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12,1412,14-prostaglandins D2 and J2 Are Potent Activators of Human Eosinophils1
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Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada; and
Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, Melbourne, FL 32901
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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12,14-PDJ2
(15d-PGJ2) is a degradation product of PGD2
that has been proposed as an anti-inflammatory compound because of
its various inhibitory effects, some of which are mediated by
peroxisome proliferator-activated receptor-
. In contrast to its
reported inhibitory effects on macrophages and other cells, we found
that this compound is a potent activator of eosinophils, inducing
calcium mobilization, actin polymerization, and CD11b expression. It is
selective for eosinophils, having little or no effect on neutrophils or
monocytes. 15d-PGJ2 has an EC50 of 
10 nM,
similar to that of its precursor, PGD2. The concentrations
of 15d-PGJ2 required to activate eosinophils are thus much
lower than those required for its anti-inflammatory effects
(usually micromolar). 15-Deoxy-12,14-prostaglandin
D2 (15d-PGD2) is also a potent activator of
eosinophils, with an EC50 about the same as that of
PGD2, whereas 12-PGJ2 is
slightly less potent. Eosinophils pretreated with PGD2 no
longer respond to 15d-PGJ2, and vice versa, but in both
cases the cells still respond to another eicosanoid proinflammatory
mediator, 5-oxo-6,8,11,14-eicosatetraenoic acid. This indicates that
the effects of 15d-PGJ2 are mediated by the
DP2/chemoattractant receptor-homologous molecule expressed
on Th2 cells that has recently been identified in eosinophils.
15d-PGJ2 is selective for the DP2 receptor, in
that it has no effect on DP1 receptor-mediated adenylyl
cyclase activity in platelets. We conclude that 15d-PGJ2
and 15d-PGD2 are selective DP2 receptor
agonists that activate human eosinophils with potencies at least 100
times greater than those for the proposed anti-inflammatory effects
of 15d-PGJ2 on other cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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12,14-PGJ2
(15d-PGJ2),4 which has been proposed as an
anti-inflammatory compound because of its various inhibitory effects,
some of which are mediated by peroxisome proliferator-activated
receptor- (PPAR). Most notably, it strongly activates PPAR
(1, 2), a transcription factor that is preferentially
expressed in adipose tissue, vascular smooth muscle cells, and
macrophages (3). PPAR plays a central role in
adipogenesis, enhances sensitivity to insulin, and inhibits the
inflammatory response (3). Stimulation of PPAR by
relatively high (low micromolar) concentrations of
15d-PGJ2, as well as by other PPAR agonists
results in suppression of the expression of cytokines (4, 5), matrix metalloproteinase-9, and inducible nitric oxide
synthase (6). In addition, activation of PPAR induces
apoptosis in a variety of cells including macrophages (7, 8). Many of the anti-inflammatory effects due to stimulation
of PPAR may be due to antagonism of the transcription factors
NF-
B, AP-1, and STAT-1 (6).
