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J. Biol. Chem., Vol. 277, Issue 29, 26012-26020, July 19, 2002
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From the
Received for publication, February 22, 2002, and in revised form, April 5, 2002
We investigated the actions of a panel of
nonsteroidal anti-inflammatory drugs on eosinophils, basophils,
neutrophils, and monocytes. Indomethacin alone was a potent and
selective inducer of eosinophil and basophil shape change. In
eosinophils, indomethacin induced chemotaxis, CD11b up-regulation,
respiratory burst, and L-selectin shedding but did not cause
up-regulation of CD63 expression. Pretreatment of eosinophils with
indomethacin also enhanced subsequent eosinophil shape change induced
by eotaxin, although treatment with higher concentrations of
indomethacin resulted in a decrease in the expression of the major
eosinophil chemokine receptor, CCR3. Indomethacin activities and cell
selectivity closely resembled those of prostaglandin
D2 (PGD2). Eosinophil shape change in
response to eotaxin was inhibited by pertussis toxin, but indomethacin- and PGD2-induced shape change responses were not. Treatment
of eosinophils with specific inhibitors of phospholipase C (U-73122), phosphatidylinositol 3-kinase (LY-294002), and p38 mitogen-activated protein kinase (SB-202190) revealed roles for these pathways in indomethacin signaling. Indomethacin and its analogues may therefore provide a structural basis from which selective PGD2
receptor small molecule antagonists may be designed and which may have utility in the treatment of allergic inflammatory disease.
Eosinophils and basophils are important effector cells in allergic
diseases such as asthma and eczema (1). Many groups have examined the
mechanisms initiating and regulating the responsiveness and activation
of these cells in the context of allergic disease (1-3). Several
agonists mediate eosinophil chemotaxis in vitro and
recruitment in vivo. Foremost among these are the
chemokines, in particular those acting via CCR3 such as eotaxin/CCL11
(4-9), eotaxin-2/CCL24 (10-12), eotaxin-3/CCL26 (13), and MCP-4/CCL13 (14-16). Additionally, eosinophils from some donors exhibit a
significant expression of CCR1 and respond effectively to its ligand
MIP-1 Basophil responses to chemoattractant ligands are more complex. These
responses are dependent both upon patterns of receptor expression that
overlap with other cell types including eosinophils and monocytes and
also the varying affinities of ligands such as MCP-1 and MCP-4 for more
than one chemokine receptor (15, 30-34).
Nonsteroidal anti-inflammatory drugs
(NSAIDs)1 can have complex
anti-inflammatory actions. A recent study revealed that, in addition to
their effects on cyclooxygenase function, they also caused varying
levels of shedding of the adhesion molecule L-selectin from the surface
of neutrophils following a reduction in intracellular ATP (35). Such
actions might modulate neutrophil recruitment; however, the ability of
NSAIDs to induce similar modulation of eosinophil and basophil function
has not been explored. We therefore examined the actions of a large
panel of NSAIDs on eosinophil and basophil function. Surprisingly, one
NSAID, indomethacin, showed a marked direct action upon leukocytes,
inducing selective responses in basophils and eosinophils consistent
with a very recent paper that has identified indomethacin as a CRTH2
agonist (36). We have therefore investigated the actions and signaling pathways of indomethacin and related these actions to other stimuli involved in selective recruitment and activation of these allergic inflammatory leukocytes.
Reagents--
All laboratory reagents were from Sigma (Poole,
UK) unless otherwise specified. The NSAIDs indomethacin, diclofenac,
flufenamic acid, etodolac, piroxicam, and flurbiprofen were also from
Sigma. The COX-2-selective NSAID NS-398 was from Cayman (Ann Arbor,
MI), and the COX-1 selective NSAID SC-560 was a generous gift from Dr.
R. A. Marks (Searle, Skokie, IL). They were dissolved at high concentration as recommended in water, PBS, Me2SO, or
ethanol. Relevant vehicle controls were without effect in any assay
tested beyond responses seen in buffer-treated cells alone. Dulbecco's modified PBS (with or without Ca2+ and Mg2+)
was from Invitrogen (Paisley, UK). Chemokines were from
Peprotech EC (London, UK). CellFix and FACSFlow were from Becton
Dickinson Immunocytometry Systems (San Jose, CA). Antibodies to CD63
(FITC conjugate) were from Autogen Bioclear (Calne, UK), antibodies to
HLA-DR (FITC conjugate) were from Sigma, and antibodies to CD123 (PE)
and CD14 (FITC) were from Becton Dickinson. Anti-CD16 (FITC or PE) and
anti-CD11b (PE) were purchased from Dako (Ely, UK). Anti-L-selectin
(PE) was from eBioscience (San Diego, CA). The anti-human CCR3
monoclonal antibody 7B11 (isotype IgG2a) was a generous gift from Dr.
