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B
Degradation Using a Peroxisome Proliferator-Activated Receptor-
-Independent Mechanism1
* Rayne Laboratory, Respiratory Medicine Unit, Medical Research Council Center for Inflammation Research, University of Edinburgh Medical School, Edinburgh, United Kingdom; and
Cell Biology Unit, GlaxoSmithKline, Stevenage, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) either delayed apoptosis or had
no effect, whereas PGD2 and its metabolite PGJ2
selectively induced eosinophil, but not neutrophil apoptosis. This
novel proapoptotic effect does not appear to be mediated via classical
PG receptor ligation or by elevation of intracellular cAMP or
Ca2+. Intriguingly, the sequential metabolites
12PGJ2 and 15-deoxy-12,
14-PGJ2 (15dPGJ2) induced
caspase-dependent apoptosis in both granulocytes, an effect that did
not involve de novo protein synthesis. Despite the fact that
12PGJ2 and 15dPGJ2 are
peroxisome proliferator-activated receptor-
(PPAR-) activators,
apoptosis was not mimicked by synthetic PPAR- and PPAR- ligands
or blocked by an irreversible PPAR- antagonist. Furthermore,
12PGJ2 and 15dPGJ2 inhibited
LPS-induced I
B degradation and subsequent inhibition of
neutrophil apoptosis, suggesting that apoptosis is mediated via
PPAR--independent inhibition of NF-B activation. In addition, we
show that TNF--mediated loss of cytoplasmic IB in eosinophils
is inhibited by 15dPGJ2 in a concentration-dependent
manner. The selective induction of eosinophil apoptosis by
PGD2 and PGJ2 may help define novel therapeutic
pathways in diseases in which it would be desirable to specifically
remove eosinophils but retain neutrophils for antibacterial host
defense. The powerful proapoptotic effects of
12PGJ2 and 15dPGJ2 in both
granulocyte types suggest that these natural products control the
longevity of key inflammatory cells and may be relevant to
understanding the control and resolution of
inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are released. At sites of inflammation, granulocytes
are exposed to a plethora of different inflammatory mediators, most of
which not only influence granulocyte responsiveness but also prolong
their functional longevity by delaying apoptosis (6). A
notable exception to this rule is the acceleration of apoptosis by the
proinflammatory mediator TNF- after exposure of neutrophils to this
cytokine (7), particularly in the presence of NF-B
inhibitors (8).
PGs, a group of C20 carboxylic acids
containing a cyclopentane ring, have been unequivocally shown to play a
prominent role in the inflammatory process; however, their effects on
granulocyte apoptosis have not been fully examined. Prostanoid
formation occurs when cyclooxygenase oxygenates arachidonate converting
it to PGG2, which is then reduced to
PGH2. PGH2, in turn, is
converted to five primary active metabolites,
PGD2, PGE2,
PGF2, PGI2, or
thromboxane A2 via distinct synthases
(9, 10, 11). PGE1 and
PGE2 can be metabolized to
PGA1 and PGA2
(12), whereas PGD2 sequentially
forms metabolites of the J series,
9-deoxy-9PGD2
(PGJ2),
12PGJ2, and
15-deoxy-12,14PGJ2
(15dPGJ2)3
(13). Both neutrophils and eosinophils synthesize, to
varying degrees, some of these PGs and are capable of responding to
specific PGs by interaction with their cognate classical
seven-transmembrane prostanoid receptors (14, 15).
Interestingly, PGD2, which is produced by both
neutrophils and eosinophils (16, 17), is also generated by
Ag-stimulated human Th2 cells (18) and is the major
arachidonic acid metabolite released from activated human mast cells
(19). Thus, this PG is considered to be an important
mediator of allergic disorders such as allergic rhinitis and is present
after Ag challenge in the airways of patients with asthma
(20). Although PGD2 binds
preferentially to the PGD2 receptor (DP
receptor), it can also bind to other PG receptors, thereby triggering
several different signaling pathways. For example, binding to the
DP receptor increases intracellular cAMP and/or cytosolic free
Ca2+ concentration
([Ca2+]i), whereas PGE
receptor (EP receptor) 1, PGF2 receptor (FP
receptor), or thromboxane A2 receptor (TP
receptor) ligation increases
[Ca2+]i but has no direct
effect on cAMP levels. Prostacyclin receptor (IP receptor) activation
also increases intracellular cAMP (9, 21, 22, 23). The
metabolites of PGD2,
12PGJ2 and
15dPGJ2, have been shown to activate
intracellular peroxisome proliferator-activated receptors (PPARs) which
are transducer proteins belonging to the steroid/thyroid/retinoid
receptor superfamily that regulate target genes by binding to PPAR
response elements (24, 25). Three isoforms of PPAR
(PPAR-, PPAR-
, and PPAR-) are present in human cells
(26, 27, 28). PPAR- is primarily expressed in tissues with
high fatty acid catabolism; PPAR- is expressed in adipose tissue,
adrenal gland, spleen, and several myeloid cell lines; and PPAR- is
highly expressed in heart, kidney, and intestine
(29, 30, 31).