15d-PGJ2 also has anti-inflammatory effects
that are independent of PPAR. It inhibits adhesion and the oxidative
burst in neutrophils by a mechanism that appears not to be mediated by
any known receptors (9). At least some
non-PPAR-mediated anti-inflammatory effects of
15d-PGJ2 are due to the chemical reactivity of
its dienone system, resulting in addition to protein thiol groups, as
has been shown for NF-B (10, 11, 12). The
anti-inflammatory effects of 15d-PGJ2
described above are not shared by its precursor
PGD2. Little information is available about the ability of 15d-PGJ2 to stimulate PGD2 receptors, and the effects of this substance on eosinophils have not previously been explored. 15d-PGJ2 has been reported to compete only very weakly for binding to the DP1 receptor that is coupled positively to adenylyl cyclase (13). We have recently identified a second receptor for PGD2 (DP2 receptor) on eosinophils, activation of which leads to cell migration, increased expression of CD11b, shedding of L-selectin, and actin polymerization (14). This receptor appears to be identical with chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), an orphan receptor for which PGD2 was recently identified as a ligand (15). We previously found that PGJ2 stimulates both DP1 and DP2 receptors on eosinophils, indicating that the 9-hydroxyl group is not required for biological activity (14). Furthermore, 13,14-dihydro-15-oxo-PGD2 is a good agonist at the DP2 but not the DP1 receptor, indicating that although the 15-hydroxyl group of PGD2 is required for activation of the DP1 receptor, it is not essential for interaction with the DP2 receptor (14, 15). This raised the possibility that 15d-PGJ2, which lacks this hydroxyl group, could have proinflammatory effects by selectively stimulating the DP2 receptor in inflammatory cells. The present study was designed to examine the effects of 15d-PGJ2 on eosinophils and to determine whether it could stimulate these cells by activating the DP2 receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All prostaglandins were purchased from Cayman Chemical (Ann
Arbor, MI). 15d-PGJ2 was the purified
14-cis isomer (
97% pure; catalog number 18570).
15-Deoxy-12,14-prostaglandin
D2 (15d-PGD2) is the
14-trans isomer. 5-Oxo-6,8,11,14-eicosatetraenoic acid
(5-oxo-ETE) was synthesized chemically as described previously
(16).
Preparation of leukocytes
All studies with eosinophils were conducted using unfractionated leukocytes from healthy volunteers. Whole blood was treated with Dextran T-500 (Amersham-Pharmacia Biotech, Piscataway, NJ) for 45 min at 4°C to remove RBC (17). The supernatant was centrifuged at 200 x g for 10 min, and the pellet was suspended in water (10 ml). After 30 s, the suspension was diluted with 2x concentrated PBS to give final concentrations of 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4 at pH 7.4. For experiments designed to measure CD11b expression, the cells were resuspended directly in PBS without prior lysis of RBC (this was accomplished later using OptiLyse (Beckman Coulter, Fullerton, CA)).
Preparation of platelets
Whole blood (20 ml) was collected in medium (2.8 ml) containing citric acid (15.5 mM), sodium citrate (90 mM), NaH2PO4 (16 mM), dextrose (161 mM), and adenine (2 mM). After centrifugation at 200 x g for 15 min, the supernatant was diluted with an equal volume of medium containing 94 mM citrate and 140 mM dextrose, pH 6.5. The mixture was centrifuged at 1000 x g for 10 min, and the pellet was suspended in PBS containing Ca2+ (1.8 mM) and Mg2+ (1 mM) to give a platelet concentration of 3 x 108 cells/ml.
Analysis of intracellular calcium levels by flow cytometry
Leukocytes (107 cells/ml) were treated
with the acetoxymethyl ester of fluo-3 (2 µM; Molecular Probes,
Eugene, OR) in the presence of Pluronic F-127 (0.02%; Molecular
Probes) for 60 min at 23°C. The mixture was then centrifuged at
200 x g for 10 min, and the pellet was resuspended in
PBS to give a concentration of 50 x 106
cells/ml. The leukocytes were then treated with PC5-labeled mouse
anti-human CD16 (3.3 µl/106 cells; Beckman
Coulter) for 30 min at 0°C. PBS (25 ml) was then added, the mixture
centrifuged as described above, and the pellet was resuspended in PBS
to give a concentration of 3 x 106
leukocytes/ml. After incubation at 23°C for 30 min, an aliquot (0.95
ml) of the leukocyte suspension was removed and treated with PBS (50
µl) containing Ca2+ (36 mM) and
Mg2+ (20 mM). After 5 min, the cells were
analyzed by flow cytometry using a FACSCalibur instrument (BD
Biosciences, San Jose, CA). A total of 106
cells was counted during a period of 3–4 min for each sample. Fluo-3
fluorescence was measured in eosinophils, neutrophils, and monocytes,
which were gated out on the basis of CD16 staining and side scatter
(Fig. 1
A). Maximal calcium
responses were determined by addition of A23187 (10 µM) at the end of
each run.
