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(Journal of Leukocyte Biology. 2005;77:800-810.)
© 2005 by Society for Leukocyte Biology

Phosphoinositide-3 kinases critically regulate the recruitment and survival of eosinophils in vivo: importance for the resolution of allergic inflammation

Vanessa Pinho*, Danielle G. Souza*, Michele M. Barsante*, Fabiana P. Hamer*, Marta S. De Freitas{dagger}, Adriano G. Rossi{ddagger} and Mauro M. Teixeira*,1

* Immunopharmacology, Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil;
{dagger} Departamento de Farmacologia, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Brazil; and
{ddagger} Respiratory Medicine Unit, MRC, Centre for Inflammation Research, University of Edinburgh Medical School, Scotland

1 Correspondence: Imunofarmacologia, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627–Pampulha, 31270-901–Belo Horizonte, MG, Brazil. E-mail: mmtex{at}icb.ufmg.br


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The phosphatidylinositol-3 kinase (PI3K) family of signaling enzymes plays a crucial role in leukocyte recruitment and activation and hence, likely regulates the induction and propagation phases of inflammation. However, little data have emerged showing a role for these processes in the resolution phase in models of in vivo inflammation. Here, we have evaluated the role of PI3K for the migration and survival of eosinophils in a model of allergic pleurisy in mice. Eosinophil accumulation in PI3K{gamma}-deficient mice was inhibited at 48 h, as compared with wild-type mice but not at earlier time-points (6 and 24 h). Experiments with adoptive transfer of bone marrow showed that PI3K{gamma} in eosinophils but not in non-bone marrow-derived cells was required for their accumulation. Systemic treatment with PI3K inhibitors before antigen challenge prevented the recruitment of eosinophils. This was associated with decreased Akt phosphorylation, interleukin-5 production, and eosinophil release from the bone marrow. Treatment with PI3K inhibitors 24 h after antigen challenge markedly cleared the accumulated eosinophils, an effect associated with inhibition of Akt phosphorylation and an increased number of apoptotic events. Altogether, our data demonstrate an important role of PI3K{gamma} for the maintenance of eosinophilic inflammation in vivo, whereas other isoforms of PI3K may be relevant for the recruitment process.

Key Words: eosinophilia • signal transduction • apoptosis • allergy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphatidylinositol-3 kinases (PI3Ks) are a family of proteins that catalyze the phosphorylation of the 3'-OH position of the inositol ring of PIs, resulting in the formation of PI(3)P, PI(3,4)P2, and PI(3,4,5)P3, collectively termed 3'-PI lipids [1 , 2 ]. These molecules work as second messengers and are important in receptor signaling by growth factors, cytokines, and hormones [1 2 3 4 ]. There are different isoforms of PI3Ks, which can be divided in three main classes. The class I PI3Ks are heterodimers consisting of a catalytic subunit and an adaptor/regulatory subunit present mainly in the cytosol of cells and can be subdivided in two subclasses. Class IA enzymes may have one of three catalytic isoforms (p110{alpha}, p110ß, or p110{delta}) associated to five regulatory isoforms and signal downstream of tyrosine kinases. Class IB enzymes are constituted of a unique catalytic subunit (p110{gamma}) associated with a regulatory subunit p101 and signal downstream of heterotrimeric G-protein-coupled receptors (GPCRs). There is also a class II PI3K, characterized by the presence of a C2 domain, and a class III PI3K, which uses only PI as a substrate [1 , 2 ].

Recently, several studies have demonstrated the importance of PI3K in cellular migration in vitro and in vivo [5 6 7 8 9 ]. Early evidence for the role of PI3K in chemotaxis was the demonstration that human T cell migration induced by regulated on activation, normal T expressed and secreted was PI3K activation-dependent [10 ]. In the immune system, PI3K may also be activated by antigen receptors, such as the B cell, the T cell, and Fc receptors (FcRs)–receptors for mitogenic and inflammatory cytokines–and chemokine receptors [11 ].