Shixin Qin (Millennium Pharmaceuticals Inc., Cambridge, MA). Relevant
isotype-matched control antibodies were used throughout. The p38
mitogen-activated protein kinase (MAPK) inhibitor SB-202190, the
MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitor
U-0126, and the PI 3-kinase inhibitor LY-294002 were purchased from
Calbiochem. The phosphatidylinositide-specific phospholipase C (PLC)
inhibitor U-73122 (37) and the control small molecule U-73343 were
supplied by Biomol (Plymouth Meeting, PA). Boyden chambers and 5-µm
pore size polycarbonate filters were from Neuro Probe Inc.
(Gaithersburg, MD).
Cell Preparation--
Peripheral blood leukocyte preparations of
granulocytes (containing eosinophils and neutrophils) and mononuclear
cells (including basophils, monocytes, and lymphocytes) were prepared
by plasma/Percoll gradients or Histopaque gradients as described
(14, 30, 38). Cells prepared by either technique gave similar results
in the highly sensitive assays of leukocyte shape change (data not
shown). In some experiments, eosinophils were further purified from
granulocyte populations by negative magnetic selection using an
antibody mixture from StemCell Technologies (Vancouver, Canada),
according to the manufacturer's instructions. Resulting populations of
eosinophils were typically >97%, with the majority of contaminating
cells being lymphocytes as judged by flow cytometry forward scatter (FSC)/side scatter (SSC) plots.
Leukocyte Shape Change Assays--
Eosinophil, monocyte,
basophil, and neutrophil shape change was assayed as described in
previous work (14, 18, 30). Stimulation of these leukocytes by
chemoattractant and chemokinetic agonists results in changes in cell
shape that are measured as changes in their ability to scatter light
when illuminated in a flow cytometer (Fig. 1) (14, 30). In all
experiments, data are displayed as percentage increase in FSC
compared with samples treated with buffer or vehicle alone. The various
vehicles used (Me2SO, PBS, ethanol, and water) were without
effect in these shape change assays at the dilutions tested.
Eosinophils were identified according to their size and granularity as
determined by their FSC/SSC characteristics and by their
autofluorescence, neutrophils were identified by FSC/SSC gating and
lack of autofluorescence (Fig. 1), basophils were identified by FSC/SSC
gating and staining with anti-HLA-DR and anti-CD123, and monocytes were
identified by FSC/SSC gating and anti-CD14 staining (14, 30).
Modulation of Surface Molecule Expression--
Leukocyte
preparations were resuspended in assay buffer (PBS plus
Ca2+/Mg2+ with 0.1% bovine serum albumin, 10 mM HEPES, and 10 mM glucose, pH 7.4) at 5 × 106 cells/ml together with agonists for the time
indicated. Following stimulation, cells were washed in
fluorescence-activated cell sorting buffer (PBS without
Ca2+/Mg2+ with 0.25% bovine serum albumin and
10 mM HEPES) and stained with the indicated antibodies (to
L-selectin, CD11b, and CCR3) on ice. The CCR3 antibody was not directly
conjugated; therefore, its binding was detected using PE-conjugated
goat anti-mouse F(ab')2 Ab (DAKO), following which the
cells were incubated with mouse IgG for 10 min prior to staining with a
FITC-conjugated anti-CD16 monoclonal antibody. For experiments
investigating changes in CD63 expression, leukocytes in mixed PMNL
suspensions were labeled with anti-CD16, pretreated with cytochalasin B
(5 µg/ml) for 5 min, and stimulated for 30 min with agonists as
indicated in the presence of the anti-CD63 monoclonal antibody. All
data were analyzed by flow cytometry. Eosinophils were identified by
FSC/SSC gating and were CD16-negative, while neutrophils were in a
similar FSC/SSC region but were CD16-positive. Data were expressed as
percentage change from a control sample incubated with buffer alone.
Respiratory Burst--
Eosinophils purified by negative magnetic
selection were stimulated with buffer or agonists at 37 °C for 20 min in the presence of 1 µM dihydrorhodamine 123 (39).
Subsequent changes in eosinophil fluorescence were measured in the FL-1
channel of the flow cytometer.
Chemotaxis--
Eosinophils were purified by negative magnetic
selection as described. Fifty µl of cells at 2 × 106/ml (in Hanks' buffered salt solution containing
Ca2+ and Mg2+, 0.25% bovine serum albumin, and
30 mM HEPES, pH 7.4) were placed onto the top filter of a
micro-Boyden chamber, with agonists in the bottom well of the plate.
The plates were incubated at 37 °C in a humidified CO2
incubator for 1 h, and the membrane was carefully removed. Cells
in the lower chamber were counted by flow cytometry as previously
described for chemotaxis using transwell filters (7). In each
chemotaxis plate, migration in response to buffer alone was determined,
and migration in response to agonists was expressed as a ratio of the
migration in response to the buffer control (chemotactic index).
Statistics--
Comparisons of two groups of data were performed
using the Mann-Whitney U test. Other data were analyzed by
analysis of variance, using Tukey's or Dunnett's post-test. Analysis
was performed using the GraphPad InStat and Prism programs
(GraphPad Software, San Diego, CA).
