immune system,
PGE2 is also mainly produced by APCs, and the
effects are almost suppressive on Th1-related immune responses and
augment Th2-related immune responses. PGE2
preferentially down-regulates IL-12R expression and inhibits the
differentiation of Th1 (25, 26).
PGE2 also suppresses LPS-induced IL-12 production
by macrophages, and IL-12 and PGE2 derived from
APCs down-regulate IFN- production by T cells (27, 28).
In B cell function, PGE2 enhances IgE production
of IL-4- and LPS-stimulated B cells in vitro (29). In
these reports, the effect of PGE2 is similar to
IL-4 in immune responses.
In this study, we examined the effect of PGE2 on IP-10 and MDC productions in vitro, and demonstrate that PGE2 up-regulates MDC production, but down-regulates IP-10 production by APCs induced with LPS and CD40 stimulation. These results suggest that PGE2 plays an important role not only for the differentiation of Th2, but also for the recruitment of Th2 via the preferential production of Th2-related chemokines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c male mice, 7–8 wk old, were purchased from Seac (Oita, Japan), and were maintained in our laboratory under specific pathogen-free condition.
Cytokines, chemokines, Abs, and other reagents
PGE2, indomethacin, dibutyryl cAMP
(dbcAMP), 3-isobutyl-1-methylxanthine (IBMX), and
o-phenylenediamine were purchased from Sigma (St. Louis,
MO). Recombinant mouse IL-12, IFN-, and MDC were purchased from R&D
Systems (Minneapolis, MN). Recombinant mouse IL-4, IL-5, IL-6, and
IL-10 were purchased from PeproTech (London, U.K.). Human TGF-,
anti-mouse MDC, and anti-mouse CRG-2 (IP-10) Abs were purchased
from Genzyme/Techne (Cambridge, MA). Anti-mouse IFN- and anti-mouse
IL-12 Abs were purchased from PharMingen (San Diego, CA). Recombinant
mouse IP-10/CRG-2 was purchased from Dako Japan (Kyoto, Japan).
Peroxidase-conjugated rabbit anti-goat IgG Ab was purchased from
MBL (Nagoya, Japan).
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). B cells (B220+ cells), macrophages (CD11b+ cells), dendritic cells (CD11c+ cells), and Th cells (CD4+ cells) were purified from spleen cells by magnetic cell sorting according to the manufacturer’s procedures (MACS; Miltenyi Biotech, Bergish-Gladbach, Germany). Purity of sorted cells was >95%, as determined by flow cytometry. In some experiments, B220-, CD11b-, and CD11c-negative cells were used as T cells (<5% Ig+, <5% Mac-1+, and >95% CD3+). The number of whole spleen cells and sorted cells was adjusted to 5 x 106/ml (whole spleen cells, B cells and T cells) or 2 x 106/ml (macrophages, dendritic cells, and macrophages/dendritic cells fraction), and used for experiments.
In vitro culture and stimulation of cells
Whole spleen cells or sorted cells were cultured in 24-well
culture plates (Falcon 3047; Becton Dickinson, Franklin Lakes, NJ) with
or without PGE2 (1–100 nM), IL-4 (10 ng/ml),
IFN- (50 ng/ml), IL-12 (50 ng/ml), IL-5 (10 ng/ml), IL-6 (10 ng/ml),
IL-10 (10 ng/ml), TGF- (4 ng/ml), dbcAMP (50 µM), IBMX (50 µM),
anti-IFN- Ab (1 µg/ml), or anti-IL-12 Ab (1 µg/ml) in
the presence of indomethacin (1 µM) for 12 h, and then they were
stimulated with LPS (10 µg/ml), anti-CD40 Ab (10 µg/ml), or
anti-CD3 Ab (1 µg/ml) for an additional 24, 48, or 72 h.
Purified CD4+ T cells were stimulated with
plate-coated anti-CD3 Ab in the presence or absence of
PGE2 or IL-4 for 48 h. In some experiments,
cells were cultured without indomethacin for 12 h, and then
stimulated. The culture supernatants were collected and used for the
assay of chemokine or PGE2, as described
below.
