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JPET #123026
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PI3Kγ inhibition plays a crucial role in early steps of inflammation by blocking
neutrophil recruitment
Chiara Ferrandi, Vittoria Ardissone, Pamela Ferro, Thomas Rückle, Paola Zaratin,
Elena Ammannati, Ehud Hauben, Christian Rommel and Rocco Cirillo
Istituto di Ricerche Biomediche ‘A. Marxer’, RBM/Merck Serono, I-10010 Colleretto
Giacosa, Italy (CF, VA, PF, PZ, EA, EH, RC)
Merck Serono Geneva Pharmaceutical Research Institute, CH-1228 Geneva,
Switzerland (TR, CR)
JPET Fast Forward. Published on May 25, 2007 as DOI:10.1124/jpet.107.123026
Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: PI3Kγ inhibition reduces neutrophil recruitment
Abbreviations: r-hRANTES: recombinant-human Regulated upon Activation, Normal
T-cell Expressed, and Secreted, PBS: Phosphate Buffered Saline, BSA: Bovine Serum
Albumin, FACS: Flow Analyzer Cell Sorter, PECs: Peritoneal Exudate Cells, NPIR:
non-phagocytic response, PIR: phagocytic response)
Corresponding author: Chiara Ferrandi
Molecular Medicine
Pharmacology Dep
RBM/Merck Serono Via Ribes 1, Colleretto Giacosa (TO)
+39 0125 222525
+39 0125 222599
Number of text pages: 33
Number of figures: 7
Number of tables: 2
Number of references: 40
Number of words in the abstract: 211
Number of words in the introduction: 716
Number of words in the discussion: 1092
Recommended section: Inflammation, Immunopharmacology, and Asthma
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Abstract
Leukocyte trafficking to inflammatory sites is a gradual process, which is
dominated in its early phases by chemokine- and cytokine-mediated neutrophil
recruitment. The chemokine RANTES has been shown to be highly expressed in the
joints of patient with rheumatoid arthritis, and to promote leukocyte trafficking into
the synovial tissue. In this study, we investigated the effect of RANTES in a murine
model of peritoneal chemotaxis, and we found that RANTES dose-dependently
induces neutrophil recruitment. Then, through morphological and histological
analyses, we observed activated neutrophils represent the major infiltrating population
in response to RANTES chemotactic stimulus. Furthermore, we demonstrated that
oral administration of either a non-isoform specific PI3K inhibitor (LY294002), or of
a selective PI3Kγ inhibitor (AS041164), blocks RANTES-induced chemotaxis and
reduces the level of AKT phosphorylation. Since the two compounds showed a
similar pharmacokinetic profile in terms of bioavailability and half-life after oral route
administration, the selective inhibition of the PI3Kγ-isoform pathway, through
AS041164 was three times more potent in reducing neutrophil recruitment. Finally, to
confirm the blockade of neutrophil infiltration that occurs in the early phase of the
inflammatory response, AS041164 was also tested in a model of carrageenan-induced
paw edema in rats. Therefore, the PI3Kγ pathway plays an important role in
controlling neutrophil chemotaxis during early steps of inflammation.
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Introduction
During inflammation neutrophils are rapidly recruited at sites of acute
infection and dominate the initial influx of leukocytes (Issekutz and Movat, 1980).
Later, during the progression of inflammation, monocytes and macrophages replace
neutrophils, suggesting a bimodal recruitment pattern involving a switch from
neutrophils to monocytes (Doherthy et al., 1988; Henderson et al., 2003). The first
step in approaching a site of insult requires neutrophils to transmigrate across
endothelial barriers, a process that depends on chemokines (Yoshie et al., 2001). In
response to a chemotactic gradient, CXC and CC chemokines activate leukocytes by
binding to seven trans-membrane receptors coupled to G proteins (Proudfoot, 2002)
linked to heterotrimeric G protein complexes (Proudfoot, 2002). Upon stimulation, the
G protein complex dissociates and subsequently recruits various signaling
components such as nucleotide exchange factors, phospholipid lipases and lipid
kinase phosphoinositide-3’OH-kinase isoforms, such as PI3K (Akasaki et al., 1999).
Neutrophils have been shown to express different chemokine receptors, including
CXCR2 and CCR1 (Lee et al., 1995; Zhang et al., 1999). Furthermore, the blockade
of the chemokine receptor CXCR2 or of its ligands IL-8 and macrophage-
inflammatory protein-2 (MIP-2), or alternatively of the CCR1/MIP-1α interaction,
have been shown to inhibit neutrophil migration in murine models of inflammation
(Tessier et al., 1997).
Neutrophils are responsible to drive inflammatory response in local tissues.
Upon binding of TLR2 or TLR4 ligands, neutrophils upregulate the expression of
chemokines, downregulate some chemokine receptors, and change their expression of
adhesion molecules and respiratory burst mediators. Consequently, they leave the
blood and migrate to sites of infection in a multistep process mediated through
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adhesive interactions that are regulated by macrophage-derived cytokines and
chemokines (Muller and Randolph, 1999).
The CC chemokine RANTES/CCL5 (ligand for CCR1, 3, and 5) is up
regulated in the joints of patients with rheumatoid arthritis (Shahrara et al., 2003) and
has been reported to play a role in the in vivo pathogenesis of experimental arthritis
(Shahrara et al., 2003; Barnes et al., 1998). Accordingly, intraperitoneal injection of
recombinant human RANTES (r-hRANTES) has been demonstrated to produce a
marked recruitment of inflammatory cells in the peritoneal cavity in mice (Proudfoot
et al., 2003).