PGD2, Eotaxin, and Indomethacin Cause Eosinophil and
Basophil Shape Change--
Shape change induced by eosinophil- and
basophil-stimulating ligands was measured as described. Fig.
1 illustrates the changes in shape in
neutrophils and eosinophils in mixed leukocyte populations as measured
by flow cytometry, showing selective eosinophil responses to eotaxin,
PGD2, and indomethacin and selective neutrophil responses to interleukin-8. In mean data, Fig.
2A shows that the chemokine eotaxin induced eosinophil and basophil shape change, interleukin-8 induced selective neutrophil shape change, and MCP-1 induced monocyte shape change as described previously. PGD2 was a potent
inducer of eosinophil and basophil shape change but had no effect on
neutrophils or monocytes. A wide range of NSAIDs have been shown to
induce neutrophil L-selectin shedding (35), and we therefore postulated that they would also induce leukocyte shape change. Surprisingly, of
the NSAIDs tested, only indomethacin induced a shape change response.
This response was observed in eosinophils and basophils but not
neutrophils or monocytes (Fig. 2A). Indomethacin induced eosinophil shape change with a maximal response at 100 nM
and showed lower potency but the same efficacy as eotaxin/CCL11. The NSAIDs ibuprofen, flurbiprofen, NS-398, SC-560, piroxicam, and etodolac
(each 50 nM to 10 µM) and acetylsalicylic
acid (5 µM to 1 mM) all failed to induce any
detectable shape change response in eosinophils or neutrophils
(n = 4-6, data not shown). Similarly, diclofenac and
flufenamic acid (each 50 nM to 10 µM) were
without effect (n = 3, data not shown).
Indomethacin Causes Eosinophil Chemotaxis--
We have
previously shown that eosinophil shape change can be induced by both
chemotactic agonists such as eotaxin/CCL11 and by chemokinetic agonists
such as interleukin-5 (14). We therefore investigated the ability of
eotaxin/CCL11 and indomethacin to induce eosinophil chemotaxis in
micro-Boyden chambers. Each agonist induced migration of eosinophils
into the bottom chamber of the chemotaxis plate (Fig. 2B).
The addition of indomethacin (1 µM) to the top of the
chemotaxis chamber alone resulted in some variable migration of
eosinophils into the lower chamber although to a lesser degree than
seen when indomethacin was added to the bottom chamber alone (Fig.
2C).
Indomethacin Up-regulates Eosinophil CD11b and Down-regulates
L-selectin Expression--
Our data showed that indomethacin acted as
an eosinophil chemoattractant agonist in a similar manner to the
chemokine eotaxin/CCL11. We therefore investigated whether indomethacin
could induce modulation of adhesion molecule expression. Fig.
3A shows that treatment of
eosinophils in mixed cell suspensions with indomethacin resulted in a
reduction in cell surface L-selectin expression, whereas neutrophil
L-selectin expression was not significantly affected. Indomethacin also
induced an up-regulation of eosinophil CD11b expression (Fig.
3B). In comparison, neutrophil CD11b expression was not
affected by indomethacin at any concentration tested (Fig. 3B).
Indomethacin Causes Respiratory Burst but Not Up-regulation of CD63
Expression--
Chemokine chemoattractants are typically good
stimulators of leukocyte shape change and chemotaxis, variable inducers
of respiratory burst, and relatively poor stimulators of degranulation.
Classical chemoattractants such as C5a are more potent inducers of
these latter responses. In keeping with these data, eosinophil
respiratory burst (Fig. 3C) and CD63 up-regulation (a
degranulation-associated marker (40) (Fig. 3D)) were
strongly induced by C5a. In contrast, indomethacin, eotaxin/CCL11, and
PGD2 all induced eosinophil respiratory burst (eotaxin = PGD2 > indomethacin (Fig. 3C)), although they were less efficacious than C5a and were unable to induce any changes in
eosinophil CD63 expression at the concentrations tested (Fig. 3D).
Indomethacin-induced Eosinophil Shape Change Is Inhibited by
Cytochalasin B but Not by Pertussis Toxin--
Eosinophil shape change
as induced by chemotactic factors is dependent upon G protein-coupled
receptors mediating regulation of the cell cytoskeleton. The majority
of studies have emphasized a role for pertussis toxin (PTX)-sensitive G
proteins in the regulation of leukocyte chemokine responses.
Pretreatment of eosinophils with PTX (in accordance with protocols
previously used to investigate CCR3 signaling (41)) suggested
differences in the signaling pathways activated by eotaxin and
indomethacin. Eosinophil shape change in response to eotaxin/CCL11 was
abolished by PTX, whereas in parallel samples, eosinophils retained
their ability to respond to both indomethacin and PGD2
(Fig. 4A).