Chemokine mRNA analysis
mRNA expression was detected by RT-PCR. Total cellular RNA was
extracted by TRIzol (Life Technologies, Rockville, MD), according to
the manufacturer’s protocol. First strand cDNA was synthesized from 5
µg of total RNA by Superscript II RNase H-reverse transcriptase (Life
Technologies), following the instruction of manufacturer using 0.25
µg of random primer (Life Technologies). One-tenth of synthesized
cDNA was amplified by PCR using 50 pmol sense and antisense primers
with 1 U of Taq polymerase (Boehringer Mannheim, Mannheim,
Germany) in a total volume of 50 µl. PCR cycles were performed for 1
min at 94°C for denaturation, 1 min at 55°C for annealing, and 2
min at 72°C for extension, and at the first cycle, denaturation was
run for 2 min at 94°C. The sequence of the sense and antisense
primers, products size, and PCR cycles are as follows: -actin,
5'-ACCAACTGGGACGACATGGAGAA-3' and 5'-GTGGTGGTGAAGCTGTAGCC-3', 380
bp, 30 cycles; MDC, 5'-CTGGGTGAAGAAGCTACTCCAT-3' and
5'-TAGAAACTTGGGAGGTGTGTGG-3', 493 bp, 35 cycles; IP-10,
5'-GGGCCAGTGAGAATGAGGGC-3' and 5'-TGAGCTAGGGAGGACAAGGAG-3', 544 bp, 35
cycles. A 10 µl of PCR products was electrophoresed using a 1.5%
agarose gel. After ethidium bromide staining (Sigma), PCR products were
visualized by UV illumination.
Assay of chemokine
The concentrations of MDC in the supernatants were measured by
competitive ELISA inhibition assay (30). In brief, ELISA
plates (Nunc-Immuno Plate MaxiSorp; Nunc, Roskilde, Denmark) were
coated with 100 µl of 250 ng/ml MDC in PBS at 4°C for 18 h.
The wells were then washed and blocked with 300 µl of RPMI 1640
containing 10% FBS at 37°C for 30 min. Standard solutions
(40,000
625 pg/ml in culture medium) or culture supernatants and
equal volume of 400 ng/ml anti-MDC Ab were mixed well in microtubes
and incubated at 37°C for 1 h. After incubation, 200 µl of
prepared standard or sample solutions was transferred to each well of
MDC-coated plates, and the plates were kept at 4°C for 18 h. The
wells were washed and filled with 100 µl of peroxidase-conjugated
rabbit anti-goat IgG Ab (diluted 1/2000 in PBS containing 0.05%
Tween 20 and 1% BSA). The plates were incubated at 37°C for 2
h, washed, and then substrate (0.04% o-phenylenediamine,
0.01% H2O2 in 50 mM
disodium hydrogen phosphate, and 150 mM citric acid, pH 5) was added to
the wells. Thirty minutes later, the enzyme reaction was stopped by the
addition of 25 µl of 8 N
H2SO4, and the absorbance
at 490 nm was measured. The results are expressed as mean ± SD of
chemokine produced (pg/ml) in triplicate cultures. Competitive ELISA
inhibition assay for IP-10 and TARC was also performed by a similar
method except for Ab concentration. The concentration of anti-IP-10
Ab and anti-TARC Ab was used at 100 ng/ml. Detection limit of these
assays is 1000 pg/ml.
PGE2 enzyme immunoassay
PGE2 in the culture supernatant was measured using STAT-PGE2 Enzyme Immunoassay Kit (Cayman Chemicals, Ann Arbor, MI), according to the manufacturer’s protocol.
Statistics
All experiments were repeated at least three times, and one representative result of each experiment is shown in figures. Statistical analyses were performed using the Student’s t test. A confidence level of <0.05 was considered significant (31).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At first, we investigated the effect of PGE2
on Th1- and Th2-related chemokine (IP-10 and MDC, respectively) mRNA
expression in murine spleen cells. Spleen cells were pretreated with
PGE2 for 12 h, then stimulated with LPS,
anti-CD3 Ab, or anti-CD40 Ab for an additional 24 h, and
mRNA expression was detected by RT-PCR. As shown in Fig. 1
, both IP-10 and MDC mRNA expressions
were up-regulated following stimulation. The pretreatment with
PGE2 suppressed the expression of IP-10 mRNA. On
the contrary, the expression of MDC mRNA was enhanced by the
pretreatment with PGE2.