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Leukocytes (5 x 107 cells/ml) were treated with PC5-labeled mouse anti-human CD16 (10 µl/106 cells; Beckman Coulter) for 30 min on ice. PBS (2 ml/106 cells) was then added, and the mixture was centrifuged at 200 x g for 10 min. The pellet was resuspended in ice cold PBS containing Ca2+ (1.8 mM) and Mg2+ (1 mM) to give a concentration of 5 x 106 cells/ml. Aliquots of the leukocyte suspension (90 µl for the concentration-response experiment; 800 µl for the time course experiment) were preincubated for 5 min at 37°C before the addition of agonist or vehicle (10 µl PBS containing Ca2+ and Mg2+ and 0.1% BSA). The incubations were terminated at various times (20 s for the concentration-response experiment) by addition of formaldehyde (37%) to give a final concentration of 8.5%. After the samples were kept on ice for 30 min, a mixture of lysophosphatidylcholine (30 µg in 23.8 µl PBS) and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (Molecular Probes; 49 pmol in 6.2 µl methanol; final concentration, 0.3 µM) was added to each sample (18), followed by incubation overnight in the dark at 4°C. The cells were then washed by addition of PBS (1 ml), followed by centrifugation at 200 x g for 10 min and resuspension of the pellet in 300 µl PBS containing 1% formaldehyde. F-actin levels were measured by flow cytometry in eosinophils (high side scatter; low CD16) and neutrophils (high side scatter; high CD16).
Measurement of CD11b expression
Leukocytes (0.5 ml; 106/ml) in PBS containing Ca2+ and Mg2+ were incubated with agonists for 10 min. The incubations were terminated by the addition of ice cold FACSFlow (BD Biosciences) and centrifugation. The cells were then incubated for 30 min at 4°C with a mixture of PE-labeled mouse anti-human VLA-4 (5 µl; BD Biosciences) and FITC-labeled mouse anti-human CD11b (10 µl Bear1; Beckman Coulter). After incubation with OptiLyse C (0.25 ml; Beckman Coulter) for 15 min, the cells were centrifuged and fixed in PBS (0.4 ml) containing 1% formaldehyde. The distribution of fluorescence intensities among 60,000 cells was measured by flow cytometry. CD11b was measured in both eosinophils (high side scatter; high VLA-4) and neutrophils (high side scatter; low VLA-4) (19). The cells gated as eosinophils contained <2% basophils, based on expression of high IgE.
Determination of cAMP levels in platelets
Platelets (3 x 107 cells in 100 µl) were incubated with prostanoids for 2 min at 37°C. The incubations were terminated by addition of ice cold ethanol (300 µl), and the precipitated proteins were removed by centrifugation (600 x g for 15 min). cAMP in the supernatants was measured using a competitive protein-binding radiometric assay (Diagnostic Products, Los Angeles, CA) according to the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intracellular calcium levels were measured by flow cytometry in
mixed leukocytes loaded with fluo-3 and treated with PC5-tagged
anti-CD16, which labeled neutrophils but not eosinophils. This
averted the potential for eosinophil activation that could occur if an
Ab to an eosinophil surface marker were used. Eosinophils (very low
CD16; high side scatter) could easily be distinguished from neutrophils
(high CD16; high side scatter) and monocytes (low CD16; low side
scatter) (Fig. 1
A). The effects of
15d-PGJ2 on intracellular calcium levels in the
above three cell types were compared with those of
PGD2 and 5-oxo-ETE (Fig. 1B).