A large body of evidence indicates a central role of the phosphoinositide 3-
kinase class IB isoform (PI3Kγ) in chemokine-induced leukocytes recruitment. Thus,
PI3Kγ deficient mice as well as mice treated with specific PI3Kγ inhibitors showed
significantly reduced 3’-phosphorylated phosphoinositides production by leukocytes,
following stimulation with various chemoattractants (e.g. fMLP, C5a, IL-8) (Hirsh et
al., 2000). Accordingly, chemokines failed to stimulate the phosphoinositides-
dependent kinase pathway (e.g. PKB/AKT) in cells isolated from PI3Kγ deficient
mice (Hirsh et al., 2000). Indeed, the recruitment of neutrophils, monocytes and
macrophages in response to chemokines in vitro and in vivo in PI3Kγ deficient mice
was significantly reduced, while the response to pleiotropic inflammatory stimuli such
as carrageenan in the air-pouch model was unaffected (Hirsh et al., 2000; Stephens et
al., 1998; Al-Aoukaty et al., 1999). Furthermore, blockade of PI3Kγ was shown to
suppress joint inflammation and damage in a mouse model of rheumatoid arthritis
(Camps et al., 2005).
The aim of the present study was to investigate the role of PI3K/PI3Kγ in r-
hRANTES-induced neutrophil chemotaxis. To this end, the non-selective PI3K
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inhibitor, LY294002 (Morpholin-4-yl-8-phenyl-chromen-4-one described in Vlahos et
al., 1994)), and the selective PI3Kγ isoform inhibitor, AS041164 (5-
Benzo[1,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione, synthesis described in
Rückle et al., 2004) (see Tables I and II for details of both inhibitors) were orally
administered into RANTES-injected mice. Finally, to confirm the blockade of
neutrophil infiltration that occurs in the early phase of the inflammatory response ,
AS041164 was also tested in a model of carrageenan-induced paw edema in rats, a
model in which neutrophils play a crucial role (Vinegar et al. 1987). According to
Vinegar et al., this model can be described as a biphasic edematous response during
the first 3 hours and two inflammatory phases can be distinguished: the first non-
phagocytic (NPIR) and the second phagocytic (PIR). Their findings show that 180
minutes after carrageenan challenge a large number of neutrophils is observed
histologically in and around the small blood vessels in the dermis initiating the PIR
phase. The agent responsible for neutrophils’ diapedesis to the injured dermal cells is
unknown. Our findings illustrate the mechanism by which RANTES promotes
neutrophil chemotaxis, and suggest PI3Kγ inhibition as a potential treatment for
inflammatory diseases that involve neutrophils recruitment.
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Materials and Methods
Animals
Female Balb/C mice of about 8-12 weeks of age (18-22 g b.w.) from Charles River
Italia (Calco, Italy) were used for RANTES-induced neutrophils peritoneal
recruitment and male Wistar rats (100-150 g b.w.) from Charles River Italia (Calco,
Italy) were used for carrageenan-induced inflammation model. Mice and rats were
housed under the following constant environmental conditions: temperature 22°C ± 2,
relative humidity 55% ± 10, 15-20 air changes per hour (filtered on HEPA 99.99%)
and artificial light with a 12-hour circadian cycle (7 a.m.-7 p.m.). All in vivo studies
were performed according to the European Council Directive 86/609/EEC and the
Italian Ministry guidelines for the care and use of experimental animals (decree #
116/92). All the experimental protocols were authorized by the Italian Ministry of
Health.
Chemicals and reagents
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless
otherwise specified. Antibodies used in flow cytometry were obtained from BD
Pharmingen (San Diego, CA, USA), except the antibody against CCR3, purchased
from R&D (Minneapolis, MN, USA). Recombinant wild-type RANTES (Proudfoot at
al., 2003), LY294002 (Morpholin-4-yl-8-phenyl-chromen-4-one described in Vlahos
et al., 1994) and AS041164 (5-Benzo[1,3]dioxol-5-ylmethylene-thiazolidine-2,4-
dione, synthesis described in Rückle et al., 2004) were from Serono Pharmaceutical
Research Institute, Geneva, Switzerland. For the in vivo studies, both compounds
were suspended in 0.5% carboxymethylcellulose/0.25% tween 20 as vehicle.
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r-hRANTES-induced neutrophil peritoneal recruitment
Mice received intraperitoneally 200 µl of lipopolysaccharide-free saline or r-
hRANTES (0.05-0.15-0.5-1.5 mg/kg, n=6/group). Four and eighteen hours post-
injection, mice were asphyxiatied by CO2 inhalation and their peritoneal cavity
washed three times with 5 ml of ice-cold PBS. The total lavage was then pooled for
individual mouse and 200 µl processed for morphological cell analysis with cytospin
(Proudfoot et al., 2003). Briefly, 200 µl of peritoneal lavage were cytocentrifuged
(45g, 10 minutes) onto slides, air dried, and stained with May-Grünwald-Giemsa.
Total cells from washing collections were counted with a Beckman Coulter ® AcT
5diffTM.
Anti-chemotactic effect of PI3K inhibitors in RANTES-induced neutrophil peritoneal
recruitment
Lipopolysaccharide-free saline or r-hRANTES (0.5 mg/kg) were injected
intraperitoneally into mice which orally pretreated (30 minutes before r-hRANTES
injection) with LY294002 (30-100-300 mg/kg, n=6/group) or AS041164 (3-10-30
mg/kg, n=6/group). Four hours after r-hRANTES injection, mice were sacrificed, the
peritoneal cavity washed and cells processed as mentioned above.
Flow cytometric analysis
Cells obtained from peritoneal lavages were washed twice in PBS, counted, and
resuspended in FACS buffer (1% BSA in PBS containing 0.01% NaN3). For
phenotype analysis, cells (0.2-1.0 x 106 cells/stain) were initially incubated with
CD16/32 (2.4G2, Fc block) for 20 min at 4°C. Subsequently, cells were incubated
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with the appropriate antibody against cell surface markers: CD45 (30-F11), Gr1
(RB6-8C5), CCR5 (C34-3448), CD62L (MEL-14, BD Pharmingen, San Diego, CA,
USA) and CCR3 (83101) from R&D Systems (Minneapolis, MN, USA). All
incubations were performed in ice for 20 minutes followed by two washes with FACS
staining buffer (1% Bovine Serum Albumin in PBS). Appropriate isotype controls
were used in all cases. For flow cytometric analysis, a typical forward and side scatter
gate was set to exclude dead cells and aggregates; a total of 104 events in the gate
were collected and analysed using a FACS Calibur and Cell Quest software (BD
Biosciences, San Jose, CA, USA).
