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Departments of
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Pathology and
Surgery, University of Florida, Gainesville, FL 32610
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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present, sCD40L-stimulated PG metabolism
is redirected to COX-2, and PGE2 synthesis increases
severalfold. Endogenous PG production by MDC does not regulate CD40,
CD80, CD86, or HLA DR expression; however, it does promote MDC
maturation, as NS-398 significantly reduces CD83 expression in I-MDC
matured with sCD40L/IFN-. PG produced through COX-2 also
autoregulate IL-12, but the effects are dependent on the MDC maturation
state. Blocking COX-2 reduces I-MDC secretion of IL-12p40, whereas it
increases IL-12p40 and p70 production by maturing MDC. COX-2-mediated
PG production impacts MDC function as maturing these cells in the
presence of NS-398 yields MDC that stimulate significantly more IFN-
in an allogeneic mixed lymphocyte response than MDC matured without
this inhibitor. These studies demonstrate that MDC express both COX
isoforms constitutively and produce prostanoids, which autoregulate
their maturation and function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of COX enzymes, prostanoid production, and the autocrine
effects of these molecules have not been reported for monocyte-derived
dendritic cells (MDC). However, previous studies described the effects
of exogenous PG on MDC maturation and function. Kalinsky et al.
(9) demonstrated that high concentration
(10-6 M) of exogenous PGE2 added to
monocytes in the presence of GM-CSF and IL-4 profoundly modulated MDC
development, as these cells did not lose CD14, expressed low levels of
CD1a, and produced significantly less IL-12p70 and higher levels of
IL-10 (9). Additionally, MDC derived under these
conditions stimulated Th2 responses, whereas MDC cultured without
exogenous PGE2 stimulated Th1 responses. Other studies
demonstrated that PGE2 (10-6 M),
when added to cultures following monocyte differentiation into immature
MDC (I-MDC), synergized with TNF-
or TNF-/IL-1/IL-6 at
10-8 M to induce maturation, immunostimulatory
capacity, and IL-12 production (10, 11). These studies
demonstrate that exogenous prostanoids markedly affect MDC maturation
and function and that the effect is highly dependent on the
developmental stage of the MDC.
Given the profound effects of prostanoids on MDC maturation and function, and because myeloid-derived cells produce large quantities of these lipid molecules, we assessed COX expression and prostanoid production by these cells. Our studies demonstrate that MDC, unlike monocytes, constitutively express both COX-1 and COX-2 and produce prostaglandins in an autocrine manner that regulate MDC maturation and function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were isolated from buffy coats from one unit of whole blood
using Histopaque Ficoll (1.077, endotoxin tested; Sigma, St. Louis,
MO). Cells were washed two times with Dulbecco’s PBS,
Ca2+, and Mg2+ free
(endotoxin tested; Cellgro, Herndon, VA) and resuspended in RPMI 1640
media with L-glutamine (Life Technologies, Grand Island,
NY) supplemented with 10% FCS (endotoxin tested; HyClone, Logan, UT),
and 1% streptomycin, penicillin, and neomycin (Sigma). PBMC were
allowed to adhere for 2 h at 37°C, 5%
CO2, 100% humidity, and nonadherent cells were
washed away with Dulbecco’s PBS. Complete RPMI 1640 tested negative
for endotoxin (<2.0 EU/ml) (E-Toxate Kit, Sigma). Adherent cells were
cultured for 6 days in complete RPMI supplemented with 500 U (50 ng/ml)
GM-CSF (Endogen, Woburn, MA) and 500-1000 U IL-4 (R&D Systems,
Minneapolis, MN) to generate I-MDC (12). To generate
mature MDC (M-MDC), day 6 I-MDC were harvested, washed, replated at
3.0 x 105 cells/ml, and supplemented with 1
µg/ml sCD40L (gift from Immunex, Seattle, WA) and/or 1000 U of
IFN- (human recombinant, Endogen). Some cultures were supplemented
with 1 µg/ml NS-398 (Cayman Chemical, Ann Arbor, MI), a specific
COX-2 inhibitor, or 10 µg/ml indomethacin (Sigma), and a COX-1 and
COX-2 inhibitor.