PGD2 stimulates calcium mobilization
(20) and a variety of other responses (14) in
eosinophils but not neutrophils, whereas 5-oxo-ETE activates both
eosinophils and neutrophils (21, 22, 23). This is confirmed in
the present study. We also found that neither
PGD2 nor 5-oxo-ETE affects calcium levels in
monocytes (Fig. 1B, top tracings). As
with PGD2, 15d-PGJ2 (1
µM) induced calcium mobilization selectively in eosinophils, but not
in either neutrophils or monocytes. The concentration-response curves
for calcium mobilization induced by 15d-PGJ2
(EC50 29 ± 15 nM) and
PGD2 EC50 60 ± 47 nM)
were similar to one another, although there was considerable
variability in the magnitude of the individual responses using this
method (Fig. 1C).
15d-PGJ2 selectively stimulates actin polymerization in eosinophils
The effects of 15d-PGJ2 on actin
polymerization in eosinophils were compared with those of
PGD2 as well as its more immediate precursors,
PGJ2 and
12-PGJ2 (Fig. 2). Actin polymerization was measured in
unfractionated leukocytes using anti-CD16-labeling to distinguish
between eosinophils and neutrophils as shown in Fig. 1A.
15d-PGJ2 (EC50 11 ± 3
nM) was only slightly less potent than PGD2
(EC50 7 ± 2 nM).
12-PGJ2
(EC50 23 ± 2 nM) and
PGJ2 (EC50 30 ± 2 nM)
were somewhat less potent than 15d-PGJ2
(p < 0.05). All four compounds induced similar
maximal responses. In contrast to eosinophils, neither
15d-PGJ2 nor PGD2 induced
actin polymerization in neutrophils (indicated by (N) in Fig. 2).
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CD11b expression was measured in mixed leukocytes by flow
cytometry. After incubation with agonists, the eosinophils were fixed
and stained with PE-labeled anti-VLA-4 to distinguish them from
neutrophils. The effects of various dehydration products of
PGD2 on CD11b expression are shown in Fig. 3A. PGD2
(EC50, 9.4 ± 2.2 nM),
15d-PGJ2 (EC50, 11.7
± 2.7 nM), and 15d-PGD2
(EC50, 8.0 ± 2.5 nM) all stimulated CD11b
expression with similar potencies. PGJ2 and
12-PGJ2 had similar
effects but were slightly less potent, with EC50
values of 30 nM. None of the prostanoids shown in Fig. 3A
stimulated CD11b levels in neutrophils (data not shown).
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To determine whether 15d-PGJ2 could activate
DP1 receptors, we compared its effect to that of
PGD2 on cAMP levels in human platelets (Fig. 4). Consistent with published data
(24), PGD2 strongly stimulated cAMP
production in platelets. In contrast, 15d-PGJ2
had no detectable effect on cAMP levels in these cells. This is
consistent with a previous study showing that
15d-PGJ2 is 300 times less potent than
PGD2 in competing for binding to HEK 293 cells
transfected with DP1 receptors
(13).
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The similarity of the responses induced by
PGD2 and 15d-PGJ2 in
eosinophils and the lack of response of neutrophils to both agonists
would be consistent with their interaction with the same receptor. To
further address this issue, we determined whether
15d-PGJ2 could desensitize eosinophils to the
effects of PGD2. For this purpose, we examined
two very rapid and transient responses, calcium mobilization and actin
polymerization. Measurement of calcium mobilization has the advantage
that it can be accomplished in real time.
15d-PGJ2 (1 µM) induced a strong calcium
response in eosinophils, as shown in Fig. 5A (tracings on
left). However, eosinophils treated with
15d-PGJ2 were completely unresponsive to
PGD2 (1 µM; Fig. 5A, upper left).
Similarly, prior treatment of eosinophils with
PGD2 desensitized them to
15d-PGJ2 (Fig. 5A, upper
middle). The selective DP1 receptor agonist
BW245C, which on its own had no effect on calcium levels, did not alter
the response to 15d-PGJ2 (Fig. 5A,
lower middle). In contrast to its inhibitory effect on
PGD2-induced calcium mobilization,
15d-PGJ2 did not affect the response to
5-oxo-ETE. Neither was 5-oxo-ETE (1 µM) able to prevent the response
to either 15d-PGJ2 (Fig. 5A,
upper right) or PGD2 (Fig. 5A, lower right).