Previously, we showed chemokine-induced eosinophil shape change to be
inhibited by pretreatment of cells with cytochalasin B (14). Here
again, we found that eosinophil shape change induced by eotaxin was
inhibited by cytochalasin B but not by buffer containing the vehicle
control, Me2SO (Fig. 4B). Similarly,
indomethacin-induced shape change also showed dependence upon cell
microskeletal elements, since it too was inhibited by pretreatment with
cytochalasin B.
Experiments using PTX (Fig. 4A) suggested that
different signaling pathways were involved in responses of eosinophils
to indomethacin and eotaxin/CCL11. In keeping with these data,
eotaxin/CCL11-induced shape change was more transient than that induced
by indomethacin and was undetectable after prolonged stimulation. In
contrast, indomethacin-induced shape change was still detectable after
prolonged stimulation, although the concentration-response curve was
right-shifted (Fig. 4C).
Eosinophil Responses to Indomethacin Involve Specific
Kinases--
Chemoattractant agonists activate multiple intracellular
signaling pathways in eosinophils, including PLC, PI 3-kinase, and p38
MAPK. We therefore investigated whether indomethacin- and eotaxin-induced eosinophil responses were modulated by antagonists of
these pathways. Fig. 5 shows that
eosinophil shape change responses induced by eotaxin/CCL11 were
markedly attenuated by inhibition of PLC using U-73122. Similarly,
responses of eosinophils to indomethacin and PGD2 were
inhibited by U-73122, but responses to eotaxin/CCL11, indomethacin, and
PGD2 were not inhibited by the control compound, U-73343.
Eosinophil shape change induced by submaximal concentrations of
eotaxin/CCL11 was inhibited by both the PI 3-kinase inhibitor LY-294002
and the p38 MAPK inhibitor SB-202190 (Fig.
6, A and B).
However, responses seen at concentrations of eotaxin/CCL11 inducing
maximal shape change were not inhibited by either LY-294002 or
SB-202190. The up-regulation of eosinophil CD11b expression induced by
eotaxin/CCL11 was also inhibited by both LY-294002 and SB-202190 (Fig.
6C) and also by MEK inhibitor U-0126 (Fig. 6C),
although interestingly U-0126 had no effect on eotaxin/CCL11-induced eosinophil shape change (Fig. 6A).
Indomethacin-induced responses in eosinophils were similarly modulated
by inhibitors of PI 3-kinase, p38 MAPK, and MEK but with two exceptions
(Fig. 6). First, inhibition of p38 MAPK reduced both the potency and
efficacy of indomethacin in assays of eosinophil shape change, whereas
the p38 MAPK inhibitor suppressed eotaxin-induced eosinophil shape
change potency alone. Second, the MEK inhibitor U-0126 caused an
insignificant inhibition of eosinophil shape change induced by
submaximal indomethacin concentrations and had no effect on
indomethacin-induced up-regulation of eosinophil CD11b expression.
Indomethacin Pretreatment Can Modulate Eosinophil CCR3
Expression--
We have previously shown that G protein-coupled
signaling can result in cross-desensitization and internalization of
other chemoattractant receptors in human neutrophils (42). Fig.
7 shows that eotaxin/CCL11 induced
internalization of its own receptor, in keeping with previous data
(43). Interestingly, pretreatment with either indomethacin or
PGD2 also resulted in a significant decrease in eosinophil
CCR3 expression (Fig. 7), whereas the chemoattractant PAF had no
effect.
Indomethacin Enhances Responsiveness to Eotaxin--
Since
indomethacin can be used to treat inflammatory diseases, we determined
whether stimulation with this compound would alter eosinophil responses
to eotaxin. We pretreated eosinophils with 100 nM
indomethacin (the EC50 of CCR3 down-regulation (Fig. 7))
for 30 min and then removed the indomethacin by a single wash step
prior to stimulation of the cells with eotaxin/CCL11. The data shown in
Fig. 8 show that the efficacy of
eotaxin/CCL11-induced shape change was increased by indomethacin
pretreatment, whether the data were shown as mean FSC (Fig.
8A) or responses were corrected for the minor increase in
eosinophil FSC base line arising from the indomethacin pretreatment
(Fig. 8B).
In preliminary studies, we found that lipid-derived mediators play
a role in the regulation of eosinophil chemokine
responsiveness,2 and others
have shown that NSAIDs such as indomethacin modulate expression of
adhesion molecules that are relevant in neutrophil recruitment (35). We
examined the actions of NSAIDs to determine whether treatment of
leukocytes with these compounds could modulate chemokine
responsiveness. Surprisingly, we found that indomethacin induced a
direct rapid shape change response in eosinophils in a manner similar
to the chemoattractant agonists eotaxin/CCL11 and PGD2,
reaching a maximum shape change response at indomethacin concentrations
of 100 nM, but had no effect on neutrophils. In addition,
we observed that indomethacin also induced shape change responses in
basophils but not monocytes. This effect was confined to indomethacin
alone out of a large panel of NSAIDs, including the
indomethacin-related NSAID, etodolac (44). From these data, we
postulated that indomethacin might act as a chemoattractant, and our
data show that it did induce concentration-dependent
chemotaxis of purified eosinophils, as did the other
eosinophil-stimulating agonists eotaxin/CCL11 (Fig. 2) and
PGD2 (data not shown).