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for 12 h, and then stimulated with LPS,
anti-CD3 Ab, or anti-CD40 Ab for an additional 48 h. IP-10
and MDC in the supernatants were assessed by competitive ELISA
inhibition assay. As shown in Fig. 2, in
spleen cells without stimulation (PBS), MDC production was enhanced by
PGE2 (1.5-fold higher), but IP-10 production
was not influenced. On the other hand, IFN- suppressed MDC
production, but enhanced IP-10 production. These findings were more
clearly observed in stimulated spleen cells. In spleen cells stimulated
with LPS (a model of bacterial stimulation), anti-CD40 Ab (a model
of T-APC interaction), or anti-CD3 Ab (a model of T-APC
interaction), PGE2 enhanced MDC production (about
3-fold higher), and this capacity was stronger than that of IL-4. On
the other hand, IP-10 production was suppressed by the pretreatment
with PGE2 or IL-4. IFN- had quite opposite
effects of IL-4 and PGE2, which was the same as
previously reported (23). We also assessed MDC and IP-10
productions in spleen cells stimulated with soluble anti-CD3 Ab
that is known to activate APC via CD40-CD40 ligand (CD40L) interaction
(32). The pretreatment with PGE2
enhanced MDC production, and suppressed IP-10 production in
anti-CD3 Ab-stimulated spleen cells the same as anti-CD40
stimulation. The amount of MDC was much higher in CD40-stimulated
spleen cells than in LPS-stimulated ones, and was moderate in
CD3-stimulated ones.
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, the amounts of MDC were increased in a
time-dependent manner, and pretreatment with PGE2
clearly enhanced MDC production at 48 and 72 h.
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A representative activity of PGE2 is the
elevation of intracellular cAMP (24). We examined whether
the enhanced production of MDC is mediated by intracellular
accumulation of cAMP in stimulated spleen cells. Spleen cells were
pretreated with 1–100 nM PGE2, dbcAMP
(membrane-permeable cAMP analogue), or IBMX (cAMP phosphodiesterase
inhibitor), and then stimulated with LPS or anti-CD40 Ab for
48 h. As shown in Fig. 4, A and B, in both stimuli, the pretreatment with
dbcAMP or IBMX also enhanced MDC production the same as
PGE2 treatment, but the degree of increase in
IBMX treatment was lower than PGE2 or dbcAMP.
This difference seems to be caused by a different mechanism of cAMP
accumulation between PGE2 and IBMX.
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and IL-12,
and an inducer of IL-10 (25, 26, 27, 28). Next, we examined
whether the enhanced production of MDC is due to a secondary effect of
the suppression of IFN- and IL-12 or the induction of IL-10. As
shown in Fig. 4
, C and D, neutralization of
IFN- or IL-12 was not sufficient to enhance MDC production in LPS-
or anti-CD40 Ab-stimulated spleen cells. Moreover, the pretreatment
with IL-10 suppressed MDC production. Another representative inhibitory
cytokine, TGF-, had no effect on MDC production. Interestingly, IL-5
also enhanced MDC production the same as IL-4, but IL-4 and IL-5 were
not detected in PGE2-treated spleen cells
stimulated with LPS or anti-CD40 Ab (data not shown). However,
IL-6, another Th2 cytokine, had no effect on MDC production. IFN-
suppressed MDC production the same as previously reported
(23), but IL-12 had no effect. Enhanced production of MDC by PGE2 is mainly mediated by B cells, macrophages, and dendritic cells
It has been reported that MDC is produced by various types of
cells, such as B cells, T cells, and macrophages (33, 34, 35, 36, 37, 38).
We investigated the producer of MDC by LPS- or anti-CD40
Ab-stimulated spleen cells in the presence of
PGE2. Spleen cells were separated into B cells
(B220+ cells), macrophages/dendritic cells
(CD11b+ or CD11c+ cells),
and T cells (B220-,
CD11b-, and CD11c- spleen
cells). Purified cell fractions were pretreated with
PGE2 or IL-4 for 12 h, and then stimulated
with LPS or anti-CD40 Ab for 48 h.