Surface and internal protein analysis
The following mAbs directed against surface or internal proteins were used: CD14, HLA-DR (Becton Dickinson, San Jose, CA), CD1a, CD86, CD80, CD40 (PharMingen, San Diego, CA), CD83 (Coulter-Immunotech, Miami, FL), and COX-2 (FITC, Cayman Chemical). Appropriate fluorochrome-labeled isotype control Abs were used. Cells were suspended in PBS with 1% BSA (reagent grade, Sigma) and 0.1% sodium azide (Sigma). For surface marker labeling, cells were incubated with 1 µg of fluorochrome-conjugated Ab/1 x 106 cells for 20 min at room temperature, then washed one time with 2.0 ml PBS and resuspended in 500 µl of 1% formaldehyde in PBS. Intracellular labeling of COX-2 was performed as previously described (13). All cells were analyzed on Becton Dickinson FACScalibur or FACSort. Flow cytometry data was analyzed and median fluorescent intensity calculated with WinMidi (Version 2.7, Joseph Trotter).
Cultured MDC were washed with PBS supplemented with protease inhibitors (1 µg/ml of each leupeptin, pepstatin, and aprotinin; Sigma) and 5 µg/ml indomethacin and frozen at -70°C. Lysates were thawed, sonicated, and centrifuged for 10 min at 14,000 rpm. Equal quantities of protein were separated by SDS-PAGE with a 10% Tris-HCl gel (Bio-Rad), and transferred to nitrocellulose (Optitran; Schleicher & Schull, Keene, NH.) Nitrocellose was probed with mAbs directed against COX-1 and COX-2 (Cayman Chemical) and secondary Abs (anti-mouse IgG-HRP; Amersham, Arlington Heights, IL). Peroxidase activity was detected by chemiluminescence (ECL Western blotting detection system; Amersham).
PGE2 and cytokine assays
Supernatants from cultures of MDC were harvested for analysis of PGE2 and IL-12. I-MDC were cultured for 6 days, washed from the plate, counted, and replated at 3 x 105 cells/ml in media containing GM-CSF and IL-4. I-MDC were cultured for an additional 48 h before supernatants were harvested for analysis. Supernatants from maturing M-MDC, were prepared by harvesting I-MDC on day 6, replating these cells at the same density in media containing GM-CSF, IL-4, and maturation stimuli. Cells were cultured for an additional 48 h and then supernatants were harvested. MDC culture supernatants from various conditions were analyzed for IL-12p70 and IL-12p40 (gift from Dr. Maurice Gately, Hoffman Roche, Nutley, NJ) by ELISA in duplicate as previously described (14). The lower limit of IL-12p40 and IL-12p70 detection in this assay is 15.6 pg/ml. Supernatants for IL-10 were measured by ELISA (Endogen, capture Ab clone 9D7 and detection Ab, clone 12G8 biotinylated). The lower limit of detection for IL-10 is 20.5 pg/ml. Measurement of PGE2 was performed using a competitive enzyme immunoassay (Cayman Chemical). To correct for PGE2 contained in the sera added to our media, we assayed in duplicate media alone (baseline). The final PGE2 concentration was calculated by subtracting the baseline from the assayed supernatant value. The limit of detection for PGE2 assay is 30 pg/ml. Final PGE2, IL-10, and IL-12 were standardized to quantity/ml/1 x 106 cells.
Allogeneic MLR
I-MDC and MDC matured with sCD40L and IFN- in the presence
and absence of NS-398 were generated according to the protocol
described above. The three MDC population were washed twice, counted
and placed in 48-well flat-bottom wells (Costar, Cambridge, MA;
2.5 x 104 cells/well) along with nylon
wool-purified allogeneic responder T cells (2.5 x
105 cells/well) in the presence or absence of
NS-398. On day 5 of the mixed lymphocyte reaction, supernatants were
harvested and analyzed for IFN- and IL-4 production. Human IFN-
was measured by specific sandwich ELISA using purified NIB42 as capture
Ab and biotinylated 4S.B3 as detection Ab (PharMingen). The lower limit
of detection for this assay is 15.6 pg/ml. Human IL-4 was also measured
by specific sandwich ELISA using 8D4–8 as capture Ab and biotinylated
MP4–25D2 as detection Ab (PharMingen) The lower limit of detection for
this assay is 7.8 pg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether MDC express COX-2, we employed an established
protocol employing GM-CSF and IL-4 to generate I-MDC from peripheral
blood monocytes (12). After 6 days in culture, I-MDC were
harvested and washed, then replated and cultured for an additional
48 h in new media containing GM-CSF and IL-4. MDC maturation was
stimulated by culturing I-MDC with either soluble trimeric CD40L
(sCD40L) in the presence or absence of human recombinant IFN- for
the same 48-h period. Cells and culture supernatants were harvested at
the 48-h time point for analysis.