Immunohistochemistry analysis
Paraformaldeyde-fixed and paraffin-embedded mesenteric tissues were sectioned at
about 4-5 µm of thickness and deparaffinized/re-hydrated for immunoperoxidase
staining using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA).
Briefly, antigen-unmasking was performed by incubation in 10 mM sodium citrate
buffer (pH 6.0); endogenous peroxidase was quenched with 1% H2O2 for 10 minutes.
Non-specific immunoglobulin binding sites was blocked by incubating for 1 hour with
normal goat serum, then sections were incubated with the primary antibody anti-
phospho-c-AKT (Ser 473) and anti-AKT (Cell Signaling Technology, Beverly, MA,
USA) overnight at 4°C. Sections were successively incubated for 30 minutes with a
biotinylated secondary antibody solution followed by 30-minute incubation with ABC
reagent (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA, USA).
Immunoglobulin complexes were visualized by incubation with 3,3’-
diaminobenzidine, then washed, counterstained with hematoxylin, cleared,
dehydrated, mounted and examined by light microscopy. 10 fields were observed for
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each sample. As negative control for the immunohistochemical staining, tissue
sections were treated with normal serum instead of primary antibodies. Conventional
histological observation was performed using hematoxylin and eosin staining.
Western Blotting analysis
Cells obtained from peritoneal lavages were lysed with ice-cold lysis buffer (62.5 mM
Tris-HCl, pH 6.8 at 25°C, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v
bromophenol blue or phenol red). After sonication, lysates were centrifuged, protein
concentration determined and 20 µg of proteins separated by electrophoresis on 10%
SDS-PAGE and transferred onto a polyvinylidene difluoride-plus membrane. After
blocking with 5% milk, the immunoblots were probed with a 1:100 dilution anti-
phospho-c-AKT (Ser 473) antibody (Cell Signaling Technology) overnight at 4°C,
followed by a 1 hour incubation at room temperature with the corresponding
secondary antibodies. The blots were visualized with ECL-plus reagent. Phospho-
AKT immunoblots were then stripped with strip buffer at 50°C for 30 minutes and
reblotted for total AKT (Cell Signaling Technology). The volume of the protein bands
was quantified by a Bio-Rad ChemiDocTM EQ densitometer and a Bio-Rad Quantity
One® software (Bio-Rad Laboratories, Hercules, CA, USA). Total AKT was used as
loading control. Phosphorylation of AKT was measured as a ratio of phospho-AKT vs
total-AKT and expressed as fold change over the saline-treated control.
Carrageenan-induced inflammation
Male Wistar rats (100-150 g of body weight) were injected subplantarly with 0.1 ml
of a 1% λ carrageenan (Sigma, St. Louis, MO, USA) suspension in sterile saline.
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Animals were fasted for approximately 15 hours prior to experiment starting. Paw
volume was determined by water displacement, i.e. measuring the volume of
displaced fluid by means of a plethysmometer (Ugo Basile, Italy) 0, 1, 3, 5 and 24
hours after carrageenan challenge. Edema volumes were determined as the difference
between paw volume at each indicated time point and pre-injection values (time 0).
Paw swelling time-dependent variations in each treatment group were then compared
to control animals receiving vehicle alone. Compounds were administered half an
hour prior to carrageenan challenge.
Statistical Analysis
All values in the text and figures are presented as mean + SEM of (n) independent
experiments. All data were analysed by one-way ANOVA followed by Tukey test. P
values < 0.05 were considered statistically significant. For peritoneal chemotaxis
experiments, a dose-response curve was plotted from the inhibition values obtained
for each dose-group at the peak effect and, when possible, the relative ED50 value was
calculated using S-Plus 2000 v. 4.6 statistical software (Mathsoft Inc., Seattle, WA,
USA).
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Results
r-hRANTES promotes rapid recruitment of Gr1+ neutrophils
In order to characterize the cell subsets recruited by r-hRANTES, peritoneal exudate
cells (PECs) were harvested 4 and 18 hours after intraperitoneal injection. Total cells
from washing collections were counted with a Beckman Coulter ® AcT 5diffTM and
stained for cytospin analysis with May-Grünwald-Giemsa (Figure 1A, 1B and 1C). A
significant dose-dependent infiltration of neutrophils, with no changes in macrophage
counts, was observed 4 hours post injection (Figure 1A). At the 18 hours time point,
neutrophils presence was less evident (significant only at 1.5 mg/kg dose), and more
macrophages were observed, although this trend was not statistically significant
compared to saline-treated mice (Figure 1A). To further characterize the cell
composition of the peritoneal infiltrate cells were analysed by flow cytometry (Figure
1D). This analysis confirmed that 4 hours r-hRANTES administration resulted in
recruitment of Gr1-positive neutrophils, characterized by a low side scatter (Figure
1D). Accordingly, a histological analysis of the mesenteric tissue confirmed the
neutrophil infiltration 4 hours post r-hRANTES injection which is not present after
saline injection (Figure 2A and 2B). These results suggest that intraperitoneal r-
hRANTES injection results in a rapid neutrophils recruitment, which gradually
resolves in the absence of additional local inflammatory signals.
lnhibition of PI3K reduces r-hRANTES-induced neutrophils recruitment
To investigate the role of PI3K in r-hRANTES-induced chemotaxis, r-
hRANTES was injected in the presence and absence of a non-selective PI3K inhibitor
(LY294002; 30-300 mg/kg p.o.) or of a selective PI3Kγ-isoform inhibitor (AS041164;
3-100 mg/kg p.o.). Both inhibitors dose-dependently decreased r-hRANTES-induced
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neutrophil recruitment (Figure 3A e 3B). ED50 values for LY294002 and AS041164
were 81.59 mg/kg (95% C.I.=26.9 to 136.28) and 27.35 mg/kg (95%=C.I. 14.26 to
68.95), respectively. Since the two compounds showed a similar pharmacokinetic
profile in terms of bioavailability and half-life after oral route administration, the
selective inhibition of the PI3Kγ-isoform pathway, through AS041164 was three
times more potent in reducing neutrophil recruitment.