PGE2-pretreated B cells produced a higher amount
of MDC as compared with nontreated cells in both LPS and anti-CD40
Ab stimulation (Fig. 5, A and
B). The amount of MDC is much higher in CD40-stimulated
macrophages/dendritic cells than other types of cells, and
PGE2 pretreatment further enhanced MDC production
in macrophages/dendritic cells treated with anti-CD40 Ab (Fig. 5D). Contrary to our expectation, however,
PGE2 pretreatment slightly reduced MDC production
in LPS-stimulated macrophages/dendritic cells (Fig. 5C).
Macrophages and dendritic cells were further purified, and MDC
production was assessed. Dendritic cells produced higher amount of MDC
than macrophages did after LPS stimulation, but
PGE2 pretreatment had no effect on MDC production
in both cells (Fig. 5E). Macrophages and dendritic cells
produced the same amount of MDC after anti-CD40 Ab stimulation, and
PGE2 pretreatment similarly enhanced MDC
production in both cells (Fig. 5F). It has been reported
that CD4+ T cells, especially Th2, are
MDC-producing cells. Although we could not detect MDC production in
LPS-, anti-CD40 Ab-, or plate-coated anti-CD3 Ab-stimulated T
cells (Fig. 5G, and data not shown), purified
CD4+ T cells produced only a small amount of MDC
following the stimulation with immobilized anti-CD3 Ab in the
presence of PGE2 (Fig. 5H). IP-10
production was also assessed. IP-10 production was not detectable in B
cells, T cells, and CD4+ T cells stimulated with
anti-CD40 Ab, LPS, or anti-CD3 Ab (data not shown).
Macrophages/dendritic cells produced high amount of IP-10 after
stimulation with LPS or anti-CD40 Ab, and
PGE2 pretreatment suppressed, but IFN-
pretreatment enhanced IP-10 production in these cells the same as
spleen cells (Fig. 5, I and J). These results
indicate that PGE2 mainly affects APCs (B cells,
macrophages, and dendritic cells) to enhance MDC production and to
suppress IP-10 production.
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Next we investigated the effect of cytokines on
PGE2-enhanced MDC production in B cells,
macrophages, and dendritic cells. As shown in Fig. 6, PGE2, IL-4, and
IL-5 alone once again enhanced MDC production. The combination of
PGE2 and IL-4 or IL-5 had additive effect on MDC
production in B cells and macrophages/dendritic cells. The combination
of PGE2 and IL-5 was the strongest in MDC
production in both cell types, especially in macrophages/dendritic
cells. IFN-, IL-10, and TGF- suppressed MDC production, as shown
in Fig. 4. These cytokines also suppressed the enhanced production of
MDC by PGE2 in B cells and macrophages/dendritic
cells. IL-6 had no effect on the PGE2-induced
enhanced production of MDC. Leukotriene B4
(LTB4), another arachidonic acid derivative,
alone or in combination with PGE2, had only a
little effect on MDC production in macrophages/dendritic cells.
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In all experiments to date, cells were pretreated with
indomethacin to inhibit endogenous production of
PGE2 after stimulation.
PGE2 was mainly produced by APCs such as
macrophages and dendritic cells. Then we investigated the effect of
endogenous PGE2 from macrophages/dendritic cells
on MDC production. Splenic macrophages/dendritic cells were pretreated
with PBS, PGE2, indomethacin, or
PGE2 plus indomethacin for 12 h, and then
stimulated with anti-CD40 Ab for an additional 48 h. As shown
in Fig. 7, indomethacin treatment
suppressed MDC production by anti-CD40 Ab stimulation. However, the
addition of PGE2 reversed the suppressed
production of MDC by indomethacin. PGE2 only
slightly enhanced MDC production by anti-CD40 Ab stimulation in the
absence of indomethacin. We also assessed the amount of
PGE2 in the supernatants after stimulation.