In a recent study, Gomez-Gaviro et al. showed that
indomethacin induced neutrophil L-selectin shedding (35) but with an IC50 in excess of 30 µg/ml (84 µM). In
order to explore the difference between NSAID actions on neutrophils
and eosinophils in more detail, we examined the ability of indomethacin
to modulate adhesion molecule expression. At the concentrations we
tested ( Chemokine agonists are generally effective stimuli of cell recruitment
but exhibit varying efficacy in their ability to cause degranulation
and respiratory burst. We showed that the classical eosinophil
chemoattractant and activator, C5a, induced potent activation of
eosinophils as seen by up-regulation of CD63 expression (a marker of
degranulation) and induction of respiratory burst. Eotaxin/CCL11,
PGD2, and indomethacin were unable to cause up-regulation of CD63, emphasizing their primary actions as mediators of cell recruitment. In keeping with the known actions of eotaxin/CCL11 (45),
we showed that indomethacin, eotaxin/CCL11, and PGD2 were all able to induce respiratory burst, although with less efficacy than C5a.
Relatively few chemoattractant receptors are expressed selectively by
eosinophils and basophils but not by neutrophils and monocytes (14, 30,
46). Indomethacin showed similar actions to eotaxin/CCL11. However, PTX
abolished shape change responses to eotaxin but had no effect on
indomethacin-induced shape change, suggesting actions on a different
receptor. Recent studies have identified a role for PGD2 as
a selective eosinophil-, basophil-, and Th2-type T cell-stimulating
activity. Eosinophils express two receptors for PGD2, DP
and CRTH2 (26-29). Of these two receptors, CRTH2 is selectively
responsible for eosinophil chemotaxis (26, 27), actin polymerization,
and CD11b up-regulation in response to PGD2 (26). We
therefore hypothesized that indomethacin was acting via a
PGD2 receptor, in keeping with its PGD2-like
activities on eosinophils, and the PTX-resistant signaling seen
in response to both indomethacin and PGD2. During the
preparation of this manuscript, a recent paper reported that
indomethacin is indeed a selective agonist for CRTH2 but is inactive at
DP (36). This paper characterized the actions of indomethacin in detail
using receptor transfectants and also found, as we have, that
indomethacin can cause eosinophil chemotaxis, although it did not
investigate other actions of indomethacin on eosinophils. In contrast,
one other recent publication identified actions of PGD2 on
eosinophil CRTH2 as inducing chemokinesis rather than chemotaxis (28). We found that indomethacin-induced signaling resulted in some chemokinetic responses, although these were variable between
experiments, and in all experiments the addition of indomethacin to the
bottom chamber of the chemotaxis plate resulted in greater leukocyte migration than when indomethacin was added to the top of the plate alone. These data suggest that indomethacin primarily induces eosinophil chemotaxis but also a variable degree of eosinophil chemokinesis. Thus, our data strongly support a role for CRTH2 as a
receptor that can induce eosinophil recruitment. As also noted by Hirai
et al. (36), these data contribute to an understanding of
various actions of indomethacin in vivo, such as its ability to induce, or failure to suppress, eosinophilic inflammation at multiple tissue sites (48, 49).
Interestingly, PGD2 signaling is thought to act via the
PTX-sensitive G protein-coupled receptor CRTH2 and the PTX-resistant receptor DP (27). Likewise, the actions of indomethacin at CRTH2 have
been shown to be PTX-sensitive in receptor transfectants and Th2-type T
cells (36). However, in primary human eosinophils, we observed that
neither PGD2 nor indomethacin responses were inhibited by
PTX under conditions where this toxin completely inhibited
eotaxin/CCL11-induced eosinophil shape change. These data suggest that
CRTH2 in eosinophils may perhaps be coupled, at least in part, to the
PTX-resistant G protein G We found that induction of eosinophil shape change in response to both
eotaxin/CCL11 and indomethacin was inhibited by an antagonist of
phosphatidylinositide-specific PLC, whose isoforms have major and
complex roles in the regulation of leukocyte chemotaxis (51, 52). PLC
has also been shown to play a role in heterologous receptor
desensitization (53) and may therefore be involved in the
indomethacin-induced modulation of CCR3 expression we observed here.
Eotaxin/CCL11-induced eosinophil responses have also been shown to
involve signaling pathways dependent upon PI 3-kinase and p38 MAPK (21,
54), both of which can couple into pathways regulating actin
polymerization and cytoskeletal change responses such as underpin
eosinophil shape change (22, 54) and leukocyte integrin-dependent adhesion (55). The roles of these
pathways in the regulation of CRTH2-induced signaling have not been
explored to date. We found that both indomethacin-induced and
eotaxin/CCL11-induced eosinophil responses were reduced by specific
inhibitors of these pathways, thus providing the first evidence for
their roles in CRTH2-mediated signaling in primary human cells. The
inability of the PI 3-kinase inhibitor to prevent agonist-induced shape change to high concentrations of chemoattractant may be consistent with
the hypothesis that the PI 3-kinase product phosphatidylinositol 3,4,5-trisphosphate is not essential for chemotaxis but rather exerts a
primarily regulatory role in this process (51). Interestingly, our data
also show that eosinophil signaling via CRTH2 may be more dependent
upon p38 MAPK than that induced by eotaxin/CCL11 acting via CCR3. An
illustration of the proposed receptors and signaling pathways for
eotaxin, PGD2, indomethacin, and NSAIDs in eosinophils is
shown in Fig. 9.
