JPET #123026 PI3Kγ inhibition plays a crucial role in...

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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.

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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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

[email protected]

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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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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