Macrophages/dendritic cells produced PGE2 about
4000 pg/2 x 106 (about 1 nM), and
indomethacin treatment completely inhibited PGE2
production (Fig. 7). These results indicate that endogenously produced
PGE2 plays a role to enhance MDC production by
macrophages/dendritic cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MDC and TARC are known as specific Th2 chemoattractants
(11, 12, 13). Several reports have shown that the preferential
chemoattractant properties of these chemokines are found not only in
vitro but also in vivo using model mice with several Th2-related
diseases. In the mouse model of airway inflammation, macrophages in
lungs (resident and infiltrating macrophages) produced MDC, and the
neutralization of MDC by the administration of specific Ab in vivo
reduced Th2 recruitment to the inflammatory sites and inhibited airway
hyperreactivity (17, 18). In NC/Nga mice, model mice of
atopic dermatitis, MDC was highly produced by dermal dendritic cells in
the lesional skin (19). In the mouse model of endotoxin
liver injury, MDC and TARC were produced by cells in the granuloma,
which was mainly composed of macrophages/dendritic cells, and
neutralization of TARC protected the mice from acute lethal liver
damage caused by Th2 (20). These reports suggest that not
only Th2 and eosinophils, but also APCs recruited to the inflamed
sites, and Th2 attractant chemokines, MDC or TARC, were mainly produced
by APCs. Many reports have indicated that Th2 cytokines, IL-4 and
IL-13, are strong inducers of MDC and TARC from APCs (12, 13, 23). However, we found that cell to cell interaction between T
cells and APCs was more important for the chemokine production.
Furthermore, MDC productions are regulated not only by cytokines, but
also by PGE2. PGE2
up-regulated MDC production by CD40-stimulated macrophages/dendritic
cells, but the enhancing effect of PGE2 on
LPS-stimulated ones was weak (Fig. 5). On the other hand, IL-4
up-regulated MDC production by both LPS- and CD40-stimulated
macrophages/dendritic cells. These differential effects probably depend
on different signal transduction pathways. LPS is a representative APC
stimulator. However, CD40 stimulation is a more appropriate model for
an activation of APCs in vivo than LPS stimulation, because chemokine
productions in vivo are not limited to endotoxin stimulation, and
higher amounts of MDC were produced by CD40 stimulation than by LPS
stimulation. We consider that, in the induction of chemokines by APCs,
T-APC interaction is more important than the stimulation with LPS
or cytokines alone, and propose that T-APC interaction, especially
CD40-CD40L interaction, is a more effective signal for the induction of
chemokine production by APCs. Kornbluth et al. also proposed the
importance of CD40-CD40L interaction in chemokine production in an HIV
infection model (44).
There are several reports indicating that PGE2
suppresses chemokine mRNA expression and chemokine production in
various types of cells, and this suppressive effect depends on the
accumulation of intracellular cAMP. For example,
PGE2 or intracellular accumulation of cAMP
suppresses RANTES production by murine mesangial cells
(39), MIP-1 production by RAW264.7 macrophage cell line
(40), IL-8 release from human neutrophils
(41), MIP-1 production by PBMC (42), and
IP-10 mRNA expression by cultured keratinocytes (43). Our
results also indicate that the IP-10 production of spleen cells is
reduced by PGE2 (Fig. 1), which is consistent
with the report on IP-10 production by keratinocytes (43),
and that the enhanced production of MDC by PGE2
also depends on the accumulation of intracellular cAMP (Fig. 2). It has
also been reported that PGE2 alters the cytokine
profile in several cells, for example, PGE2
suppresses IL-12, but enhances IL-10 production via accumulation of
intracellular cAMP (27). However, neutralization of
IFN- or IL-12 and the addition of IL-10 had only a little effect on
MDC production (Fig. 2). Furthermore, cAMP-enhancing agents such as
dbcAMP and IBMX also enhanced MDC production (Fig. 4). These results
suggest that PGE2-induced elevation of cAMP is an
important signal for MDC production. The accumulation of intracellular
cAMP is not restricted to the regulation of cytokine and chemokine
productions. Several reports have indicated that the elevation of cAMP
enhanced the expression of costimulatory molecules, B7-2, on
macrophages, and these macrophages preferentially activated Th2
(45, 46). Another report showed that the cAMP accumulation
in naive T cells inhibited Th1 development in vitro (25).