We first analyzed the MDC from these cultures for COX-1 and COX-2
expression by intracellular flow cytometry (13) and
immunoblotting. As seen in Fig. 1
, A and B, I-MDC stimulated with IFN- only,
sCD40L only, and sCD40L/IFN- constitutively express COX-1 and COX-2.
We were also able to detect intracellular COX-2 expression by flow
cytometry (see Fig. 1C) and further establish expression in
MDC. This is in marked contrast to monocytes that express COX-1
constitutively (data not shown) but require LPS induction for COX-2
expression (Fig. 1D). Of interest, whereas monocyte COX-2 is
readily suppressed by 500 U/ml of IL-4 (Fig. 1D, lane
3), the same concentration of IL-4 present in MDC cultures does
not regulate COX-2 in either I- or M-MDC (Fig. 1, A and
B). We also find that IL-10 does not suppress COX-2 (data
not shown). These findings with MDC are in marked contrast to several
studies demonstrating that LPS-induced monocyte COX-2 expression is
readily down-regulated by antiinflammatory cytokines IL-4, IL-10, and
IL-13 (8). However, our results are similar to findings by
Maloney et al. (15) that showed COX-2 induced by LPS or
GM-CSF in neutrophils was not down-regulated by IL-4 or IL-10. These
data suggest that GM-CSF, IL-4, or factors produced in culture by
monocytes or the differentiating process induces COX-2 in manner that
provides resistance to cytokine regulation.
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Next, we assessed COX-1- and COX-2-mediated prostanoid production
by MDC populations. We analyzed the supernatants of I- and maturing
M-MDC cultured in the presence and absence of NS-398, a specific COX-2
inhibitor, or indomethacin, a COX-1 and -2 inhibitor, added during the
last 48 h of cell culture. We find that I-MDC spontaneously
produce thromboxane (TBX) > PGE2 > prostacyclin
but no PGD2 (data not shown). NS-398 and indomethacin
significantly reduced PGE2 production to a similar degree,
suggesting that prostanoid synthesis occurs predominantly through COX-2
in I-MDC (Fig. 2). It is possible that
small numbers of residual monocytes, 
1% of our cultures, produced
large quantities of prostanoids and accounted for COX-2-mediated PG.
Although this possibility exists, monocytes do not express COX-2 during
culture without activation. Furthermore, the expression of this enzyme
is readily suppressed in monocytes by the presence of IL-4 in the
culture (see Fig. 1
D).