In this study, we demonstrate differential effects of
PGD2 on granulocyte apoptosis; selectively
inducing eosinophil but not neutrophil apoptosis. This intriguing
result prompted us to examine the actions of this PG and its
metabolites more closely. We show that the PGD2
metabolites, 12PGJ2 and
15dPGJ2, are powerful inducers of
caspase-dependent granulocyte apoptosis. These data could not be
mimicked using synthetic PPAR- agonists such as the
thiazolidinediones BRL49653 and ciglitazone (32), nor
could the PPAR- antagonist GW9662 (33) prevent
induction of cell death by 15dPGJ2. PGs of the J
series also activate PPAR-; however, a synthetic ligand to this
isoform, pirinixic acid (WY-14643), had no effect on granulocyte
apoptosis. We also rule out a significant proapoptotic role for the
other classical cell surface PG receptors in both neutrophils and
eosinophils. We have recently shown that NF-B plays a critical role
in the regulation of granulocyte apoptosis where specific inhibition of
NF-B can directly induce granulocyte apoptosis and can enhance
apoptosis induced by TNF- and block the delayed apoptosis induced by
LPS (8). In view of the suggestion that PPAR- ligands
may inhibit NF-B activation, we examine whether the effects of
12PGJ2 and
15dPGJ2 are mediated via inhibition of this
transcription factor. We demonstrate in the neutrophil that
PGD2 metabolites inhibit LPS-induced degradation
of IB (the inhibitory subunit of NF-B) and block LPS-mediated
inhibition of apoptosis. Furthermore, TNF--induced IB
breakdown in the eosinophil is also inhibited by
PGD2 metabolites. We conclude that the PPAR-
ligands 12PGJ2 and
15dPGJ2 influence granulocyte apoptosis by
interfering with the prosurvival NF-B pathway, an effect that is
independent of PPAR- ligation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils and eosinophils were isolated from the peripheral blood of normal donors by dextran sedimentation followed by centrifugation through discontinuous Percoll gradients (Amersham Pharmacia Biotech, Little Chalfont, U.K.) (34, 35). Only neutrophil preparations with a cell purity of >98% were used. Eosinophils were separated from contaminating neutrophils using immunomagnetic separation with sheep anti-mouse IgG-Dynabeads (Dynabeads M-450; Dynal, Merseyside, U.K.) coated with the murine anti-neutrophil Ab 3G8 (anti-CD16; a gift from Dr. J. Unkeless, Mount Sinai Medical School, New York, NY). Cells were mixed with washed Ab-coated magnetic beads at a bead-neutrophil ratio of 3:1 on a rotary mixer at 4°C for 20 min, and the beads were removed magnetically by two 3-min stationary magnetic contacts (Dynal Magnetic Particle Concentrator, MPC-1) to yield an eosinophil population of >98% purity. After purification, cells were washed twice in PBS without Ca2+ and Mg2+ before resuspending in IMDM (Life Technologies, Paisley, U.K.) supplemented with 10% autologous serum (unless otherwise stated in figures). Both cell types were cultured in flat-bottom Falcon flexible well plates (BD Biosciences, Oxford, U.K.) at 37°C in a 5% CO2 atmosphere; or in 2 ml round-bottom Eppendorf tubes in a shaking water bath at 37°C, with neutrophils 5 x 106/ml and eosinophils 2 x 106/ml. Cells were cultured in the absence or presence of test agents as described in the figures. All experiments were performed at least three times in triplicate.
Assessment of granulocyte apoptosis
Morphology. Cells were cytocentrifuged, fixed in methanol, stained with DiffQuik (Gamidor, Abingdon, U.K.) and counted using oil immersion microscopy (x100 objective) to determine the proportion of cells with distinctive apoptotic morphology (8). At least 500 cells were counted per slide with the observer blinded to the experimental conditions. The results were expressed as the mean percent apoptosis ± SEM
Annexin V binding and propidium iodide staining. A separate and independent assessment of apoptosis was performed by flow cytometry using FITC-labeled recombinant human annexin V that binds to phosphatidylserine exposed on the surface of apoptotic cells and propidium iodide as an index of loss of cell membrane integrity (8). Stock annexin V (Bender MedSystems, Vienna, Austria) was diluted 1/200 with binding buffer and then added (25 µl) to 75 µl of the recovered cell samples. After a 10-min incubation at 4°C, these samples were treated with propidium iodide (final concentration, 10 µg/ml) for 2 min before flow cytometric analysis using an EPICS XL2.