These reports and our findings indicate that cAMP regulates Th1/Th2
balance at a number of different stages. Interestingly, a
representative Th2 activator, IL-4, does not participate in the
elevation of cAMP (47). This finding indicates that
PGE2 engages Th2 activation in a quite different
way from IL-4.
We determined cell types of MDC production and found that all cell
types, such as T cells, B cells, macrophages, and dendritic cells,
produced MDC following appropriate stimuli and were up-regulated by
PGE2 (Fig. 5). Several reports have indicated
that MDC is produced by T cells and is regulated by Th1/Th2 cytokines
such as IL-4 and IFN-, and that T cell-derived MDC is very important
in several diseases (37, 38). We also found that T cells,
especially CD4+ T cells, produced MDC after CD3
ligation in the presence of PGE2 the same as the
MDC production by APCs (Fig. 5). However, the amount of MDC produced by
CD4+ T cells was much lower than that of APCs (B
cells, macrophages, and dendritic cells). Therefore, we consider that
the main cell types of MDC production are APCs. MDC production by B
cells is also an interesting finding, and ABCD-1, a mouse homologue of
human MDC, was found to be expressed in B cells and dendritic cells
(33, 34). It is well known that B cells participate in the
development of Th2 subset, and it has been reported that CD40
stimulation is necessary for the induction of Th2 by B cells (6, 48). However, we suggest that T-B interaction also induces T
cell recruitment via B cell-derived chemokines, and
PGE2 up-regulate chemokine production by B
cells.
We also found an interesting phenomenon regarding IL-5. IL-5 is a
well-known cytokine generally detected in allergic diseases and
produced by Th2, mast cells, and eosinophils (1). Our
results indicate that IL-5 is also an enhancing factor for MDC
production by APCs (Figs. 4 and 6). Surprisingly, IL-5 in cooperation
with PGE2 induced a much higher amount of MDC
from APCs, which was stronger than the combination of IL-4 and
PGE2 (Fig. 6). These results suggest that IL-5,
PGE2, or the combination of both factors are more
important than IL-4 in the recruitment of Th2.
LTB4, another arachidonic acid derivative that is
one of the chemical mediators detected in allergic response, alone and
in combination with PGE2, had only a little
effect on MDC production (Fig. 6).
In conclusion, we found a novel function of PGE2
in Th2 activation. PGE2 preferentially enhanced
MDC, but suppressed IP-10 production. These effects depended on the
accumulation of intracellular cAMP, but not on the suppression of Th1
cytokines, IFN- and IL-12, and the induction of suppressive
cytokines, IL-10 and TGF-. APCs produced especially higher amounts
of MDC via CD40 stimulation and PGE2, suggesting
that T-APC interaction is very important signal for the induction of
MDC. PGE2 has been known as one of the
immunosuppressive factors. PGE2 mainly inhibited
Th1 development at the induction phase of Th subset differentiation.
There are also several reports that PGE2
production is enhanced in Th2-related diseases (49, 50, 51).
From our findings, we propose that PGE2 also
affects Th2 activation at an effector phase, that is,
PGE2 up-regulates Th2-related chemokine
production and enhances Th2 recruitment to the inflammatory sites.
Large amounts of PGE2 are produced by suppressor
macrophages and endothelial cells in chronic inflammation
(52). Therefore, we consider that
PGE2 plays an important role for the progression
of Th2-type inflammation and Th2-related diseases. Further
clarification of the regulation mechanism of not only cytokine, but
also chemokine and PGE2 productions will be
required to understand the precise mechanism of immunological diseases
such as autoimmune or allergic diseases.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Uki Yamashita, Department of Immunology, University of Occupational and Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan.
3 Abbreviations used in this paper: CXCR, CXC chemokine receptor; CD40L, CD40 ligand; dbcAMP, dibutyryl cAMP; IBMX, 3-isobutyl-1-methylxanthine; IP-10, IFN-inducible protein-10; LTB4, leukotriene B4; MDC, macrophage-derived chemokine; MIP, macrophage-inflammatory protein; TARC, thymus and activation-regulated chemokine.
Received for publication August 7, 2000. Accepted for publication November 10, 2000.
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)/dendritic cells (DC) (C, D,
I, and J) were pretreated with
PGE2, IL-4, or IFN-