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). MDC cultured in
these conditions synthesize 2-fold more PGE2 but use
primarily COX-1 as indomethacin, but not NS-398, markedly reduced PG
production. We also evaluated the effects of IFN- on sCD40L mediated
maturation as this cytokine in combination with sCD40L strongly
influenced MDC function and development, especially secretion of
IL-12p70 (16). When MDC were matured with sCD40L in
combination with IFN-, a 3- to 4-fold increase in COX-2-mediated
PGE2 production occurred, which was reduced to I-MDC levels
in the presence of NS-398 (Fig. 2). IFN- also stimulated a 2-fold
increase in COX-2-dependent PGE2 production from I-MDC
(data not shown). The effects of IFN- on COX-2-mediated PG
production by in I-MDC and maturing MDC may be related to the increased
access of COX-2 to substrate as this cytokine readily stimulates AA
release through G-protein-mediated activation of PLA2
(17). We also analyzed the production of TBX and
prostacyclin in MDC undergoing maturation with the sCD40L/IFN-
stimulus. We found that the production of these prostanoids increased
in proportion to PGE2 with this stimulus and were reduced
to the same degree with NS-398 (data not shown). These data suggest
that the synthesis of prostanoids through COX-2 is the primary pathway
for I-MDC, whereas stimulation of I-MDC by sCD40L in the absence of
IFN- switches AA metabolism to COX-1-dependent pathway. However,
when inflammatory stimuli such as IFN- or LPS and TNF- (data not
shown) are present, COX-2-mediated PG synthesis again predominates. The quantity of PGE2 produced by MDC (10-9 M) is relatively small in comparison to LPS-activated monocytes, which produce micromolar quantities of PGE2. It is not readily evident why quantitative differences in PG metabolism exist between these two types of myeloid cells. Based on the Western blots, we do not find that monocytes express a greater mass of COX-2 than MDC (data not shown). Therefore, it may be that the presence of IL-4 in MDC cultures limits PLA2 activity and substrate availability (18). However, culturing MDC in the absence of IL-4 for 24 h increased PGE2 production, but the prostaglandin levels remained in the nanomolar range (data not shown). Alternatively, higher levels of AA may be liberated when monocytes are stimulated with LPS. However, stimulation of maturing MDC with LPS leads to only nanomolar quantities of PGE2 (data not shown). Thus the quantitative set point for production of prostanoids by MDC appears to be substantially lower than that of macrophages or monocytes.
COX-2 mediated PG synthesis promotes I-MDC maturation
To establish whether endogenous PG affect differentiation of I-MDC
from monocytes, we analyzed surface Ag expression of CD1a, CD14, CD40,
CD80, CD86, CD83, and HLA-DR on these cells cultured in the presence
and absence of NS-398 (Fig. 3). Our data
demonstrate that blocking endogenous COX-2-mediated prostanoid
production did not affect expression of CD1a, HLA-DR, or the expression
of the costimulatory molecules during differentiation from monocytes to
I-MDC (Fig. 3). These data are consistent with Kalinski et al.
(9) who reported that MDC exposed to
10-9 M exogenous PGE2, equivalent to
levels produced by I-MDC, did not affect MDC differentiation from
monocytes.
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in the presence of either NS-398 or
indomethacin. Again, we found that blocking COX-2 in MDC stimulated
with sCD40L/IFN- did not modify expression of CD40, CD80, CD86, or
HLA-DR (Fig. 4). We also find that
indomethacin used to block COX-1, the predominant enzyme metabolizing
arachidonate in MDC matured with sCD40L alone, likewise did not affect
expression of these same Ags (data not shown). Previous studies
demonstrated that micromolar concentrations of PGE2
enhanced I-MDC maturation when used in cell culture in combination with
LPS, TNF-, or a mixture of inflammatory cytokines (10, 19). However, in the present studies, we did not find that
reducing PG limited MDC maturation based on the expression of these
Ags. It appears that large quantities of PGE2, such as that
produced by macrophages, are required to modulate surface molecules
such as CD86.
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stimulation (Fig. 4). The
predominant effect of prostanoids appears to be mediated through COX-2
as the addition of indomethacin did not enhance this effect (data not
shown). When MDC were stimulated with sCD40L alone, substantially lower
levels of CD83 expression were achieved. As MDC stimulated in this
manner produce PGE2 primarily through COX-1 we blocked PG
production with indomethacin and evaluated CD83 expression. Unlike
I-MDC matured with sCD40L and IFN-, the COX inhibitor did little to
affect CD83 expression in these conditions. These data are consistent
with the previous reports suggesting that PGE2 increases
CD83 expression on MDC. However, these studies employed micromolar
concentrations of PGE2 to enhance CD83 expression
(10). Although lower doses of PGE2 equivalent
to that made by MDC were not tested in these reports, it may be that
sCD40L provides a qualitatively different stimulus than LPS, TNF-,
or a combination of inflammatory cytokines such that nanomolar levels
of PG are effective. Based on the present findings, it appears that
lower levels of endogenous PG uniquely stimulate expression of CD83 in
contrast to other maturation Ags, e.g., CD86. Endogenous prostanoid production affects secretion of IL-12
MDC secretion of the Th1-polarizing cytokine, IL-12, has been
extensively studied (9, 11, 16, 20, 21). To examine the
effect of endogenous PG on secretion of IL-12p40 and IL-12p70, we
prepared MDC and assayed both forms of this cytokine in the
supernatants in the presence and absence of COX inhibitors. We chose to
study IL-12 production during maturation of MDC using sCD40L alone and
in combination with IFN-, the latter combination stimulating
production of biologically active IL-12p70 (16).