Eosinophils are effector cells that play an important role in the pathophysiology of allergic disease [12 13 14 ]. In allergic diseases, such as asthma, eosinophils are a crucial source of cytotoxic proteins, lipid mediators, oxygen metabolites, and cytokines, which may contribute to the severity of disease [15 ]. Thus, there is a great interest in the understanding of mechanisms involved in the recruitment, activation, and survival of eosinophils in inflammatory sites. Eosinophil recruitment to sites of allergic inflammation depends on the concerted action of a variety of molecules, including chemokines, which act through GPCRs, and may result in the activation of PI3K isoforms, especially PI3K{gamma} [4 , 11 , 16 ]. Several studies have now shown that the deficiency of PI3K{gamma} impairs the migration of neutrophils and macrophages in vitro and in a septic peritonitis model [6 , 8 ]. PI3K activation may also be important for the induction of survival in cells via the phosphorylation of Akt/protein kinase B (PKB) [2 , 11 , 17 , 18 ]. However, little is known about the role of PI3K in the recruitment and/or clearance of eosinophils in sites of allergic reactions. Moreover, the role of PI3K{gamma} isoforms for eosinophil migration has not been determined.

In this study, we have investigated the role of PI3K for the recruitment and survival of eosinophils into the pleural cavity of antigen-challenged and -sensitized mice. Initial experiments investigated the role of the PI3K{gamma} isoform by using PI3K{gamma}-deficient mice (PI3K{gamma}–/–). In addition, we investigated the role of PI3K in the recruitment and survival of eosinophils in the allergic pleurisy model by using wortmannin and LY294002, inhibitors of all isoforms of PI3K [19 ].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
PI3K{gamma}–/– male C57Bl/6 x sv129 mice and their wild-type (WT) littermate control mice (+/+; 18–22 g) were used throughout these experiments, and those with drugs were conducted in BALB/c mice (18–22 g). PI3K{gamma}–/– mice (in a C57Bl/6xsv129 background) were generated as described previously [8 ]. The mice were a kind gift of Dr. Josef M. Penninger and supplied by Taconic Farms, Inc. (Germatown, NY). PI3K{gamma} gene deletion was confirmed by polymerase chain reaction using specific primers (WT primers: sense, 5-TCAGGCTCGGAGATTAGGTA-3; antisense, 5-GCCCAATCGGTGGTAGAACT-3; PI3K{gamma}–/– primers: sense, 5-GGACACGGCTTTGATTACAATC-3; antisense, 5-GGGGTGGGATTAGATAAATG-3, as described previously; ref. [8 ]). Animals were bred and housed in a temperature-controlled room with free access to water and food. All experimental protocols have been subjected to evaluation and were approved by the local animal ethics committee.

Drugs and reagents
Recombinant murine eotaxin was purchased from Peprotech (London, UK). Eotaxin was dissolved in water, diluted further in phosphate-buffered saline (PBS; pH 7.4) containing 0.01% bovine serum albumin (BSA), and stored at –20°C until use. Wortmannin was purchased from Calbiochem (San Diego, CA). Wortmannin was diluted in dimethyl sulfoxide (DMSO) and stored at –70°C until use. LY294002 was purchased from Alamone Laboratories (Jerusalem, Israel), diluted in DMSO, and stored at –70°C until use. Ovalbumin (OVA) was purchased from Sigma Chemical Co. (St. Louis, MO). Annexin-V was a gift of Dr. Gustavo Amarantes-Mendes (Universidade de São Paulo, Brazil).

Sensitization
Animals were immunized with OVA, adsorbed to an aluminium hydroxide gel, as described previously [20 ]. Briefly, mice were injected subcutaneously on days 1 and 8 with 0.2 ml solution containing 100 µg OVA and 70 µg aluminum hydroxide (Reheiss, Dublin, Ireland).