PI3Kγ inhibition reduces the level of AKT phosphorylation
To further characterize the mechanism by which these inhibitors block
neutrophil recruitment, r-hRANTES-injected mice were treated with LY294002 (100
mg/kg p.o.), AS041164 (30 mg/kg p.o.) or with the vehicle. Four hours post r-
hRANTES injection PECs were harvested, counted, and analyzed by western blot for
the level of phosphorylation of the downstream PI3K substrate Ser/Thr kinase AKT.
As previously noted, both compounds significantly decreased the number of recruited
neutrophils (Fig. 3). Furthermore, in vivo treatment with both inhibitors resulted in a
significantly decreased r-hRANTES-induced AKT phosphorylation (Figure 4).
Accordingly, morphological analysis of mesenteric tissue revealed that r-hRANTES
injection increased the number of phospho-AKT positive neutrophils (Figure 5B, 5F)
compared to saline-injected mice (Figures 5A, 5E). Moreover, a complete abrogation
of immune cell chemotaxis was observed in r-hRANTES-injected mice treated with
LY294002 (Figures 5C, 5G) or AS041164 (Figures 5D, 5H). Finally, a concomitant
flow cytometric analysis of PECs defined a Gr1-positive neutrophil population in r-
hRANTES-injected saline-treated mice, which was not observed in mice treated with
either of the PI3K inhibitors (Figures 5J, 5K & 5L).
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To further characterize the cellular infiltrate in the peritoneal cavity, we
performed a flow cytometric analysis of PECs harvested from r-hRANTES-injected
mice. This analysis confirmed that r-hRANTES administration resulted in infiltration
of Gr1-positive neutrophils. In addition, recruited neutrophils displayed a low side
scatter, were positive for L-selectin (CD62L), and negative for CCR3 and CCR5
(Figure 6). In contrast, peritoneum-resident neutrophils isolated from saline-treated
mice showed a high side scatter, mild expression of the lineage marker Gr1, were
negative for CD62L, but strongly expressed the two RANTES receptors CCR3 and
CCR5. Importantly, the administration of both PI3K inhibitors was able to inhibit the
recruitment of this RANTES-induced neutrophil population (Figure 6).
Inhibition of PI3K significantly reduce paw swelling after carrageenan challenge
The administration of AS041164 in a carrageenan-induced inflammation
model in rat showed a significant inhibition of paw swelling. In particular (Figure 7),
AS041164 at the dose of 100 mg/kg p.o. induced a significant reduction of paw
thickness that was maximal 3 hours after the carrageenan injection (p<0.01), during
the PIR phase when a massive infiltration of neutrophils into the dermal tissue is
reported. Indomethacin (at 5 mg/kg p.o.) was included in the experimental design as
internal reference control since efficacy of non-steroidal anti-inflammatory drugs is
described in this model (Otterness and Moore, 1988). Indomethacin administration
exerted an inhibitory effect on paw edema, in fact animals belonging to that treatment
group presented a significantly reduced (p<0.001) swelling again 3 hours after
carrageenan injection.
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Discussion
In this study, we used a murine model of peritoneal chemotaxis to show that r-
hRANTES injection induces a dose-dependent neutrophil recruitment, via the PI3Kγ
pathway. This evidence is confirmed by morphological and histological analyses
showing that neutrophils are the major recruited population during the early phase of
inflammation. In addition, a cytofluorimetric analysis of cellular peritoneum infiltrate
shows that the RANTES-recruited neutrophil population display a different phenotype
and can be distinguished from resident cells. Resident neutrophils display a high side
scatter and mild expression of the lineage marker Gr1. They are negative for L-
selectin (CD62L), however, they strongly express the two RANTES receptors CCR3
and CCR5 (Proudfoot, 2002). On the contrary, RANTES-recruited neutrophils
presents a low side scatter and high Gr1 expression, they are L-selectin positive and
CCR3- and CCR5-negative. Molecules of the selectin family such as L-selectin are
constitutively expressed on leukocytes and shed from the cell surface upon activation
(Lee et al., 2006). It is therefore possible that neutrophils transiently recruited by
RANTES in the absence of additional inflammatory signals do not become activated
in the absence of local inflammatory stimuli.
Neutrophils are not only the most abundant cell type in inflamed joints, they
are also important in the initiation and perpetuation of the inflammation in both
human rheumatoid arthritis and mouse models (Kistis and Weissmann, 1991; Wipke
et al., 2001). Neutrophils and inflammatory monocytes are recruited rapidly into sites
of infection. The initial influx of leukocytes into inflamed tissues is dominated by
neutrophils. This dominance may reflect the higher frequency of neutrophils in
peripheral blood compared to monocytes ( Muller and Randolph, 1999). Later in
inflammation, monocytes/macrophages gradually replace neutrophils as the
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predominant leukocyte subtype. Recruited neutrophils are thought to mediate this
switch by releasing soluble factors into the early inflammatory milieu, thus promoting
monocyte recruitment (Issekutz and Movat, 1980; Doherty et al., 1988; Henderson et
al., 2003). Neutrophils and monocytes migrate across the endothelium into tissues, in
response to endothelial cell–bound factors, such as chemokines, that deliver activating
and chemoattracting signals (Proudfoot, 2002). This critical role of chemokines in the
recruitment of myeloid cells is well established in murine in vivo models (Baggiolini
et al., 1997). CC chemokines, including RANTES, are known to act on monocytes,
lymphocytes, basophils, and eosinophils, but do not usually target neutrophils.