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B). F-actin levels were measured by flow cytometry
immediately before and at several times after addition of
15d-PGJ2. 15d-PGJ2 strongly
stimulated actin polymerization in eosinophils, inducing a maximal
response after 20 s. This declined by 50% by 6 min. Addition
of PGD2 at that time had no effect on polymerized
actin levels in eosinophils, whereas addition of 5-oxo-ETE resulted in
a strong and rapid response, comparable with that induced by
15d-PGJ2. Effects of other structural modifications of PGD2 on eosinophil activation
We also examined the effects of other PGD2
analogs on CD11b expression in eosinophils (Fig. 3B).
Introduction of a 17 double bond had
relatively little effect on biological potency, given that
PGD3 has an EC50 of 10
± 2 nM. Reduction of the 5 double bond has a
greater effect, increasing the EC50 to 92 ±
40 nM (PGD1). However, this may be an
underestimate, because the maximal response may not have been reached
at the highest concentration used. The presence of the 11-oxo group is
essential for activation of the DP2 receptor,
because the reduction product 11
-PGF2
is
>100 times less potent than PGD2. The carboxyl
group also appears to be required for activation of eosinophils via the
DP2 receptor, in that the 1-hydroxyl derivative
of PGD1 (PGD1 alcohol;
1,9,15s-trihydroxyprost-13E-en-11-one) had no detectable
effect on CD11b expression in these cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(27, 28); and direct
interaction with protein thiol groups as a result of its reactive
dienone system (10, 12, 28). The stimulatory effects of
15d-PGJ2 on eosinophils
(EC50 10 nM) occur at substantially lower
concentrations than those required for its anti-inflammatory
effects (generally low micromolar). Moreover, its effects on
eosinophils are very rapid, requiring only a few seconds in the case of
calcium mobilization and actin polymerization. In contrast, the
anti-inflammatory effects of 15d-PGJ2 are
usually observed over a period of several hours. It would therefore
seem unlikely that either of these mechanisms could explain the effects
of 15d-PGJ2 on eosinophils. Furthermore, the
anti-inflammatory effects of 15d-PGJ2 are not
shared by its precursor PGD2, whereas this PG is
also a potent activator of eosinophils. The selectivity of
15d-PGJ2 for eosinophils compared with
neutrophils and monocytes also suggests a more specific mechanism of
action than observed for its anti-inflammatory effects.
Although nearly all of the published studies on
15d-PGJ2 have focused on its
anti-inflammatory properties, Zhang et al. (49) have
recently shown that this compound has differential effects on chemokine
production by monocytes, suppressing monocyte chemoattractant protein-1
release, but enhancing the production of IL-8. RANTES expression was
unaffected. These effects were mediated by PPAR and required
micromolar concentrations of 15d-PGJ2 and
prolonged incubation times. In contrast to
15d-PGJ2, 15d-PGD2 had no
effect on IL-8 secretion by monocytes. This study thus demonstrates
that 15d-PGJ2 has the potential to induce both
pro- and anti-inflammatory responses. However, the concentration
dependence, structural requirements, and time dependence for these
responses are quite different from the rapid and potent stimulatory
effects of 15d-PGJ2 on eosinophils observed in
the present study.
The similarity between the effects of 15d-PGJ2 and PGD2 on eosinophils would suggest that the former compound might act by stimulating DP2 receptors on these cells. This issue was addressed by performing a series of cross-desensitization experiments. The selective cross-desensitization between 15d-PGJ2 and PGD2, but not with another eicosanoid proinflammatory mediator, 5-oxo-ETE, provides compelling evidence that the actions of 15d-PGJ2 on eosinophils are indeed mediated by the DP2 receptor. The DP2-mediated proinflammatory effects of 15d-PGJ2 could therefore potentially be observed only in cells possessing this receptor, which include eosinophils (14, 15), basophils, and Th2 cells (15). Whether or not 15d-PGJ2 can also activate the latter two types of cells remains to be determined. In contrast, the anti-inflammatory effects of this compound, although requiring substantially higher concentrations, can be observed in a wide variety of cells.