Leukocyte migration into the pleural cavity induced by antigen
Sensitized WT or PI3K{gamma}–/– mice were challenged by intrapleural (i.pl.) administration of antigen (OVA) or PBS. The cells present in the pleural cavity were harvested 6, 24, or 48 h after antigen challenge by injecting 2 ml PBS and total cell counts performed in a modified Neubauer chamber using Turk’s stain. For the experiments evaluating leukocyte apoptosis, infiltrating leukocytes were examined 2, 6, and 48 h after antigen challenge. Differential cell counts were performed on cytospin preparations (Shandon III) stained with May-Grumwald-Giemsa using standard morphologic criteria to identify cell types. The results are presented as the number of cells per cavity.

Production of chimeric mice
Chimeric mice, which lacked PI3K{gamma} on bone marrow-derived or on non-bone marrow-derived cells, were produced by transplanting bone marrow cells into irradiated recipient animals. Femurs and tibias of WT or PI3K{gamma}–/– donor mice were dissected and flushed with 2 ml incomplete RPMI medium. The resultant cell suspension was washed twice and resuspended in PBS. Recipient WT or PI3K{gamma}–/– mice were irradiated with 900 rad and then injected intravenously with 3 x 107 bone marrow cells in 0.2 ml PBS from WT or PI3K{gamma}–/– donor mice. Blood of bone marrow-chimeric mice was tested for the total number of circulating leukocytes at 7, 14, and 21 days after reconstitution. All mice were treated intraperitoneally (i.p.) with antibiotic (ciprofloxacin, 100 µg/animal/day) for 10 days. After 21 days, the total and differential number of leukocytes had normalized (data not shown), and mice were submitted to protocols of sensitization and challenge with OVA or PBS.

Collection of bone marrow and peripheral blood cells
Bone marrow cells were isolated from the left femur. The femoral head and condyles were removed, and the displaceable cells were recovered by flushing the marrow cavity of the femur shaft with 1 mL PBS containing heparin (10 U/ml). Blood samples were collected from the brachial plexus. Total cell counts were performed in a modified Neubauer chamber using Turk’s stain. Differential cell counts were performed on cytospin preparations (Shandon III) stained with May-Grumwald-Giemsa using standard morphologic criteria to identify cell types. The results are presented as the number of cells/femur or cells/ml blood.

Treatment with inhibitors of PI3K
The role of PI3K on eosinophil recruitment in the allergic pleurisy was also investigated by using selective inhibitors of PI3K, wortmannin, and LY294002. Wortmannin was administered systemically (i.p.) or locally (i.pl. injection) at the dose of 1.0 mg/kg, 60 min prior to or 24 h after the i.pl. administration of OVA. This dose was shown to be effective in other experimental systems [21 ]. Moreover, preliminary experiments showed the dose of 1.0 mg/kg to be maximally effective at inhibiting eosinophil recruitment in the model (data not shown). LY294002 was administered systemically or locally at the dose of 1.0 mg/kg, 60 min prior to or 24 h after antigen challenge. Drugs were dissolved in DMSO and further diluted in PBS. Control animals received drug vehicle.

Assessment of eosinophil apoptosis
Morphology
Apoptosis was assessed as described previously [22 ]. Briefly, cells (5x104) collected 48 h after antigen challenge were cytocentrifuged, fixed, and stained with May-Grunwald-Giemsa and counted using oil immersion microscopy (x100 objective) to determine the proportion of cells with distinctive apoptotic morphology. Twenty-five fields were counted per slide, and results are expressed as the mean ± SEM of the number of apoptotic cells in 25 fields.