Traditionally, human PMN have been thought to express receptors for the CXC
(Baggiolini, 1998) or CX3C (Baggiolini et al., 1997) families and to preferentially
migrate towards chemokine ligands from these two families (Pan et al., 1997).
However, different studies have provided conflicting evidences on whether human
PMNs express CCR receptors. Although Xu et al. (Xu et al., 1995) found that resting
human PMNs possess binding sites for the CC chemokines macrophage inflammatory
protein (MIP)-1α and monocyte chemoattractant protein (MCP)-3, it is generally
accepted that CC chemokines have no direct functional effect on resting human PMN.
In contrast to findings in human PMNs, experiments performed in several murine
models of inflammation have shown that CC chemokines do play a role in PMN
chemotaxis (Gao et al., 1997). For example, in vivo studies of LPS- and endotoxemia-
associated lung injury in mice have demonstrated that neutralization of MIP-1α
attenuates neutrophil infiltration into inflammatory sites (Gao et al., 1997).
Furthermore, PMN isolated from inflammatory exudates in mice display chemotactic
migration and calcium flux responses to MIP-1α (Gao et al., 1997). Furthermore, a
growing body of literature demonstrates that cytokines regulate the expression of
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chemokine receptors on a variety of cell subsets. Thus, pro-inflammatory mediators
such as TNF-α, IL-1β, and LPS have been shown to down-regulate CCR2 expression
by human monocytes (Sica et al., 1997), while the immunomodulatory cytokine IL-10
up-regulates CCR1, CCR2, and CCR5 on these cells (Sozzani et al., 1998). PI3K has
a central role in the regulation of inflammatory responses, whereas the isoforms α and
β are almost ubiquitously expressed and regulate a variety of cell functions as
proliferation and survival, the δ and γ isoforms are mostly expressed in the
hematopoietic system and have been shown to regulate immune responses (Bi et al.,
2002). Furthermore, it has been demonstrated that PI3Kγ plays a central role in
chemokine-induced recruitment of leukocytes (Sasaki et al., 2000). In fact,
PI3Kγ knock out mice showed a reduced chemoattractant-induced respiratory burst,
defective migration of macrophages and neutrophils to inflammation sites, as well as
impairments in adaptive immunity in general and in T-cells activation in particular
(Del Prete et al., 2004). In addition, impairment in the capacity of chemokines to
stimulate the phosphoinositides-dependent kinase cascades (e.g. PKB/AKT) has been
shown in cells that lack PI3Kγ expression (Hirsh et al., 2000). Accordingly, PI3Kγ -/-
mice are resistant to collagen II-specific antibody-induced arthritis and the
administration of a selective PI3Kγ inhibitor was shown to suppress the progression of
joint inflammation and damage in a mouse model of collagen-induced arthritis
(Camps et al., 2005).
In the present work, we evaluated the effect of two PI3K inhibitors with
different specificity and similar pharmacokinetic profiles, namely LY294002
(Morpholin-4-yl-8-phenyl-chromen-4-one described in Vlahos et al., 1994), a non-
isoform selective PI3K inhibitor with low affinity for the γ-isoform (Ward et al.,
2003), and AS041164 (5-Benzo[1,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione,
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synthesis described in Rückle et al., 2004), a selective PI3Kγ inhibitor (see Tables I
and II for more details). We found that AS041164-mediated inhibition of RANTES-
induced chemotaxis is three times more potent compared to that of LY294002,
suggesting that the PI3Kγ isoform has a specific role in RANTES-induced
chemotaxis. To further support these data, the level of AKT phosphorylation
following RANTES injection was investigated in the presence and absence of the two
inhibitors. We observed that both LY294002 and AS041164 decreased the level of
AKT phosphorylation in PECs. Finally, we demonstrated that inhibition of
PI3Kγ pathway in vivo results in the reduction of inflammatory swelling in the model
of carrageenan-induced paw edema and that maximal effect is obtained at time-points
in which neutrophils are massively recruited.
Together, our data suggest that the mechanism by which RANTES-induces
neutrophil recruitment involves PI3K/AKT pathway activation, via class 1B G-protein
coupled receptors. The administration of both PI3K inhibitors specifically blocks
RANTES-mediated cellular recruitment as well as inflammatory infiltrates induced by
a flogistic agent such as carrageenan. Interestingly suppression of joint inflammation
in murine models of arthritis by PI3Kγ inhibition has been reported to occur through a
reduced presence of neutrophils in joint lesions (Camps et al., 2005).
In conclusion, the present findings suggest that PI3Kγ plays an important role
in controlling neutrophil chemotaxis in early steps of inflammation. Therefore,
pharmacological interventions targeting PI3Kγ could represent an interesting and
promising new therapeutic approach to treat inflammatory diseases.
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Acknowledgments
We wish to thank Alberto Franzino for his technical support and Amanda Proudfoot
for providing us all the biochemical tools for the validation of the chemotaxis
peritoneal recruitment model.
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References
Akasaki T, Koga H and Sumimoto H (1999). Phosphoinositide 3-kinasedependent and
-independent activation of the small GTPase Rac2 in human neutrophils. J.
Biol. Chem. 274:18055-18059.
Al-Aoukaty A, Rolstad B and Maghazachi AA (1999). Recruitment of Pleckstrin and
Phosphoinositide 3-Kinaseγ into the Cell Membranes, and Their Association
with Gß γ After Activation of NK Cells with Chemokines. J Immunol.
162:3249-3255.
Baggiolini M (1998). Chemokines and leukocyte traffic. Nature 392:565-568.
Baggiolini M, Dewald B and Moser B (1994). Interleukin-8 and related chemotactic
cytokines: CXC and CC chemokines. Adv. Immunol. 55:97-179.