We have also observed that indomethacin may modulate responses of
eosinophils to other ligands. Pretreatment of eosinophils with 100 nM indomethacin enhanced the efficacy of eotaxin/CCL11 and
suggests that indomethacin may enhance eosinophil responses to
proinflammatory ligands. However, at higher concentrations, indomethacin treatment had marked effects upon CCR3 expression. Such
effects could be mediated by release of CCR3 ligands from the
eosinophil after indomethacin stimulation but are also in keeping with
the processes of heterologous desensitization between seven-transmembrane G protein-coupled chemoattractant receptors observed in leukocytes by ourselves and others (42, 53). The consequences of these effects on eosinophil responses to ligands such
as eotaxin remain to be fully explored.
Indomethacin has other potential actions in eosinophils, most notably
through its anti-inflammatory ability to inhibit COX-1 and COX-2
isoenzymes (Fig. 9). Published IC50 values for indomethacin on these enzymes vary within low to high nanomolar ranges (56-59), and
thus inhibition of eosinophil COX isoenzymes within the experiments here may have occurred. However, no other NSAID from an extensive panel
of drugs including nonselective NSAIDs and drugs with specific actions
on either COX-1 or COX-2, tested across a broad range of
concentrations, induced any similar responses in human eosinophils. These data, in combination with the observed similarities in signaling between PGD2 and indomethacin in this study and the
identification of indomethacin as a ligand for the eosinophil-expressed
PGD2 receptor, CRTH2 (36), strongly suggest that the
proinflammatory actions of indomethacin on eosinophils and basophils
are not mediated by COX inhibition.
Therefore, we have shown that indomethacin is a potent chemoattractant
ligand acting on eosinophils, with actions also upon basophils but not
neutrophils or monocytes. We have characterized the actions of
indomethacin on eosinophils and found that it exerts similar effects to
PGD2, supporting evidence that most chemoattractant-like PGD2 signaling is mediated in eosinophils by CRTH2. We have
also shown that signaling of this receptor shows different sensitivity to PTX compared with signaling in response to eotaxin and that it
is coupled into pathways including PLC, p38 MAPK, and PI
3-kinase in primary human eosinophils. Many studies have examined the
actions of indomethacin in inflammatory disease, focusing on its roles as a cyclooxygenase inhibitor, but the independent actions of indomethacin as a potent eosinophil- and basophil-stimulating compound
may alter our understanding of the actions of this drug. Development of
structural analogues of indomethacin may generate both new CRTH2
activators and perhaps antagonists that will be of use in the treatment
of human allergic inflammatory disease.
*
This work was supported by National Asthma Campaign Project
Grant 99/012 (to V. E. L. S.), a grant from the Medical Research Council UK (to I. S.), a grant from the Wellcome Trust (to
A. Hartnell and T. J. W.), a grant from the National Asthma Campaign (to T. J. W.), a grant from the Royal Society (to T. J. W. and B. A. P.), and Austrian Science Fund FWF Grant P15453 (to
A. Heinemann).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
These two authors contributed equally to this work as
principal investigators.
**
Present address: Division of Genomic Medicine, University of
Sheffield, M Floor, Royal Hallamshire Hospital, Sheffield S10 2JF,
United Kingdom.
Published, JBC Papers in Press, April 29, 2002, DOI 10.1074/jbc.M201803200
2
V. E. L. Stubbs, A. Hartnell,
T. J. Williams, A. Heinemann, and I. Sabroe, unpublished data.
The abbreviations used are:
NSAID, nonsteroidal
anti-inflammatory drug;
PTX, pertussis toxin;
PMNL, polymorphonuclear
leukocytes (comprising eosinophils and neutrophils);
PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate;
PE, phycoerythrin;
MAPK, mitogen-activated protein kinase;
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase;
PLC, phospholipase C;
PI 3-kinase, phosphatidylinositol
3-kinase;
FSC, forward scatter;
SSC, side scatter;
COX, cyclooxygenase.