DNA fragmentation assay. Cells were lysed, DNA was extracted and run on an agarose gel containing ethidium bromide, and DNA fragmentation (laddering) was visualized as described (36).
Assessment of membrane integrity and cell recovery
To ensure that the cell death observed was due solely to apoptosis, the membrane integrity of treated and untreated cells was assessed using the vital dye trypan blue. In addition, cells were counted at the start of culture and at the end of the indicated period. Under all conditions and treatments used, there was no loss of cell membrane integrity, and cell loss was minimal.
Measurement of [Ca2+]i
Freshly isolated granulocytes were washed (three times) in HBSS (Ca2+ and Mg2+ free) before being resuspended at 107/ml in HBSS (Ca2+ and Mg2+ free), for incubation with fura 2-acetoxymethyl ester (final concentration, 2 µM) for 30 min at 37°C (37, 38). The cells were then washed (twice) to remove fura 2-acetoxymethyl ester and left in HBSS (Ca2+ and Mg2+ free) for a further 10 min for optimal deesterification, before finally resuspending the granulocytes at 2 x 106/ml in HBSS (containing Ca2+ and Mg2+). Changes in fluorescence upon agonist addition were determined using a PerkinElmer (Wellesley, MA) LS50B fluorometer, with dual wavelength excitation (340 and 380 nm) and emission at 510 nm, fitted with a thermostated cuvette compartment and stirring attachment, to ensure complete mixing of reagents. [Ca2+]i was calibrated as previously described (37, 38).
Western blotting for IB
Cell samples (5 x 106/ml) were
incubated in a shaking water bath at 37°C with the agents of interest
as described in the figure legends. After treatment, cells were
immediately placed on ice, and all lysates were prepared at 4°C. To
minimize problems with proteolysis, lysates were prepared by methods
normally used for EMSA preparations (8). Lysates were run
on a 9% SDS gel and, after transfer, blocked by 5% milk protein
before an overnight incubation with primary IB Ab (New England
Biolabs, Beverly, MA) diluted 1/500. After washing, blots were
incubated with HRP-conjugated anti-biotin Ab diluted at 1/2500 and
developed using standard ECL reagents (Amersham, Arlington
Heights, IL).
Other materials
Further specific materials were obtained as follows: LPS
(Escherichia coli 0127:B8) (Sigma, Poole, U.K.);
benzylocarbonylvalylalanylaspartyl fluoromethylketone (z-VAD-fmk;
Bachem U.K., Saffron Walden, U.K.);
12PGJ2,
15dPGJ2, PGJ2,
PGD2, PGA2,
PGA1, U46619, ciglitazone, and WY-14643
(Biomol; Affinity Research Products, Mamhead, U.K.); recombinant human
TNF- (R&D Systems, Abingdon, U.K.). GW9662 was a gift from T.
Willson (Glaxo Wellcome, Research Triangle Park, NC), and BRL49653 was
a gift from K. Chaterjee (University of Cambridge, Cambridge, U.K.).
All other reagents were obtained from Sigma U.K. and were of the
highest purity.
Statistical analysis
The results are expressed as mean ± SEM of the number (n) of independent experiments each using cells from separate donors with each treatment performed in triplicate. Statistical analysis was performed by ANOVA with comparisons between groups made using the Newman-Kuels procedure. Differences were considered significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of PGs on granulocyte apoptosis are shown in Table I
. PGE2 and
11-deoxyPGE1 delayed neutrophil apoptosis as did
the dehydration product of PGE1,
PGA1. PGA2 had no
significant effect on apoptosis in either cell type, whereas
PGF2 decreased the rate of constitutive
apoptosis in both neutrophils and eosinophils. The thromboxane
A2 mimetic U46619 had no direct effect on cell
death. Only PGD2 increased the constitutive rate
of eosinophil apoptosis
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PGD2 did not enhance the rate of
constitutive neutrophil apoptosis at 20 h (Fig. 1A) or at earlier time points
(e.g., 2, 3, 4, or 6 h) when basal levels of apoptosis are much
lower (data not shown). However, PGD2
significantly increased the rate of eosinophil apoptosis, after both 20
and 40 h in culture, with levels of apoptosis in treated cells
being 
4 times higher than those in control untreated cells (Fig. 1B). As a further control in these experiments, when
neutrophils were cultured at the same density as eosinophils (2 x
106/ml), PGD2 still did not
induce neutrophil apoptosis (control, 85.9 ± 4.6;
PGD2 (10 µM), 87.9 ± 1.3
(n = 3, each experiment performed in triplicate)). To
directly demonstrate the efficacy of PGD2 on
eosinophils, apoptosis was induced by PGD2 and
compared with apoptosis induced by maximal concentrations of
dexamethasone (1 µM), an established accelerator of eosinophil
apoptosis (39), during concurrent experiments on cells
from the same donor. PGD2 (10 µM) induced
apoptosis to a greater extent than dexamethasone at both time points
(20 and 40 h) tested (Fig. 1B). In addition, this
increase in apoptosis appeared to be restricted to eosinophils because,
as shown in Table II, further studies
using other immune cells determined that PGD2 did
not induce cell death in Jurkat T lymphocytes or human PBL. Only
the J series metabolite
12PGJ2 substantially
induced apoptosis in the Jurkat cell line. Furthermore,
PGD2 did not influence the constitutive rates of
apoptosis in either peripheral blood monocytes or monocyte-derived
macrophages after 20 h of culture (data not shown).