Baggiolini M, Dewald B and Moser B (1997). Human chemokines: an update. Annu.
Rev Immunol. 15: 675-705.
Barnes DA, Tse J, Kaufhold M, Owen M, Hesselgesser J, Strieter R, Horuk R and
Perez HR (1998). Polyclonal antibody directed against human RANTES
ameliorates disease in the Lewis rat adjuvant-induced arthritis model. J Clin
Invest. 101:2910-2919.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 25, 2007 as DOI: 10.1124/jpet.107.123026
at ASPE
T Journals on January 4, 2020
jpet.aspetjournals.orgD
ownloaded from
JPET #123026
21
Bi L, Okabe I, Bernard DJ and Nussbaum RL (2002). Early embruonic lethality in
mice deficient in the p110beta catalytic subunit of PI 3-kinase. Mamm.
Genome 13:169-172.
Brando B, Barnett D, Janossy G, Mandy F, Autran B, Rothe G, Scarpati B, D'Avanzo
G, D'Hautcourt JL, Lenkei R, Schmitz G, Kunkl A, Chianese R, Papa S and
Gratama JW. (2000) Cytofluorometric methods for assessing absolute
numbers of cell subsets in blood. European Working Group on Clinical Cell
Analysis Cytometry 42:327-346
Camps M, Ruckle T, Ji H, Ardissone V, Rintelen F, Shaw J, Ferrandi C, Chabert C,
Gillieron C, Françon B, Martin T, Gretener D, Perrin D, Leroy D, Vitte PA,
Hirsch E, Wymann MP, Cirillo R, Schwarz MK and Rommel C (2005).
Blockade of PI3Kγ suppresses joint inflammation and damage in murine
models of rheumatoid arthritis. Nat. Med. 11:936-943.
Del Prete A, Vermi W, Dander E, Otero K, Barberis L, Luini W, Bernasconi S, Sironi
M, Santoro A, Garlanda C, Facchetti F, Wymann MP, Vecchi A, Hirsch E,
Mantovani A and Sozzani S (2004) Defective dendritic cell migration and
activation of adaptative immunity in PI3Kgamma-deficient mice. EMBO J.
23:3505-3515.
Doherty DE, Downey GP, Worthen GS, Haslett C and Henson PM. (1988) Monocyte
retention and migration in pulmonary inflammation: requirement for
neutrophils. Lab. Invest. 59:200-213.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 25, 2007 as DOI: 10.1124/jpet.107.123026
at ASPE
T Journals on January 4, 2020
jpet.aspetjournals.orgD
ownloaded from
JPET #123026
22
Gao JL, Wynn TA, Chang Y, Lee EJ., Broxmeyer HE., Cooper S, Tiffany HL,
Westphal H, Kwon-Chung J and Murphy PM (1997) Impaired host defense,
hematopoiesis, granulomatous inflammation and type 1-type 2 cytokine
balance in mice lacking CC chemokine receptor 1. J. Exp. Med. 185:1959-
1968.
Henderson RB, Hobbs JAR, Mathies M and Hogg N (2003). Rapid recruitment of
inflammatory monocytes is indipendent of neutrophil migration. Blood
102:328-335.
Hirsch E, Katanaev LV, Garlanda C, Azzolino O, Pirola L, Silengo L, Sozzoni S,
Mantovani A, Altruda F and Wymann MP (2000) Central role for G protein-
coupled phosphoinositide 3-kinase gamma in inflammation. Science
287:1049-1053.
Issekutz AC and Movat HZ (1980). The in vivo quantitation and kinetics of rabbit
neutrophil leukocyte accumulation in the skin in response to chemotactic
agents and Escherichia coli. Lab Invest. 42:310-317.
Kernacki KA, Barrett RP, Hobden JA and Hazlett LD (2000). Macrophage
inflammatory protein-2 is a mediator of polymorphonuclear neutrophil influx
in ocular bacterial infection. J. Immunol. 164:1037-1045.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 25, 2007 as DOI: 10.1124/jpet.107.123026
at ASPE
T Journals on January 4, 2020
jpet.aspetjournals.orgD
ownloaded from
JPET #123026
23
Kistis E. and Weissmann G (1991). The role of the neutrophil in rheumatoid arthritis.
Clin. Orthop. 265:63-72.
Lee D, Schultz JB, Knauf Pa and King MR (2006) Mechanical sheddingof L-selectin
from the neutrophil surface during rolling on sialyl Lewis X under flow. J.
Biol. Chem. 282: 4812-4820.
Lee J, Cacalano G, Camerato T, Toy K, Moore MW and Wood WI (1995)
Chemokine binding and activities mediated by the mouse IL-8 receptor. J.
Immunol. 155:2158-2564.
Mehrad B, Strieter RM, Moore TA, Tsai WC, Lira SA and Standiford TJ (1999) CXC
chemokine receptor-2 ligands are necessary components of neutrophil-
mediated host defense in invasive pulmonary aspergillosis. J. Immunol.
163:6086-6094.
Moore TA, Newstead MW, Strieter RM, Mehrad B, Beaman BL and Standiford TJ
(2000) Bacterial clearance and survival are dependent on CXC chemokine
receptor-2 ligands in a murine model of pulmonary Nocardia asteroids
infection. J. Immunol. 164:908-915.
Muller WA and Randolph GJ (1999) Migration of leukocytes across endothelium and
beyond: molecules involved in the transmigration and fate of monocytes J.
Leukoc. Biol. 66: 698-704.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 25, 2007 as DOI: 10.1124/jpet.107.123026
at ASPE
T Journals on January 4, 2020
jpet.aspetjournals.orgD
ownloaded from
JPET #123026
24
Otterness IG and Moore PF (1988) Carrageenan foot edema test. Methods Enzymol.
162:320-327.