Indomethacin Causes Prostaglandin D2-like and
Eotaxin-like Selective Responses in Eosinophils and Basophils*
,,,
, and¶**
Leukocyte Biology Section, Biomedical
Sciences Division, Imperial College Faculty of Medicine, Imperial
College of Science, Technology and Medicine, South Kensington, London
SW7 2AZ, United Kingdom and the § Department of Experimental
and Clinical Pharmacology, Universitatsplatz 4, Graz A-8010, Austria
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/CCL3 (14). Similar results have now been seen by other groups
(17), and we have shown that responses of eosinophils to chemokines can
be blocked by chemokine receptor antagonists with theoretical benefits
for the treatment of allergic inflammation (18). However, activated
complement fragments such as C5a and C3a, formylated peptides
(formyl-methionyl-leucyl-phenylalanine), and a wide variety of lipid
mediators can also induce similar responses in eosinophils (19-22).
Recently, interest has arisen in the potential for PGD2, a
mediator known to have actions on eosinophils (23, 24) and which is
generated in the asthmatic lung (25), to stimulate eosinophil,
basophil, and Th2-type T cell functions in allergic disease through its
action on two cell surface receptors, DP and CRTH2 (26-29).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Chemokine- and NSAID-induced leukocyte shape
change responses. Illustrative flow cytometry plots of
leukocyte shape change in response to agonists. Eosinophils and
neutrophils form overlapping populations when identified by forward
light scatter (reflective of cell size) and side light scatter
(reflective of cell granularity) in the illuminating laser
(top left plot) but can be identified
and plotted separately by measurement of autofluorescence, which is
more pronounced in eosinophils (measured in FL-2 as shown, with
percentages of eosinophils indicated by the R1 gate and neutrophils by
the R2 gate). Changes in cell shape upon stimulation are reflected in
changes in the forward light scatter, and illustrative shape changes
induced by buffer, the eosinophil-selective chemokine eotaxin,
PGD2, indomethacin, and the neutrophil-selective chemokine
interleukin-8 (IL-8), are shown, with the corresponding FSC
values inset in the plots. For each stimulus, the PMNL
population containing eosinophils and neutrophils is shown on the
left, and alongside this are the plots of the eosinophils
(middle plot) and neutrophils
(right-hand plot), separated on the basis of
their autofluorescence.
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Fig. 2.
Chemokine and NSAID-induced leukocyte shape
change responses and chemotaxis. A shows the induction
of shape change in eosinophils (i), basophils
(ii), neutrophils (iii), and monocytes
(iv) and in mixed cell suspensions in response to selected
chemokines, PGD2, and indomethacin, stimulated for 4 min at
37 °C. Data shown are the mean of at least four experiments ± S.E. B shows the chemotactic response of eosinophils
purified by negative magnetic selection to indomethacin and eotaxin.
Data shown are the mean of six experiments ± S.E. C
shows the ability of these ligands to induce chemokinesis and
chemotaxis. Cells were treated with indomethacin (10 µM)
or eotaxin (100 nM) in the top of the chemotaxis chamber or
in the bottom well as indicated. Data shown are from five experiments,
with one donor (indicated by the open squares)
being tested twice.
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Fig. 3.
Actions of indomethacin on eosinophils and
neutrophils. PMNL were stimulated with indomethacin for 30 min.
Samples were stained to analyze L-selectin (A) and CD11b
(B) expression and double-stained with anti-CD16 to identify
neutrophils and eosinophils. Data were quantified as percentage change
from basal (buffer-treated) expression levels and are the mean of four
experiments ± S.E. In separate experiments, induction of
respiratory burst (C) was investigated by treating purified
eosinophils with agonists for 20 min, and CD63 up-regulation
(D) was investigated by stimulating eosinophils in
mixed cell suspensions for 30 min. Changes in anti-CD63 binding or
dihydrorhodamine 123 fluorescence are shown as the mean of four
experiments (CD63 up-regulation) and three experiments (respiratory
burst) ± S.E.
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Fig. 4.
Modulation of eosinophil shape change by
pertussis toxin and cytochalasin B and time dependence of
signaling. Eosinophils in mixed PMNL populations were pretreated
with PTX (1 µg/ml for 60 min (A, i-iii) or
cytochalasin B (5 µg/ml for 5 min; B, i-ii)
and stimulated with indicated ligands for 4 min, and shape change was
measured as described. C depicts the eosinophil shape change
induced in mixed cell populations by stimulation with eotaxin/CCL11 or
indomethacin for the times indicated. Data shown are the mean of six
experiments ± S.E. (A and B) and four
experiments (C, indomethacin) or three experiments
(C, eotaxin) ± S.E., and significant inhibition of
shape change in the presence of PTX or cytochalasin B is indicated by
p < 0.05 (*).
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Fig. 5.
Modulation of eosinophil chemoattractant
responses by inhibition of PLC. Eosinophils in mixed PMNL
populations were pretreated with 3.6 µM U-73122 PLC
inhibitor and inactive control compound U-73343 for 30 min at 37 °C,
after which shape change responses to the indicated ligands were
determined as described in the presence of the inhibitors. Data are
shown as the percentage of the maximal shape change response and are
the mean of seven (eotaxin), six (indomethacin), or four
(PGD2) experiments ± S.E. Significant inhibition of
agonist-induced responses are indicated by p < 0.05 (*).
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Fig. 6.