The physiological significance of 15d-PGJ2 is
under debate (30, 31). It is a degradation product of
PGD2 that can be formed spontaneously or in the
presence of albumin, so that it could potentially occur in vivo
(32). The first step in its formation is loss of the
9-hydroxyl group of PGD2, which is followed by
rearrangement of the 13 double bond to give
12-PGJ2 and subsequent
loss of the 15-hydroxyl group (Fig. 6).
As yet, there is no evidence for its formation by an enzymatic process
(33). However, substantial levels of
15d-PGJ2 have been detected by mass spectrometry
in human urine (34) and in pleural exudate fluid after
administration of carrageenin to rats (25).
15d-PGJ2 immunoreactivity has also recently been
detected in foam cells in human atherosclerotic lesions as well as in
RAW 264.7 cells, a murine macrophage-like cell line (35).
Even less is known about the physiological significance of
15d-PGD2, although this compound is formed in
much larger amounts than 15d-PGJ2 after
incubation of PGD2 with albumin
(32).
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11 nM) is nearly as potent as
PGD2 (EC50 8 nM) in
stimulating actin polymerization and CD11b expression in eosinophils,
and at least as potent in inducing calcium mobilization. In contrast,
Hirai et al. (15) reported that
15d-PGJ2 has a
Ki of 2300 nM compared with 61 nM for
PGD2 in displacing
[3H]PGD2 from specific
binding sites in K562 cells transfected with CRTH2. There are several
possible explanations for this discrepancy. First,
15d-PGJ2 is a rather unstable molecule that
exists in several isomeric forms, of which the 14-cis form
is the most abundant (33). Although it is possible that
this instability could lead to altered biological responses, in our
hands responses to 15d-PGJ2 have been quite
consistent over a period of 1 year, suggesting that storage is not a
major problem. Another possibility could be that the receptor
responsible for the action of 15d-PGJ2 is not
identical with the DP2/CRTH2 receptor, or that
the DP2 receptor and CRTH2 are different from one
another. This scenario would require the presence of two distinct
PGD2 receptors on eosinophils (i.e., a third DP
receptor), both of which mediate cell activation, and only one of which
would be highly sensitive to 15d-PGJ2. Although
we cannot completely exclude this possibility, it would seem rather
unlikely in view of the fact that 15d-PGJ2
completely desensitizes eosinophils to PGD2,
suggesting that a single DP receptor on eosinophils is responsible for
activation of these cells by both 15d-PGJ2 and
PGD2. Moreover, other structural requirements for
interaction with CRTH2 and the DP2 receptor are
quite similar to one another:
13,14-dihydro-15-oxo-PGD2 is a very good ligand
for both receptors; the DP1 receptor agonist
BW245C is inactive at both receptors (14, 15), as is the
PGD2 metabolite
11-PGF2, as shown both by the present study
and by Hirai et al. (15). In addition, neither receptor is
activated by any prostanoids unrelated to PGD2.
Finally, it is possible that the binding properties of
DP2/CRTH2 ligands are different in transfected
K562 cells than in human eosinophils. This hypothesis is difficult to
address directly in view of severe limitations in the numbers of
available eosinophils. However, it is conceivable that K562 cells may
lack some of the G proteins present in eosinophils, which may result in
differences in the conformation of the receptor that could affect the
structural requirements for binding.
We also observed a similar discrepancy for
12-PGJ2, which differs
from PGJ2 only in the position of the double bond
in the 
side chain of the molecule.