Pan Y, Lloyd C, Zhou H, Dolich S, Deeds J, Gonzalo JA, Vath J, Gosselin M, Ma J,
Dussault B, Woolf E, Alperin G, Culpepper J,. Gutierrez-Ramos JC and
Gearing D (1997) Neurotactin, a membrane-anchored chemokine upregulated
in brain inflammation. Nature 387:100-617.
Proudfoot AE, Handel TM, Johnson Z, Lau EK, LiWang P, Clark-Lewis I, Borlat F,
Wells TN and Kosco-Vilbois MH (2003) Glycosaminoglycan binding and
oligomerization are essential for the in vivo activity of certain chemokines.
Proc Natl Acad Sci 100:1885-1890.
Proudfoot, AEI (2002) Chemokine receptors: multifaced therapeutic targets. Nature
Reviews Immunol. 2:106-115.
Rückle T, Jiang X, Gaillard P, Church D and Vallotton T (2004). Azolidinone-vinyl
fused-benzene derivatives. WO2004007491 (Applied Research Systems Ars
Holding N.V.)
Sasaki T, Irie-Sasaki J, Jones RJ, Oliveira-dos-Santos AJ, Stanford WL, Bolon B,
Wakeham A, Itie A, Bouchard D, Kozieradzki I, Joza N, Mak TN, Ohashi PS,
Suzuki A and Penninger JM (2000) Function of PI3Kgamma in thymocyte
development, T cell activation and neutrophil migration. Science 287:1040-
1046.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 25, 2007 as DOI: 10.1124/jpet.107.123026
at ASPE
T Journals on January 4, 2020
jpet.aspetjournals.orgD
ownloaded from
JPET #123026
25
Shahrara S, Amin MA, Woods JM, Haines GK and Kock AE (2003). Chemokine
receptor expression and in vivo signaling pathways in the joints of rats with
adjuvant-induced arthritis. Arthritis Rheum. Dec 48:3568-3583.
Sica A, Saccani A, Borsatti A, Power CA, Wells TN, Luini W, Polentarutti N,
Sozzani S and Mantovani A (1997) Bacterial lipopolysaccharide rapidly
inhibits expression of C-C chemokine receptors in human monocytes. J. Exp.
Med. 185:969-974.
Sozzani S, Ghezzi S, Iannolo G, Luini W, Borsatti A, Polentarutti N, Sica A, Locati
M, Mackay C, Wells TN, Biswas P, Vicenzi E, Poli G and Mantovani A
(1998) Interleukin 10 increases CCR5 expression and HIV infection in human
monocytes. J. Exp. Med. 187:439-444.
Stephens L, Anderson, Stokoe D, Erdjument-Bromage H, Painter GF, Holmes AB,
Gaffney PRJ, Reese CB, McCormick F, Tempst P, Coadwell J and Hawkins
PT (1998) Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-
trisphosphate-dependent activation of protein kinase B. Science 279:710-714.
Tessier PA, Naccache PH, Clark-Lewis I, Gladue RP,. Neote KS and McColl SR
(1997) Chemokine networks in vivo: involvement of C-X-C and C-C
chemokines in neutrophil extravasation in vivo in response to TNF-α. J.
Immunol. 159:3595-3602.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 25, 2007 as DOI: 10.1124/jpet.107.123026
at ASPE
T Journals on January 4, 2020
jpet.aspetjournals.orgD
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JPET #123026
26
Vinegar R., Truax JF, Selph JL, Johnston PR, Venable AL and McKenzie KK. (1987)
Pathway to carrgeenan-induced inflammation in the hind limb of the rat.
Federation Proc. 46:118-126
Vlahos CJ, Matter WF, Hui KY and Brown RF (1994) A specific inhibitor of
phosphatidylinositol 3-kinase, 2-(4- morpholinyl)-8-phenyl-4H-1-benzopyran-
4-one (LY294002) J. Biol. Chem. 269:5241-5248
Ward S, Sotsios Y, Dowden J, Bruce I and Finan P (2003) Therapeutic potential of
Phosphoinositide 3-Kinase inhibitors. Chemistry & Biology 10:207-213.
Wipke BT and Allen PM (2001) Essential role of neutrophils in the initiation and
progression of a murine model of rheumatoid arthritis. J. Immunol 167:1601-
1608.
Xu LL, McVicar DW, Ben-Baruch A, Kuhns DB, Johnston J, Oppenheim JJ and
Wang JM (1995) Monocyte chemotactic protein-3 (MCP3) interacts with
multiple leukocyte receptors: binding and signaling of MCP3 through shared
as well as unique receptors on monocytes and neutrophils. Eur. J. Immunol.
25:2612-2617.
Yoshie O, Imai T and Nomiyama H (2001) Chemokines in immunity. Adv. Immunol.
78:57-110.
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 25, 2007 as DOI: 10.1124/jpet.107.123026
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JPET #123026
27
Zhang S, Youn BS, Gao JL, Murphy PM and Kwon BS (1999) Differential effects of
leukotactin-1 and macrophage inflammatory protein-1α on neutrophils
mediated by CCR1. J. Immunol. 162:4938-4942.
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Legends for Figures
Figure 1. r-hRANTES-induced recruitment of neutrophils and macrophages to the
mouse peritoneal cavity. A. Peritoneal exudate cells (PECs) were harvested and
counted with a Beckman Coulter ® AcT 5diffTM 4 and 18 hours post r-hRANTES
injection. Each point represents the mean + SEM of 3 separate experiments
(n=6/group). * p<0.05; *** p<0.001 vs saline-treated control group. B. Cell
morphology by cytospin analysis of peritoneal lavages collected 4 hours after r-
hRANTES injection (magnification 400x). Blue arrows indicate neutrophils. C.