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PGD2 can be metabolized to biologically
active breakdown products. Therefore, to further investigate the
possible mechanism of PGD2-mediated acceleration
of eosinophil apoptosis, we used a stable
PGD2 mimetic
(5Z,13E)-(9R,11R,15S)-9-chloro-15-hydroxy-16,17,18,19,20-pentano-3-oxa-5,13-prostadienoic
acid (ZK 118.182)) (40). This compound allowed
investigation of the consequences of activating the DP receptor without
the production of active PGD2 metabolites. In
contrast to PGD2, ZK 118.182 significantly
inhibited apoptosis in neutrophils (Fig. 1
C) while
increasing eosinophil apoptosis at 40 h to a small but significant
extent; however, the effect was minimal in comparison to levels of cell
death induced by PGD2 (Fig. 1D). Thus,
these disparate results suggest that ligation of the DP receptor is
unlikely to account for the proapoptotic effects observed with
PGD2.
The increase in eosinophil apoptosis is not explained by binding to other surface PG receptors
PGD2 at the concentrations used in this
study, has the ability to bind other PG receptors, namely, the EP, FP,
IP, and TP receptors (9). We therefore examined the
effects of agonist binding to these receptors to determine whether the
increase in apoptosis observed with PGD2 could be
reproduced. The FP receptor binds PGF2, which
inhibits several functional activities in human neutrophils (41, 42); however, little is known of its effects on eosinophils. As
shown in Table I, PGF2, significantly
increased neutrophil survival, and by 40 h of culture, eosinophil
apoptosis was almost 50% of control values. Had
PGD2 been acting via the FP receptor, apoptosis
should have been inhibited in both eosinophils and neutrophils.
The use of U-46619, a thromboxane A2 receptor
agonist, demonstrated that activation of putative TP receptors on
granulocytes had no significant effect on apoptosis in either
neutrophils or eosinophils (Table I), again suggesting that
PGD2 is not acting via this receptor. Activation
of the DP, IP, EP2, EP3,
and EP4 receptors has been demonstrated to
increase intracellular cAMP levels in many cell types including
granulocytes (9). To mimic elevation of cAMP, we used the
stable, cell-permeable analog of cAMP, dbcAMP (dbcAMP), and show that
this compound powerfully delays apoptosis in both neutrophilic (Fig. 1E) and eosinophilic (Fig. 1F) granulocytes.
Therefore, elevation of cAMP by ligation of certain PG receptors cannot
explain the dramatic augmentation of apoptosis induced by
PGD2.
Ligation of the EP1 receptor has no reported
effects on intracellular cAMP levels, but it does increase
[Ca2+]i in some cell
systems. To investigate directly whether PGD2 can
influence [Ca2+]i in
granulocytes, we performed studies using fura 2-loaded cells.
Neutrophils (Fig. 2) and eosinophils
(Fig. 3) respond to stimuli such as
platelet-activating factor and leukotriene B4,
which act on specific G protein-coupled receptors by a rapid and
reversible elevation of
[Ca2+]i. We found that
PGD2 at 10 µM induced a rapid and reversible
elevation of [Ca2+]i in
eosinophils but not in neutrophils. These differential effects were
mirrored by the PGD2 sequential metabolites
PGJ2, 12PGJ2, and
15dPGJ2. Interestingly, in eosinophils there was
both homologous and heterologous desensitization between the different
PGD2 metabolites and indeed
PGD2 itself (Fig. 3 and data not shown). It
has been recently reported that PGD2
metabolites are potent selective activators of human eosinophils
inducing calcium mobilization, actin polymerization, and CD11b
expression by interacting with DP2
(43). We have previously reported that increasing levels
of [Ca2+]i inhibits
neutrophil apoptosis while accelerating this process in
eosinophils (44, 45). However, use of calcium ionophores
and other pharmacological agents (e.g., thapsigargin) that elevate
[Ca2+]i also cause
degranulation in the eosinophil and cause necrosis rather than
apoptosis by 40 h. Neither degranulation nor necrosis was observed
in either cell type when treated with PGD2 at any
time point examined. This suggests that increases in
[Ca2+]i were not
responsible for the proapoptotic effect of this PG. Therefore, the
proapoptotic effect of PGD2 on eosinophils does
not appear to be mediated via any known classical cell surface PG
receptor or by increasing intracellular cAMP or
[Ca2+]i.