Consistent with previous reports, we found that I-MDC produced only
IL-12p40 and did not produce IL-12p70 (19, 20, 22). When
I-MDC were cultured in the presence of NS-398 for 48 h, IL-12p40
was significantly reduced (Fig. 5). The
inhibition of IL-12 by indomethacin was not different from that of
NS-398, suggesting the effects of prostanoids on this cytokine are
predominantly mediated by the COX-2 isoform (data not shown). These
results are consistent with those of Rieser et al. (11)
who showed an increase in total IL-12 when I-MDC were exposed to
PGE2 or other compounds which increase intracellular cAMP.
In marked contrast, I-MDC undergoing maturation for 48 h with
sCD40L and IFN- in the presence of COX-2 inhibitor, significantly
increased IL-12p40 production (p = 0.007) and
increased, but not significantly, IL-12p70 production
(p = 0.068; Fig. 5). These findings mirror
previous studies that showed addition of PGE2 to cell
culture suppressed IL-12p70 production by maturing MDC (20, 23). Preliminary studies in our laboratory showed
PGE2 to be the predominant prostanoid suppressing IL-12
production. Prostacyclin had similar but lesser effects than
PGE2 on secretion of IL-12, whereas TBX and metabolites of
PGD2 had little to no effect (manuscript in preparation).
Collectively, these data further demonstrate that prostanoids produced
via COX-2 modulate MDC function and markedly affect the secretion of
IL-12. However, the effect is dependent on the state of differentiation
of these cells.
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Previous studies in murine macrophages demonstrated that IL-10
production in LPS-stimulated macrophages occurred through a
cAMP/PGE2-dependent mechanism (24). We
therefore evaluated the production of IL-10 in I-MDC and maturing MDC.
We find that I-MDC do not produce detectable levels of IL-10, whereas
M-MDC matured with soluble sCD40L alone or with and IFN- produce low
levels that are not significantly reduced with NS-398 or indomethacin
(Fig. 6). These experiments do not
suggest that PG produced by MDC stimulate IL-10 production.
Furthermore, they demonstrate that PG-mediated suppression of IL-12 in
maturing MDC is mediated directly by endogenous PGE2
directly and not through its effect on IL-10.
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Blocking COX-2-mediated prostanoid production in maturing MDC
increased IL-12p70 secretion (Fig. 5), and leads to a sustained
capacity of M-MDC to produce higher levels of IL-12p70 with subsequent
stimulation (data not shown). Because of these effects, we questioned
whether M-MDC generated in the presence of NS-398 would promote Th1
responses to a greater degree than M-MDC matured in the absence of this
specific inhibitor. To address this question we evaluated the ability
of I-MDC and M-MDC matured with sCD40L and IFN- in the presence or
absence of NS-398 to activate allogeneic T cell IFN- or IL-4
production. As shown in Fig. 7, MDC
matured with sCD40L and IFN- in the presence of NS-398 stimulated
significantly more IFN- in the allogeneic MLR than did MDC matured
without NS-398 (p < 0.0048). We did not detect
IL-4 in any of the culture supernatants (lower limit of detection is
7.8 pg/ml). These data suggest that COX-2 mediated PG production by MDC
during the maturation process determines their subsequent capacity to
produce IL-12 and to promote Th1 responses. Another possibility may be
that blocking prostanoid production during maturation affects M-MDC
factors other than IL-12 that promote Th1 responses. We attempted but
were unable to detect IL-12p70 in these cell cultures. This may be due
to the low numbers of MDC (2.5 x 104 cells)
in each condition or because of cytokine consumption. Finally, the
addition of NS-398 at the beginning of the mixed lymphocyte response
had no effect on IFN- production stimulated by I-MDC or either M-MDC
population and suggests IL-12 and IFN- production by M-MDC and T
cells, respectively, are not affected by prostanoids produced during
the allogeneic mixed lymphocyte response. Alternatively, it may be that
the levels of prostanoids produced by MDC or T cells are too low to
affect the response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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leads to higher levels of
PGE2, but production reverts back to the COX-2 pathway. The
observation that PG synthesis fluctuates from one COX isoform to the
other is not a novel finding. Previous studies demonstrated that this
phenomenon occurs as a consequence of the coupling of COX isoforms to
distinct PLA2 isoenzymes, e.g., cytoplasmic
PlA2 to COX-2, and linkage of apparently discrete pools of
AA to either COX-1 or COX-2 (25). Supporting these
published studies, we find that mouse macrophages expressing both COX-1
and COX-2 produce PGE2 only through COX-1 when AA added to
cultures, whereas IFN- stimulates only COX-2-mediated PG synthesis
(manuscript in preparation).