Beckman Coulter ® AcT 5diffTM analysis of peritoneal lavages after 4 hours. Blue
arrows indicate neutrophils. D. Cytofluorimetric analysis on peritoneal cells from
peritoneal lavages collected 4 hours following saline or r-hRANTES injection (0.5
mg/kg, i.p.). We identified neutrophil population using the physical gate, side scatter
vs forward scatter, designed to highlight physical properties as dimension and
complexity of leukocytes and the leukogate, side scatter vs CD45, in order to show
the characteristic positivity of lymphocytes, monocytes and PMNs for CD45 (Brando
et al., 2000). Using these two gates and high fluorescence for the neutrophil lineage
marker Gr1, we identified the two neutrophils population.
Figure 2. Hematoxylin and eosin staining of mesenteric tissue 4 hours after saline (A)
or r-hRANTES injection at 0.5 mg/kg i.p. (B). Magnification 100x and 400x in the
upper and lower panels, respectively.
Figure 3. Effect of LY294002 (A) and AS041164 (B) on r-hRANTES-induced
neutrophil recruitment in the peritoneal cavity. Cell counts were determined 4 hours
after rhRANTES challenge. Each point represents the mean + SEM of 3 separate
experiments (n=6/group). *, **, *** p<0.05, p<0.01, p<0.001 vs saline-treated control
group.
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Figure 4. Western blot analysis of phospho-AKT and total AKT in cells obtained
from peritoneal lavages after r-hRANTES injection in the presence and in the absence
of LY294002 or AS041164. Total AKT was used as loading control. Phosphorylation
of AKT was measured by band densitometry analysis and indicated as a ratio of
phospho-AKT vs total-AKT. Data have been expressed as fold change over the saline-
treated control. The fold change represents the mean + SEM of 3 independent
experiments. * p<0.05; ** p<0.01”
Figure 5. Hematoxylin and eosin staining of mesenteric tissue (A-D),
immunohistochemistry staining on phospho-AKT (Ser 473) (E-H) and FACS analysis
of Gr1 cell surface marker on cells obtained from peritoneal lavages (I-L) The dot plot
colour code is the following: lymphocytes are represented in green, monocytes in red
and neutrophils in blue. Effect of saline (A, E, I) and of r-hRANTES at 0.5 mg/kg i.p.
alone (B, F, J) and in the presence of LY294002 at 100 mg/kg p.o. (C, G, K) or
AS041164 at 30 mg/kg p.o. (D, H, L). Blue arrows indicate neutrophils.
Figure 6. Cytofluorimetric analysis on peritoneal cells from peritoneal lavages
collected 4 hours following r-hRANTES injection. Cell baseline surface markers
expression of Gr1, L-selectin, CCR3, CCR5 and CD45 on peritoneal cells without r-
hRANTES administration (lane A). Effect of r-hRANTES administration (0.5 mg/kg,
i.p.) in the absence (lane B) and in the presence of LY294002 at 100 mg/kg p.o. (lane
C) or AS041164 at 30 mg/kg p.o. (lane D).
Figure 7. Effect of AS041164 and indomethacin on carrageenan-induced
inflammation model in rats. Paw swelling is expressed as variation with respect to
pre-injection basal values. Each point represent the mean + SEM of 2 separate
experiments (n=10/group). *p<0.05, ** p<0.01, *** p<0.001 AS041164 (black closed
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circle) and Indomethacin (grey diamond) treated animals compared to vehicle-treated
animals.
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Table 1
PI3K isoforms selectivity profile of AS041164 and LY294002.
Protein kinases were assayed for their ability to phosphorylate the appropriate
peptide/protein substrates. PI3Kγ lipid kinase assay, based on neomycin-coated
Scintillation Proximity Assay (SPA) bead technology (Amersham Biosciences,
Piscataway, NJ, USA), was performed in 96 well plates using ATP/[γ33P]ATP and
PtdIns (Sigma) as substrates, as described (Camps et al., 2005). IC50-value
derminations for selectivity evaluation were run at ATP concentrations corresponding
to experimental Km values found for each isoform. Kinase assays for IC50
determinations with PI3Kα, PI3Kβ and PI3Kδ were carried out as described (Camps
et al., 2005).
Inhibitor (IC50 µM) PI3K γ PI3K α PI3K β PI3K δ
AS041164 0.07 0.24 1.45 1.70
LY294002 6.60 0.73 0.31 1.05
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Table 2
Kinase selectivity profile of AS041164 and LY294002.
Kinase Activity
(% Control)
Kinase Activity
(% Control)
KINASE AS041164 LY294002 KINASE AS041164 LY294002
C-RAF (h) 104 61 p70S6K (h) 92 90
MEK1 (h) 104 105 CHK1 (h) 101 105
MKK6 (h) 104 107 PKBα (h) 96 91
SAPK2a (h) 103 93 cSRC (h) 98 94
SAPK2b (h) 91 101 CDK2/cyclinA (h) 97 75
SAPK3 (h) 81 81 CaMKII (r) 92 97
SAPK4 (h) 96 89 PRK2 (h) 112 76
MAPKAP-K2 (h) 107 91 CHK2 (h) 105 97
MSK1 (h) 94 99 Fyn (h) 85 99
MKK4 (m) 113 86 JNK3 (r) 109 82
MKK7β (h) 103 113 CDK1/cyclinB (h) 81 84
JNK1α1 (h) 108 101 MAPK1 (h) 95 101
JNK2α2 (h) 107 96 PKCβII (h) 116 101
PDK1 (h) 103 98 PKCγ (h) 108 98
SGK (h) 97 101 ZAP-70 (h) 107 81
GSK3β (h) 97 56 CK1 (y) 87 47
PKA (b) 97 96 MAPK2 (m) 100 84
PKCα (h) 101 101 AMPK (r) 104 95
ROCK-II (r) 103 107 Lck (h) 108 179
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h = human, r = rat, y = yeast, m = mouse, b = bovine
All assays were performed in duplicate at 1µM compound concentration, and in each
case, the kinase activity is expressed as a percentage of that in controls. ATP was
10µM in all assays. For method details refer to Camps et al. (Camps et al., 2005).
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