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12PGJ2, and 15dPGJ2 induce
granulocyte apoptosis
PGJ2, an active PGD2
metabolite, is also capable of selectively triggering the DP receptor
(13, 22). As demonstrated in Fig. 4A, PGJ2
produced results similar to those of PGD2 in
neutrophils, causing no significant change in the rate of constitutive
apoptosis at 20 h. However, PGJ2
significantly increased eosinophil apoptosis at 20 and 40 h, but
this increase was markedly less than that induced by the parent
compound, PGD2, when used at equimolar
concentrations (Fig. 4B).
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12PGJ2 and
15dPGJ2 could increase levels of constitutive
granulocyte apoptosis. Fig. 4, C and D,
illustrates that these metabolites are efficacious inducers of
programmed cell death in both cell types causing increases in
eosinophil apoptosis comparable with those observed with
PGD2. However,
12PGJ2 and
15dPGJ2 also had powerful proapoptotic effects on
neutrophils, increasing control rates from 66 to 90% at 20 h.
These data support the possibility that neutrophils and eosinophils may
metabolize PGD2 differently. The proportion of
cells exhibiting classical pyknotic nuclei of apoptotic neutrophils and
eosinophils increases markedly in cells treated with
15dPGJ2 and
12PGJ2 even at the early
time points of 3–4 h (Fig. 6 and data not shown). When DNA was
extracted from granulocytes treated with PGD2
metabolites for short period of culture (e.g. 4 h) and run on an
agarose gel, a typical DNA ladder pattern was evident whereas there was
no DNA "laddering" observed in control cells (data not shown).
Moreover, the proportion of granulocytes binding FITC-labeled annexin V
(indicative of cell surface changes associated with apoptosis) was
increased when cells were treated with PGD2 and
its metabolites (Fig. 5). A smaller
increase in propidium iodide staining was also observed with the PGs.
We believe that this increase is likely due to secondary necrosis, in
that failure of apoptotic eosinophil clearance results in these cells
quickly undergoing necrosis and it is possible that actual processing
of eosinophils during the double staining technique can contribute to
an abnormally high level of propidium iodide staining. This latter
point is further supported by the observation that we failed to observe
any significant uptake of the vital dye trypan blue when the cells are
treated with these PGs.
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12PGJ2 and 15dPGJ2 is
independent of synthesis of a death protein but dependent on activation
of caspases
To investigate whether the proapoptotic effect of
12PGJ2 and
15dPGJ2 requires synthesis of protein(s) (e.g., a
death-inducing protein), cells were cultured with a protein synthesis
inhibitor, cycloheximide. Although cycloheximide itself is a potent
accelerator of granulocyte apoptosis at 20 h (46),
experiments were performed at a 3-h time point at which
cycloheximide alone has little influence on this process.
Interestingly, the proapoptotic effect of 15dPGJ2
was apparent even at this short incubation period (Fig. 6A). Cycloheximide did not
inhibit the induction of cell death but rather produced an additive
increase in the levels of apoptosis produced by this metabolite. The
increase in apoptosis induced by 15dPGJ2 was
attenuated when cells were cotreated with the pan-caspase inhibitor
z-VAD-fmk but not by the vehicle control (0.02% DMSO) (Fig. 6B). z-VAD-fmk also blocked the induction of eosinophil
apoptosis by both metabolites (data not shown). Taken together, these
results clearly demonstrate that the proapoptotic nature of the
PGD2 metabolites likely does not result from the
synthesis of a death-inducing protein as has been suggested for the
proapoptotic effect observed in other cells (47) but is,
however, critically dependent on the activation of caspases.
Synthetic PPAR- activators do not induce granulocyte apoptosis
Because 12PGJ2 and
15dPGJ2 are known ligands for PPAR-, we
investigated the effects of other known PPAR- activators on
granulocyte apoptosis. The synthetic PPAR- ligands, BRL49653 and
ciglitazone, used at concentrations ranging from 1 nM to 100 µM, did
not affect the rate of constitutive apoptosis in either cell type (Fig. 6C). Moreover, GW9662, an irreversible PPAR- antagonist
(33), did not prevent the induction of apoptosis induced
by 15dPGJ2 (Fig. 6D). Because some
reports indicate that PGD2 metabolites can also activate
PPAR- at higher concentrations (48), we also incubated
granulocytes with pirinixic acid (WY-14643), a PPAR- agonist. This
compound did not influence apoptosis in either cell type over the wide
range of concentrations examined (1–300 µM) (Fig. 6C).