Annexin-V binding and propidium staining
Assessment of apoptosis was also performed by flow cytometry using fluorescein isothiocyanate (FITC)-labeled annexin-V, which binds to phosphatidylserine exposed on the surface of apoptotic cells and propidium iodide as an index of loss of cell membrane integrity. Stock annexin-V was diluted 1:1000 with binding buffer and then added to 100 µl 2.5 x 105 cells collected after 2 and 6 h after wortmannin treatment. Following 10 min incubation at room temperature, these samples were treated with 5 µl propidium iodide (concentration, 50 µg/ml) and analyzed using Becton Dickinson (San Jose, CA) FACScan and CELLQuest software. Results are expressed as cells undergoing the early stage of apoptosis, quantified by staining with annexin-V but not propidium iodide.

Measurement of eotaxin and interleukin (IL)-5
Frozen supernatants obtained from pleural cavity washes after 6 h of the challenge with OVA (1 µg/cavity) were used for eotaxin and IL-5 detection. The concentration of eotaxin and IL-5 protein in pleural effluents was measured by enzyme-linked immunosorbent assay (ELISA), using commercially available antibody pairs and as specified by the supplier (R&D Systems, Minneapolis, MN).

Measurement of anti-OVA serum antibodies
Ninety-six-well ELISA plates (Nunc, Roshilde, Denmark) were coated overnight at 4°C with 2 µg per well OVA in coating buffer. Plates were blocked with 200 µl/well PBS containing 0.25% casein for 1 h at room temperature. After washing with PBS containing 0.1% Tween-20, serum samples were diluted in PBS/0.25% casein (starting at 1:100). Plates were incubated for 1 h at 37°C, washed six times, and incubated with peroxidase-conjugated goat anti-mouse antibody for 1 h at 37°C. After six washes, the reaction was developed at room temperature with 100 µl/well o-phenylenediamine (1 mg/ml), 0.04% H2O2 in sodium citrate buffer. The reaction was interrupted by the addition of 20 µl per well of 2N H2SO4. Absorbance was measured at 492 nm by an ELISA reader (Bio-Rad, Hercules, CA). ELISA was computed by optical densities between 1:100 and 1:6400 of serum dilutions in individual mice.

Immunoprecipitation
Cells (5x106 cells/ml) obtained from the pleural cavity of challenged mice were lysed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1.5 mM EDTA, 1% Triton X-100, 10% glycerol, 10 µg/µl aprotinin, 10 µg/µl leupeptin, 2 µg/µl pepstatin, and 1 mM phenylmethylsulfonyl fluoride. Lysates (2 µg/µl) were incubated overnight at 4°C with anti-Akt antibody (1:200, Santa Cruz Biotechnology, CA). Blocking peptide (Santa Cruz Biotechnology) was used as a negative control for anti-Akt antibody. Then, protein A/G-agarose (20 µl/mg protein, Santa Cruz Biotechnology) was added, and samples were incubated at 4°C under rotation for 2 h. The contents of Akt and phosphorylated Akt were analyzed by Western blotting, as described subsequently.

Immunoblotting analysis for Akt
Lysates (30 µg) were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were transferred to polyvinylidene difluoride (PVDF) filters (Hybond-P, Amersham Biosciences, Little Chalfont, UK). Rainbow markers (Amersham Biosciences) were run in parallel to estimate molecular weights. Membranes were blocked with Tween-Tris-buffered saline (TBS; 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% Tween-20) containing 1% BSA. Primary antibodies used in Western analysis were monoclonal antiphosphoserine (1:1000, Sigma Chemical Co.) and polyclonal anti-Akt (1:1000, Santa Cruz Biotechnology). The PVDF filters were next washed three times with Tween-TBS, followed by 1 h incubation with appropriate secondary antibody conjugated to biotin (Santa Cruz Biotechnology). Then, the filters were incubated with streptavidin-conjugated horseradish peroxidase (1:1000, Caltag Laboratories, South San Francisco, CA). Immunoreactive proteins were visualized by 3,3'-diaminobenzidine (Sigma Chemical Co.) staining. The bands were quantified by densitometry, using Scion image software (Scion Co., Frederick, MD).