The regulation of COX-2 expression in MDC is unlike that of the
precursor monocyte population, as MDC are highly resistant to
suppression by the anti-inflammatory cytokines IL-4 and IL-10. The
reason for the marked alteration in COX-2 regulation is not apparent
but may be related to the continuous presence of GM-CSF in vitro.
Another possibility is that long-term culture or long-term exposure to
IL-4 may also diminish the MDC response to this cytokine.
Alternatively, studies by Hart et al. (15) demonstrated
that freshly isolated monocytes were more responsive to IL-4 induced
TNF- suppression than macrophages cultured for 7 days. These authors
concluded that the monocytes’ responses to immunoregulatory cytokines
such as IL-4 may not mirror responses by their differentiated or
activated counterparts (15). This suggestion is also
supported by the studies of Maloney et al. (26) that
demonstrated neutrophil expression of COX-2 was likewise resistant to
IL-4, IL-10, and IL-13. Therefore, the pathway of myeloid
differentiation or maturation may dictate the responsiveness of COX-2
to anti-inflammatory cytokines.
The production of PG appears to autoregulate some aspects of MDC
maturation (e.g., CD83) and function (e.g., IL-12 production by MDC).
Our finding that endogenous prostanoids generated through COX-2 in
vitro did not interfere with the expression of HLA-DR and costimulatory
molecules on I-MDC was expected because previous studies showed that
less that 10-9 M PGE2 had little
effect on these differentiation Ags for MDC. Although we find that
endogenous production of prostanoids does not modify HLA-DR or the
costimulatory molecules CD40, CD80, and CD86; however, COX-2 PG
markedly modulate the expression of the maturation Ag, CD83, in
sCD40L/IFN- stimulated MDC. It appears that the threshold for
prostanoid regulation of CD83 differs markedly from that of HLA-DR and
costimulatory molecules. In the case of costimulatory molecules, cells
producing higher levels of PGE2 than MDC, perhaps
macrophages, within the local environment may be required to affect the
up-regulation of these molecules as previously described
(10).
In this study, we find a divergent regulation of IL-12 by
COX-2-mediated prostanoid production. We find that endogenously
produced PG increased the IL-12p40 in I-MDC but did not stimulate
IL-12p70 production. This prostanoid-mediated enhancement of IL-12p40
production by I-MDC may serve to limit the Th1 immune response as IL-12
p40 homodimers function as a receptor antagonist (27, 28).
As MDC mature in the presence of IFN- the level of COX-2-mediated
prostanoid production increases which effectively suppresses IL-12p70
and p40. This is in agreement with studies of others demonstrating that
addition of PGE2 to cell culture reduces IL-12 production
by M-MDC (23, 29). Thus, endogenous prostanoids appear to
play an important role in limiting the capacity of M- MDC to become a
potent Th1 promoting APCs by down regulating the production of
biologically active IL-12p70 by these cells. The mechanism responsible
for the interesting divergence in prostanoid-mediated regulation of
IL-12 has not been defined. However, modulation of surface or nuclear
receptors for PGE2, e.g., EP1, EP2, EP3, or EP4 could be
responsible for these changes in the response of MDC as they mature.