12-PGJ2 has cytotoxic
effects on a variety of tumor cell lines (EC50
1 µM) (36). In the present study, we found that this
compound is approximately as potent as PGJ2 in
stimulating CD11b expression and actin polymerization
(EC50 25–30 nM). In contrast,
12-PGJ2 was reported to
have a Ki of 7100 nM in competing with
[3H]PGD2 for binding to
CRTH2-transfected cells, compared with a
Ki of 460 nM for
PGJ2 (15). Thus, the binding assay
using transfected K562 cells appears to be more sensitive to structural
changes in the side chain of PGD2 (with the
exception of 13,14-dihydro-15-oxo-PGD2) than
biological assays with eosinophils. However, it is important to bear in
mind that it is the response of peripheral eosinophils to
prostaglandins that is more relevant to the potential in vivo effects
of these compounds. In contrast to its stimulatory effects on
eosinophils, 12-PGJ2
does not stimulate DP1 receptor-mediated
activation of adenylyl cyclase in platelets (37).
15d-PGD2 is also a potent stimulator of
eosinophils, having the lowest EC50 (8 nM) for
stimulation of CD11b expression of all the compounds tested. Although
it has received much less attention than the cyclopentenone
prostaglandins 12-PGJ2
and 15d-PGJ2, it is a major product of
albumin-catalyzed degradation of PGD2, being
formed in amounts similar to those of
12-PGJ2 and considerably
greater than those of 15d-PGJ2 (32).
Therefore, if the dehydration pathway for PGD2 is
confirmed to be important physiologically,
15d-PGD2 may be an important proinflammatory
mediator. This compound has been reported to be 100 times less potent
than PGD2 in inhibiting platelet aggregation
(38), consistent with the requirement for a 15-hydroxyl
group for activation of the DP1 receptor. Thus
15d-PGD2, like 15d-PGJ2, is
a selective DP2 receptor agonist.
We have also examined the effects of other structural modifications of
PGD2 on eosinophil activation (Fig. 6). The high
potency of PGD3 (EC50 10
nM) in stimulating CD11b expression is consistent with the ability of
the DP2 receptor to recognize agonists with
substantially modified side chains. This is consistent with our
previous finding that 13,14-dihydro-15-oxo-PGD2
is a highly potent DP2 agonist (14).
In contrast, modification of the carboxyl side chain has a much greater
effect. Saturation of the 5 double bond
reduces potency by >10-fold, whereas reduction of the carboxyl group
to a hydroxyl group has a much more substantial effect. The 11-oxo
group of PGD2 is also clearly essential for a
potent biological response, because its reduction to a hydroxyl group,
as in 11-PGF2, reduces potency by
100-fold.
Both the DP1 and DP2
receptors are highly selective for PGD2 over all
of the other prostanoids formed directly from the endoperoxide
intermediate PGH2. However, the two receptors
differ markedly in their selectivities for metabolites/degradation
products of PGD2. PGJ2, the
initial dehydration product of PGD2, is
equipotent with PGD2 in stimulating the
DP1 receptor, but less potent in activating the
DP2 receptor, suggesting that the 9-hydroxyl
group is somewhat more important for recognition by the
DP2 receptor. However, alterations in the
side chain of PGD2 are much better tolerated by
the DP2 receptor. Metabolism of prostanoids by
15-hydroxyprostaglandin dehydrogenase normally results in termination
of their biological activities, but this is clearly not the case for
stimulation of the DP2 receptor. Migration of the
13 double bond to the 12-position, as in
12-PGJ2, dramatically
reduces activation through the DP1 receptor
(39) but has no effect on activation via the
DP2 receptor. Further dehydration, resulting in
loss of the 15-hydroxyl group appears, if anything, to slightly augment
DP2 receptor-mediated biological activity, as
15d-PGJ2 is more potent than
12-PGJ2 and
15d-PGD2 is at least as, if not more, potent than
PGD2. Thus, in contrast to the
DP1 receptor, neither metabolism of
PGD2 by the dehydrogenase pathway nor degradation
by the dehydration/isomerization pathway results in loss of ability to
activate eosinophils via the DP2 receptor. This
would suggest that activation of this receptor could be much more
prolonged than that of other prostanoid receptors, which would cease to
be stimulated after the rapid biological inactivation of their natural
ligands.