Statistical analysis
All results are presented as the mean ± SEM. Normalized data were analyzed by one-way ANOVA, and differences between groups were assessed using the Student-Newman-Keuls post-test. A P value <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Role of PI3K{gamma} for eosinophil accumulation in a model allergic pleurisy
To investigate the importance of PI3K for eosinophil recruitment, sensitized WT and PI3K{gamma}–/– mice were challenged (i.pl.) with OVA. The i.pl. injection of OVA in sensitized WT and PI3K{gamma}–/– mice (1 µg/cavity) induced a significant recruitment of eosinophils at 6 and 24 h after antigen challenge (Fig. 1A ). However, the number of eosinophils in PI3K{gamma}–/– mice was diminished by 82% after 48 h, as compared with WT mice. At this time-point, a significant accumulation of mononuclear cells was also observed in WT but not in PI3K{gamma}–/– mice (Table 1 ). The accumulation of macrophages and neutrophils at 6 and 24 h after challenge was not altered in PI3K{gamma}–/– mice when compared with WT mice (data not shown). The baseline levels of leukocytes in blood and bone marrow of WT and PI3K{gamma}–/– mice were not different (Table 2 ). Furthermore, analysis of the cellular infiltrate in bone marrow of WT and PI3K{gamma}–/– mice at 6, 24, and 48 h after antigen challenge demonstrated that there were no differences in the number of eosinophils between the two groups in the various time-points (Table 2) . There were no morphological figures typical of apoptosis in the pleural cavity of immunized and challenged WT and PI3K{gamma}–/– mice at 24, 36, and 48 h (data not shown). There were few apoptotic figures of eosinophils in bone marrow of WT or PI3K{gamma}–/– mice (data not shown).



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  		Figure 1. Eosinophil accumulation induced by antigen challenge of sensitized PI3K{gamma}–/– and WT mice. (A) Immunized WT or PI3K{gamma}–/– mice or (B) immunized chimeric mice were challenged with an i.pl. injection of the OVA (1 µg/cavity) or PBS and eosinophil accumulation assessed at the time-point described in the figure. Chimeric mice, which lacked PI3K{gamma} on bone marrow-derived or on non-bone marrow-derived cells, were produced by transplanting bone marrow cells into irradiated recipient animals. Results are expressed as the means ± SEM of five mice in each group. *, P < 0.01, when compared with PBS-injected mice and #, P < 0.01, when compared with OVA-injected WT mice.

 

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  		Table 1. Recruitment of Leukocytes (x105 Cells/Cavity) Induced by Antigen Challenge of Sensitized PI3K{gamma}–/– and WT Mice

 

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  		Table 2. Number of Eosinophils in Bone Marrow and Blood at 6, 24, and 48 h after Antigen Challenge of Sensitized PI3K{gamma}–/– and WT Mice

 
Although PI3K{gamma} is mainly expressed in leukocytes, studies have demonstrated that this PI3K isoform may also be expressed by endothelial cell [23 ]. Thus, to verify the relevance of PI3K{gamma} expressed on leukocytes and on non-bone marrow-derived cells for the process of eosinophil accumulation in the pleural cavity, we made use of bone marrow transplantation. PI3K{gamma}–/– mice were reconstituted with bone marrow from PI3K{gamma}+/+ (WT mice) donors so that they had PI3K{gamma}-positive leukocytes but PI3K{gamma}-negative non-bone marrow cells, and irradiated WT mice were reconstituted with bone marrow from PI3K{gamma}–/– donors so that they had PI3K{gamma}-positive non-bone marrow cells but PI3K{gamma}-negative leukocytes. In control groups, irradiated PI3K{gamma}–/– mice were reconstituted with bone marrow from PI3K{gamma}–/– donors, and irradiated WT mice were reconstituted with bone marrow from WT donors. The i.pl. injection of OVA in sensitized WT and PI3K{gamma}–/– mice whose bone marrow had been reconstituted with WT bone marrow induced a significant recruitment of eosinophils after 48 h (Fig. 1B) . In contrast, the eosinophil accumulation induced by i.pl. injection of OVA in sensitized WT and PI3K{gamma}–/– mice whose bone marrow had been reconstituted with PI3K{gamma}–/– bone marrow was significantly reduced, suggesting that PI3K{gamma} on bone marrow-derived cells was required for eosinophil accumulation into the pleural cavity 48 h after antigen challenge (Fig. 1B) . After bone marrow transfer, the number of circulating leukocytes in blood was analyzed every 7 days, and we failed to observe any differences between the groups (data not shown).