Our data strongly suggest the powerful induction of apoptosis by
12PGJ2 and
15dPGJ2 is not mediated via PPAR- or PPAR-
activation.
12PGJ2 and 15dPGJ2 may
induce apoptosis in granulocytes by inhibition of IB proteolysis
Recent work has demonstrated that
15dPGJ2 can inhibit activation of NF-B
(49), a transcription factor that we have
recently shown to be critically involved in regulating granulocyte
survival (8). Activation of NF-B by LPS may explain the
powerful inhibition of granulocyte apoptosis by this proinflammatory
bacterial product. As shown in Fig. 7A,
15dPGJ2 completely prevented LPS-induced delay of
apoptosis, indicating that the cyclopentenone PGs may be inhibiting
NF-B activation. We therefore examined this possibility directly by
Western blot analysis of IB degradation. As shown in Fig. 7B, 15dPGJ2 and
12PGJ2 did not cause
IB degradation, but both metabolites inhibited the proteolytic
breakdown of IB in response to LPS stimulation. Further studies
determined that neither PGD2 nor
PGJ2, which do not induce apoptosis in
neutrophils, could prevent IB degradation in these cells (data
not shown). It is therefore apparent that the proapoptotic effect of
12PGJ2 and
15dPGJ2 may be mediated by inhibition of NF-B
activation. This is further supported by the demonstration that the
inhibition of LPS by
12PGJ2 in neutrophils
(Fig. 8A) and TNF--mediated
IB breakdown by 15dPGJ2 in eosinophils
(Fig. 8B) is concentration dependent.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, B and C)
rather than induce apoptosis. PGD2 causes a rapid
transient increase in
[Ca2+]i in human
eosinophils (Ref. 23 ; see Fig. 3) and primes eosinophils
for enhanced release of LTC4 in response to the
calcium ionophore A23187 (23). Although fura 2-loaded
eosinophils exposed to PGD2 and its metabolites
exhibit a rapid transient elevation of
[Ca2+]i (Fig. 3), we
believe that an increase in
[Ca2+]i is not
responsible for the PGD2-mediated eosinophil
proapoptotic effect because: 1) fura 2-loaded neutrophils do not
exhibit a Ca2+ transient when exposed to the
PGD2 metabolites although these metabolites
induce apoptosis in neutrophils; 2) elevation of eosinophil
[Ca2+]i by
pharmacological agents such as A23187 and thapsigargin causes
degranulation and necrosis at time points when
PGD2 induces apoptosis; 3) receptor-directed
stimuli such as IL-5 that increase eosinophil
[Ca2+]i inhibit rather
than induce apoptosis (52, 53); 4)
PGD2 and PGJ2 exhibit
similar Ca2+ responses whereas
PGD2 is a more powerful inducer of eosinophil
apoptosis; and 5) chemotactic agents such as eotaxin that cause an
increase in [Ca2+]i
(54) do not induce eosinophil apoptosis (C. Ward and A. G.
Rossi, unpublished observations). A newly described receptor
CRTH2/DP2 has been found to mediate
[Ca2+]i flux and
responsiveness to PGD2 (55, 56) and
indeed Ca2+ mobilization in response to
PGD2 metabolites (43) in
eosinophils; however, it is unlikely that
[Ca2+]i flux, and hence
this receptor, are involved in the proapoptotic effect of the
PGD2 metabolites. Furthermore, this
receptor binds PGE2 and
PGF2 with similar affinities to
15dPGJ2, and as illustrated in Table I both these
PGs inhibit rather than induce eosinophil apoptosis. This receptor is
not present on neutrophils; therefore, it cannot be responsible for the
proapoptotic effects of
12PGJ2 and
15dPGJ2 observed in these cells.
PGD2 shows a promiscuous PG receptor binding
profile (57, 58); and in the absence of direct evidence
for the expression of IP, FP, or TP receptors on granulocytes, it is
possible that other receptors (e.g., EP) are mediating the
proapoptotic activities of PGD2. However, the
results obtained using a wide variety of PGs or PG analogs demonstrate
that the proapoptotic effect of PGD2 could not be
reproduced through triggering of any of the classical cell surface PG
receptors for which it has a known affinity (Table I; Figs. 1 and 2).