Although eicosanoids were initially considered to be primarily
proinflammatory mediators, it is becoming clear that their role in
inflammation is quite complex. Both PGD2 and
15d-PGJ2 have proinflammatory effects mediated by
the DP2 receptor. PGD2 also
has anti-inflammatory effects via DP1
receptors and can thereby inhibit the activation of inflammatory cells,
including neutrophils (40) and Langerhans cells
(41). However, the DP1 receptor also
appears to mediate proinflammatory effects, given that its deletion
resulted in reduced inflammation and airway hyperresponsiveness after
Ag challenge of sensitized knockout mice (42). Although
15d-PGJ2 does not activate the
DP1 receptor, relatively high concentrations of
this substance can inhibit macrophages and other cells by both PPAR
and non-receptor-mediated mechanisms (4, 6, 29). Like
PGD2, PGE2 also has both
pro- and anti-inflammatory effects in various models of
inflammation (43, 44, 45). In contrast, most of the
eicosanoids formed by the 5-lipoxygenase pathway, including leukotriene
B4, the cysteinyl-leukotrienes, and 5-oxo-ETE,
appear to have strictly proinflammatory effects (46), with
the exception of the lipoxins, which are highly potent
anti-inflammatory agents at nanomolar or subnanomolar
concentrations (47, 48).
In conclusion, we have shown that 15d-PGJ2 is a potent and selective DP2 receptor agonist that stimulates a variety of responses in eosinophils, including calcium mobilization, actin polymerization, and CD11b expression. 15d-PGD2 has similar effects on CD11b expression. In contrast to PGD2, 15d-PGJ2 has no effect on DP1 receptor-mediated activation of adenylyl cyclase. The proinflammatory effects of this compound occur much more rapidly and at far lower concentrations than its anti-inflammatory effects on macrophages and other cells, and this should be taken into consideration if such compounds are considered for development as anti-inflammatory agents. Thus 15d-PGJ2 can induce both pro- and anti-inflammatory responses, depending on the target cells, its concentration, and the time of exposure.
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2 Current address: Flow Cytometry Unit, Immunology Laboratory, Lyon-Sud University Hospital, 69495 Perre-Bénite, France.
3 Address correspondence and reprint requests to Dr. William S. Powell, Meakins-Christie Laboratories, Department of Medicine, McGill University, 3626 St. Urbain Street, Montreal, Quebec, Canada H2X 2P2. E-mail address: William. Powell{at}McGill.ca
4 Abbreviations used in this paper: 15d-PGJ2, 15-deoxy-12,14-PGJ2; 15d-PGD2, 15-deoxy-12,14-PGD2; PPAR, peroxisome proliferator-activated receptor-; 5-oxo-ETE, 5-oxo-6,8,11,14-eicosatetraenoic acid; CRTH2, chemoattractant receptor-homologous molecule expressed on Th2 cells.
Received for publication November 20, 2001. Accepted for publication February 1, 2002.
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Y. Kobayashi, S. Ueki, G. Mahemuti, T. Chiba, H. Oyamada, N. Saito, A. Kanda, H. Kayaba, and J. Chihara Physiological Levels of 15-Deoxy-{Delta}12,14-Prostaglandin J2 Prime Eotaxin-Induced Chemo |
) and 15d-PGJ2 (•) on calcium
mobilization in eosinophils. The values are expressed as percentages of
the maximal responses to PGD2 (649 ± 73 nM above
baseline) and are means of experiments on leukocytes from four
different donors.
; n = 6), 15d-PGJ2 (•, 
;
15dJ2; n = 6),
; 
; dD2; n = 6),
;
n = 10). B, Effects of other
prostaglandins: PGD2 (