Preliminary studies in the laboratory indeed suggest that maturation
stimuli regulate EP receptor expression and this defines the response
of MDC to PGE2.
Previous reports have suggested that COX-2 is important for high level
production of prostanoids by particular cells types and is the form of
the enzyme associated with inflammation. It is of interest when MDC are
exposed to IFN- in culture that PGE2 production is
enhanced and COX-2 is the predominant isoform of the enzyme used to
produce prostanoids. These findings suggest that a default setting for
I-MDC and maturing MDC is to increase COX-2-mediated PGE2
production when involved in inflammation, when acting as an APC for
established Th1 responses, or when encountering other IFN- producing
cells (e.g., NK or NKT cells). Under these circumstances, MDC are thus
programmed, via COX-2 expression, to suppress IL-12 production and thus
autoregulate their capacity to further stimulate Th1 cells. Indeed,
results from the allogeneic MLR support such a role for PG. These data
suggest the production of endogenous PG during maturation directs MDC
functional development such that their capacity to produce IL-12 and
stimulate Th1 responses is significantly limited.
Prostaglandin production by MDC appears to play an important and
focused role in the function of MDC. From the findings of this study it
appears that MDC tend to produce lower levels of PGE2 than
that produced by monocytes or macrophages. The lower level of PG
produced by MDC may be of practical importance as these lipid molecules
work in an autocrine fashion modulating MDC function, and perhaps in
regulating T cells within their microenviroment in a paracrine fashion.
Working in this manner, the effects of prostanoids would be contained
and would limit the untoward effects of these molecules. In this
context, determining regulation of prostanoid receptors on MDC and T
cells thus becomes critical to understanding the effects of these lipid
molecules on their target cells. Furthermore, the production of PG by
MDC may provide these cells with a self-contained "signal 3" as
proposed by Kalinski et al. (30), which would polarize the
MDC away from stimulating Th1 responses, perhaps more toward a Th2
promoting APC. Our findings also have important implications regarding
the effects of COX inhibitors, particularly the new class of
COX-2-specific drugs, on the immune response. The potent
anti-inflammatory action of these drugs may in part be limiting MDC
maturation. These studies also raise a potential concern regarding the
possibility that COX-2-specific drugs could potentiate Th1 responses.
Indeed our in vitro experiments suggest that by removing the
suppressive autocrine effects of PG on IL-12 production in maturing
M-MDC these cells stimulate significantly higher levels of IFN-
production by T cells. Further study in vivo is required to establish
the effect of these drugs on the biology of MDC.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael Clare-Salzler, Department of Pathology, Box 100275, 1600 SW Archer Rd, Gainesville, FL 32610-0275.
3 Abbreviations used in this paper: AA, arachidonic acid; PLA2, phospholipase A2; MDC, moncyte-derived dendritic cells; I-MDC, immature MDC; M-MDC, mature MDC; COX, cyclooxygenase; sCD40L, soluble CD40 ligand; TBX, thromboxane.
Received for publication April 13, 2000. Accepted for publication July 28, 2000.