It is unlikely that the DP receptor is involved in mediating the
proapoptotic effect in eosinophils because the stable
PGD2 mimetic ZK118.182 induced minimal increases
in eosinophil apoptosis and inhibited this process in the neutrophil. A
plausible explanation for the observed differential proapoptotic
effects of PGD2 could be that eosinophil and
neutrophil granulocytes metabolize PGD2
differently, resulting in distinct functional outcomes. It is possible
that neutrophils fail to metabolize PGJ2 to the
12PGJ2 and
15dPGJ2 sequential metabolites and are thus
protected from the proapoptotic effects of these products. It is also
possible that eosinophils readily metabolize PGD2
into the active PGD2 proapoptotic metabolites or
alternatively that eosinophil uptake of PGD2 and
PGJ2 may differ from the process in neutrophils.
These intriguing possibilities await further investigation.
Interestingly, a very recent study has specifically and conclusively
shown, using a newly described mAb raised against
15dPGJ2, that this metabolite is present in the
cytoplasm of macrophages in human atherosclerotic plaques
(59). Thus, these authors have set a precedent for the
detection of PGD2 metabolites in vivo and
specifically demonstrated that this metabolite can be generated during
an inflammatory response in an important human disease. Although we and
many others have used low micromolar concentrations of
PGD2 and its metabolites, we believe that such
levels could be achieved in vivo at their site of action. There is
convincing evidence demonstrating that certain PGs can reach
concentrations in the micromolar range at sites of acute inflammation
(60) and in certain biological fluids, e.g., seminal fluid
(61). Actual concentrations of PGD2
metabolites at relevant sites in vivo await confirmation. Because the
metabolites can be produced intracellularly and extracellularly it
would be difficult to estimate or assess actual concentrations at their
target sites.
The mechanisms by which
12PGJ2 and
15dPGJ2 induce granulocyte apoptosis involve
caspase activation because death was inhibited by z-VAD-fmk. Although
12PGJ2 and
15dPGJ2 may have their primary targets in the
nucleus, where they regulate the expression of specific genes, e.g.,
via binding to PPAR-, we found that other synthetic agonists to
PPAR- and PPAR- could not mimic the proapoptotic effects of
12PGJ2 and
15dPGJ2. Although the compound WY-14643 is an
effective activator of PPAR-, PPAR- is also activated by this
agent at concentrations of 100 µM (32). Despite the use
of WY-14643 concentrations up to 300 µM (Fig. 3C),
apoptosis was not affected in either cell type. In addition, the
thiazolidinediones BRL49653 and ciglitazone, potent activators of
PPAR-, did not induce apoptosis in either neutrophils or eosinophils
despite the use of concentrations as high as 100 µM. Moreover, when
PPAR- was blocked using the irreversible antagonist, GW9662
(32), the induction of apoptosis mediated by either of the
PPAR- ligands was unaffected despite the use of concentrations in
excess of those known to block PPAR- activation. Taken together,
these data strongly indicate that the proapoptotic effects of
12PGJ2 and 15dPGJ on
granulocytes signal independently of PPAR- (or PPAR-) and point
to the existence of another mechanism. Evidence supporting this
possibility has been reported by Thieringer et al. (62),
who showed that 15dPGJ2 but not other PPAR-
agonists could inhibit cytokine production in primary human
monocyte-derived macrophages and RAW 264.7 cells. However, the
signaling mechanisms of cyclopentenone PGs appear to be cell type
specific (49). For example, in activated macrophages,
15dPGJ2 as well as BRL49653 antagonize AP-1,
STAT, and NF-B in a PPAR--dependent manner (63),
whereas in human monocytes there is no inhibitory effect of PPAR-
agonists on the induced expression of TNF- and IL-6, products that
are controlled by these transcription factors (64). In
human monocyte-derived macrophages, both BRL49653 and
15dPGJ2 induced apoptosis (PPAR--dependent)
(65); and in endothelial cells, both
15dPGJ2 and ciglitazone caused cell death via a
caspase and PPAR--dependent mechanism (66). These
putative PPAR- ligands can therefore act in some cells in a
PPAR--independent manner. Studies in human neutrophils have shown
that 15dPGJ2 inhibits the
2 integrin-dependent respiratory burst via a
PPAR--independent pathway and also suggest the presence of an as yet
unidentified receptor (67). Data obtained suggested that
such a receptor may act via increases in cytosolic cAMP. Our data,
however, indicate that such a mechanism could not be responsible for
the proapoptotic effect because an elevation of cAMP strongly inhibits
granulocyte apoptosis (Fig. 1, E and F and Refs.
68 and 69).