Modulation of eosinophil chemoattractant
responses by inhibition of PI 3-kinase, p38 MAPK, and MEK.
Eosinophils in mixed PMNL populations were pretreated with specific
kinase inhibitors (10 µM SB-202190 p38 MAPK inhibitor, 2 µM U-0126 MEK inhibitor, and 20 µM
LY-294002 PI 3-kinase inhibitor) for 60 min at 37 °C, after which
shape change responses (4-min stimulation; A) were
determined as described in the presence of the inhibitors. B
shows in more detail the percentage inhibition of the eosinophil shape
change in response to 0.16 nM eotaxin or 16 nM
indomethacin after pretreatment with the indicated inhibitors
(statistical analysis performed on raw data prior to calculation of
percentage inhibition). C shows the effects of these
inhibitors on the up-regulation of CD11b expression (induced by 30-min
ligand stimulation) as a percentage of the CD11b expression in cells
stimulated for 30 min in the presence of buffer alone. Inhibitors
remained present throughout the assay. Data shown are the mean of four
experiments ± S.E. Significant inhibition of agonist-induced
responses is indicated by p < 0.05 (*).
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Fig. 7.
Modulation of eosinophil CCR3
expression. Eosinophils in mixed PMNL populations were incubated
with indomethacin, eotaxin/CCL11, PGD2, PAF, or buffer for
30 min at 37 °C, following which CCR3 expression was determined as
described. Data are shown as the percentage change in CCR3 expression
from buffer-treated cells and are the mean of five experiments ± S.E. Significant changes from buffer-treated cells are indicated by
p < 0.05 (*).
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Fig. 8.
Indomethacin pretreatment modulates
eosinophil eotaxin/CCL11 responsiveness. Eosinophils in mixed PMNL
populations were incubated with indomethacin (100 nM) or
buffer for 30 min at 37 °C, washed, and treated with eotaxin/CCL11
for 4 min at 37 °C, and the resulting eosinophil shape change was
measured. The data shown are the mean of four experiments ± S.E.,
and significant differences in eotaxin/CCL11 responses between cells
pretreated with indomethacin or buffer are indicated by
p < 0.05 (*).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10 µM), we were unable to detect significant
indomethacin-induced changes in neutrophil L-selectin expression. In
contrast, indomethacin caused small but significant decreases in
eosinophil L-selectin levels. Indomethacin was also efficacious in the
up-regulation of eosinophil CD11b expression, yet had no significant
effect on neutrophil CD11b expression, again mirroring effects of
eosinophil-selective chemoattractants such as eotaxin/CCL11 and
PGD2 (26, 45, 46). One other group has described an ability
of indomethacin to induce up-regulation of neutrophil CD11b
up-regulation, but again this effect was maximal only at high
concentrations (100 µM) (47). Thus, our data potentially separate two actions of indomethacin: one that may be common to other
NSAIDs, is dependent upon changes in intracellular ATP levels, and
mediates L-selectin shedding (35) and one that is specific to
indomethacin and is consistent with chemoattractant-like actions.
q/11 which is also involved in
eosinophil chemotactic responses to other ligands, although
interpretation of PTX-dependence in investigation of agonist-mediated
responses must be carried out with caution (50). An alternative
hypothesis, that the actions of both indomethacin and PGD2
on eosinophils are mediated predominantly via the PTX-resistant DP
receptor rather than CRTH2, is unlikely, given the published specificity of indomethacin and the previous data on the selective roles of CRTH2 in mediating eosinophil PGD2 responses
(26-28, 36). In keeping with this, signaling via DP has been shown to
modulate eosinophil apoptosis only (28), and we found that the
DP-selective agonist BW245C was unable to cause eosinophil shape change
(n = 7, data not shown).
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Fig. 9.
Proposed receptors and signaling pathways
involved in eosinophil responses to eotaxin, PGD2, and
indomethacin. Eotaxin and related chemokines activate the G
protein-coupled receptor CCR3. PGD2 binds to two cell
surface G protein-coupled receptors: DP and CRTH2 (26, 28). BW245C is a
selective agonist of DP, whereas DK-PGD2 and indomethacin
are selective agonists of CRTH2 (36). CCR3 and CRTH2 ligation results
in activation of similar signaling pathways (although with potential
differences in dependence upon PTX-sensitive G proteins; see
"Results"), leading to functional responses including
chemotaxis, respiratory burst, shape change, and adhesion molecule
expression. In contrast, stimulation of DP acts via alternative
signaling pathways to modulate responses such as eosinophil apoptosis
(28). Indomethacin, together with other NSAIDs, also has the potential
to exert actions primarily via inhibition of COX isoenzymes, although
NSAIDs as a group have varying abilities to inhibit other
proinflammatory pathways such as those activated by MAPK family members
(57).
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Experimental and Clinical Pharmacology, Universitatsplatz 4, A-8010
Graz, Austria. E-mail: akos.heinemann@kfunigraz.ac.at.
ABBREVIATIONS
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