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  M. Lehner, A. Stilper, P. Morhart, and W. Holter Plasticity of dendritic cell function in response to prostaglandin E2 (PGE2) and interferon-{gamma} (IFN-{gamma}) J. Leukoc. Biol., April 1, 2008; 83(4): 883 - 893. [Abstract] [Full Text] [PDF]
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 W. R. Winnall, U. Ali, M. K. O'Bryan, J. J. Hirst, P. A.F. Whiley, J. A. Muir, and M. P. Hedger Constitutive Expression of Prostaglandin-Endoperoxide Synthase 2 by Somatic and Spermatogenic Cells Is Responsible for Prostaglandin E2 Production in the Adult Rat Testis Biol Reprod, May 1, 2007; 76(5): 759 - 768. [Abstract] [Full Text] [PDF]
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 T. Yokoyama, O. Aramaki, T. Takayama, S. Takano, Q. Zhang, M. Shimazu, M. Kitajima, Y. Ikeda, N. Shirasugi, and M. Niimi Selective cyclooxygenase 2 inhibitor induces indefinite survival of fully allogeneic cardiac grafts and generates CD4+ regulatory cells J. Thorac. Cardiovasc. Surg., October 1, 2005; 130(4): 1167 - 1174. [Abstract] [Full Text] [PDF]
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F. Baratelli, K. Krysan, N. Heuze-Vourc'h, L. Zhu, B. Escuadro, S. Sharma, K. Reckamp, M. Dohadwala, and S. M. Dubinett PGE2 confers survivin-dependent apoptosis resistance in human monocyte-derived dendritic cells J. Leukoc. Biol., August 1, 2005; 78(2): 555 - 564. [Abstract] [Full Text] [PDF]
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 M. Zeyda, M. D. Saemann, K. M. Stuhlmeier, D. G. Mascher, P. N. Nowotny, G. J. Zlabinger, W. Waldhausl, and T. M. Stulnig Polyunsaturated Fatty Acids Block Dendritic Cell Activation and Function Independently of NF-{kappa}B Activation J. Biol. Chem., April 8, 2005; 280(14): 14293 - 14301. [Abstract] [Full Text] [PDF]
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 J. A. Long, M. Fogel-Petrovic, D. A. Knight, P. J. Thompson, and J. W. Upham Higher Prostaglandin E2 Production by Dendritic Cells from Subjects with Asthma Compared with Normal Subjects Am. J. Respir. Crit. Care Med., September 1, 2004; 170(5): 485 - 491. [Abstract] [Full Text] [PDF]
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 A. M. Blum, A. Metwali, D. E. Elliott, D. J. Berg, and J. V. Weinstock CD4+ T cells from IL-10-deficient mice transfer susceptibility to NSAID-induced Rag colitis Am J Physiol Gastrointest Liver Physiol, August 1, 2004; 287(2): G320 - G325. [Abstract] [Full Text] [PDF]
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 A. R. R. Goncalves, C. K. Fujihara, A. L. Mattar, D. M. A. C. Malheiros, I. L. Noronha, G. de Nucci, and R. Zatz Renal expression of COX-2, ANG II, and AT1 receptor in remnant kidney: strong renoprotection by therapy with losartan and a nonsteroidal anti-inflammatory Am J Physiol Renal Physiol, May 1, 2004; 286(5): F945 - F954. [Abstract] [Full Text] [PDF]
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M. Hewison, L. Freeman, S. V. Hughes, K. N. Evans, R. Bland, A. G. Eliopoulos, M. D. Kilby, P. A. H. Moss, and R. Chakraverty Differential Regulation of Vitamin D Receptor and Its Ligand in Human Monocyte-Derived Dendritic Cells J. Immunol., June 1, 2003; 170(11): 5382 - 5390. [Abstract] [Full Text] [PDF]
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S. Al-Darmaki, H. A. Schenkein, J. G. Tew, and S. E. Barbour Differential Expression of Platelet-Activating Factor Acetylhydrolase in Macrophages and Monocyte-Derived Dendritic Cells J. Immunol., January 1, 2003; 170(1): 167 - 173. [Abstract] [Full Text] [PDF]
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E. Scandella, Y. Men, S. Gillessen, R. Forster, and M. Groettrup Prostaglandin E2 is a key factor for CCR7 surface expression and migration of monocyte-derived dendritic cells Blood, July 30, 2002; 100(4): 1354 - 1361. [Abstract] [Full Text] [PDF]
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C. C. Sombroek, A. G. M. Stam, A. J. Masterson, S. M. Lougheed, M. J. A. G. Schakel, C. J. L. M. Meijer, H. M. Pinedo, A. J. M. van den Eertwegh, R. J. Scheper, and T. D. de Gruijl Prostanoids Play a Major Role in the Primary Tumor-Induced Inhibition of Dendritic Cell Differentiation J. Immunol., May 1, 2002; 168(9): 4333 - 4343. [Abstract] [Full Text] [PDF]
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0.05 as calculated by one-way ANOVA.
).
B, M-MDC (
) and M-MDC matured in the presence of
NS-398 (
). MDC were washed extensively before use and cultured with
allogeneic T cells in either the absence (left) and
presence (right) of NS-398. Each lymphocyte response was
performed in duplicate cultures. Supernatants from each culture well
were analyzed for IFN-