Because we have recently shown that activation of an inducible form of
NF-B is crucial to granulocyte survival (8), the
reported inhibition of this transcription factor by these metabolites
(49, 63, 70) made an attractive hypothesis to explain the
increases in cell death observed in our studies. Thus, we examined the
effects of 12PGJ2 and
15dPGJ2 on the activation of NF-B in
granulocytes and found that both metabolites could inhibit LPS-induced
degradation of IB in neutrophils with concentrations reported in
other studies (49, 63, 70). Interestingly, we show for the
first time that TNF--mediated loss of cytoplasmic IB in
eosinophils is also inhibited, in a concentration-dependent manner, by
15dPGJ2. Thus, we have shown in granulocytes that
this inhibition was concentration dependent and correlated well with
the proapoptotic effects observed. Recently, it has been demonstrated
that the cyclopentenone PG, PGA1, inhibits IB
kinase (IKK) activity in Jurkat, HeLa, and COS cells transfected with
IKK. Moreover, in HeLa cells, which do not express PPAR-,
15dPGJ2 inhibits IKK and NF-B activation by
TNF- (70). This group shows that A- and J-type PGs
inhibited IKK activity and thus that a reactive -unsaturated
carbonyl group in the cyclopentane ring was critical for IKK inhibition
(70). However, as shown in Table I, neither
PGA1 nor PGA2, both of
which contain this reactive carbonyl group (48, 70),
induced apoptosis in neutrophils; indeed by 20 h,
PGA1 had produced a small but significant
decrease in neutrophil programmed cell death. Because we have
previously shown that NF-B activation is crucial for survival in
these cells (8), these data also suggest a degree of
specificity in the interaction of cells with these PGs and that the
effects of PGs on NF-B may be dependent on cell type.
Knowledge of the mechanisms by which these metabolites exert their
proapoptotic effects may be central to understanding why such products
influence inflammation. For example, 15dPGJ2
suppresses adjuvant-induced arthritis in the rat (71),
whereas in a rat model of pleurisy, increased levels of
15dPGJ2 and PGD2 have been
shown to correlate with the resolution of inflammation
(72). PPAR- ligands also affect the acquired immune
response; e.g., ciglitazone and 15dPGJ2 inhibit
helper T cell responses by inhibiting IL-2 secretion (73).
However, whether this phenomenon is concurrent with apoptosis and
clearance of inflammatory cells from the respiratory system, is
currently unknown. Our results depicting PGD2 as
a powerful selective inducer of eosinophil apoptosis may have
implications where induction of apoptosis of a specific inflammatory
cell type may be of importance in the control of eosinophilic type
inflammation. The mechanism involved is likely to depend on the
metabolism of this parent prostanoid to products such as
PGJ2 and the PPAR- ligands
12PGJ2 and
15dPGJ2, which also induce eosinophil and
neutrophil apoptosis. However, this proapoptotic effect is not mediated
by activation of PPAR- or PPAR-. Interestingly, cyclopentenone
PGs have been shown to bind irreversibly to nascent proteins in the
endoplasmic reticulum (74). In granulocytes, this
mechanism could interfere with the function of survival proteins needed
to prevent activation of the apoptotic pathway, a possibility that is
currently under investigation.
In conclusion, we have shown for the first time that
PGD2 is a powerful, selective accelerator of
eosinophil apoptosis, the effects being more significant that those
described previously for corticosteroids (50). A
preliminary investigation of the underlying mechanisms have shown that
its sequential metabolite PGJ2 also induces
eosinophil cell death but that the sequential natural metabolites
12PGJ2 and
15dPGJ2, which inhibit the degradation of
IB in granulocytes, also accelerate apoptosis in both neutrophils
and eosinophils. The proapoptotic effects of these cyclopentenone PGs
are caspase dependent but do not involve ligation of PPAR- or
PPAR- receptors. Our experiments suggest that the mechanism involves
inhibition of NF-B activation, a central event in the control of
granulocyte apoptosis (8), and thus these metabolites may
fundamentally influence the resolution phase of inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Adriano G. Rossi, Rayne Laboratory, Respiratory Medicine Unit, Medical Research Council Center for Inflammation Research, University of Edinburgh Medical School, Teviot Place, Edinburgh, EH8 9AG U.K. E-mail address: a.g.rossi{at}ed.ac.uk
3 Abbreviations used in this paper: 15dPGJ2, 15-deoxy-12, 14-PGJ2; [Ca2+]i, cytosolic free Ca2+ concentration; IKK, IB kinase; PGJ2, 9-deoxy-9-PGD2; DP receptor, PGD2 receptor; IP receptor, prostacyclin receptor; TP receptor, thromboxane A2 receptor; FP receptor, PGF2 receptor; EP receptor, PGE receptor; PPAR, peroxisome proliferator-activated receptor; z-VAD-fmk, benzylocarbonylvalylalanylaspartyl fluoromethylketone; dbcAMP, dibutyryl cAMP.
Received for publication December 26, 2001. Accepted for publication April 17, 2002.
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is vehicle control-treated
cells. A, PGD2 (10 µM)-treated cells
(
); B, dexamethasone (1 µM)
(
),
PGD2 (10 µM) (