Previous studies have shown that PI3K{gamma}–/– mice exhibited low titers of immunoglobulin G1 (IgG1) antibodies to hapten-conjugated OVA, suggesting that PI3K{gamma} is required to generate functional T helper cell-dependent responses to antigen in vivo [8 ]. In our model, the titers of total anti-OVA Ig and IgG1 are shown in Figure 2A and 2B , respectively. Thus, whereas WT mice exhibited high, total anti-OVA-specific Ig and IgG1 titers, these antibodies were reduced in PI3K{gamma}–/– mice.



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  		Figure 2. Titers of anti-OVA Ig in nonsensitized and in sensitized PI3K{gamma}–/– and WT mice. PI3K{gamma}–/– or WT mice were immunized with OVA and blood serum samples obtained 7 days after the last immunization. The titers of (A) total and (B) IgG1 anti-OVA Ig were assessed by ELISA. Results are expressed as means ± SEM absorbance of five mice in eight serial dilutions. *, P < 0.01, when compared with WT mice.

 
Next, we evaluated the production of eotaxin and IL-5, important mediators of eosinophil accumulation, in the allergic pleurisy model [24 , 25 ]. At 6 h after antigen challenge, there was no difference in the production of eotaxin (OVA-WT mice, 252±40 pg/pleural cavity; OVA-PI3K{gamma}–/–, 247±58 pg/pleural cavity; n=5) or IL-5 (OVA-WT mice, 27.9±10.2 pg/pleural cavity; OVA-PI3K{gamma}–/–, 38.0±14.6 pg/pleural cavity; n=5) in the pleural cavity of WT and PI3K{gamma}–/– mice. There was no difference in basal production of eotaxin or IL-5 between WT or PI3K{gamma}–/– mice (data not shown).

Effects of treatment with PI3K inhibitors (wortmannin and LY294002) on recruitment of eosinophils
The next experiments were designed to investigate whether a pharmacological strategy, i.e., inhibitor of PI3Ks, could mimic the phenotype observed in PI3K{gamma}–/– mice. Preliminary dose-response studies demonstrated that the best dose of systemically administered wortmannin to affect eosinophil accumulation was 1 mg/kg (OVA+vehicle, 4.5±0.2x105 eosinophil/cavity; OVA+wortmannin, 0.1 mg/kg: 4.9±0.9x105 eosinophil/cavity; OVA+wortmannin, 0.3 mg/kg: 4.2±0.6x105 eosinophil/cavity; OVA+wortmannin, 1 mg/kg: 1.4±0.3x105 eosinophil/cavity). Thus, as seen in Figure 3A , sensitized mice that had been treated systemically with the PI3K inhibitor wortmannin (1 mg/kg), 1 h before OVA challenge, had a significant reduction in the recruitment of eosinophils into the pleural cavity when compared with vehicle-treated controls. A significant reduction in the recruitment of total cells, mononuclear cells, and neutrophils was also observed with the wortmannin treatment (Table 3 ). In contrast to its effects when given systemically, wortmannin failed to affect the allergen-induced recruitment of eosinophils when given into the pleural cavity (locally) 1 h before the challenge (Fig. 3B) . The reduction in the number of eosinophils was also seen when LY294002 (1.0 mg/kg), which is a structurally distinct PI3K inhibitor, was injected systemically but not when injected locally 1 h before challenge in sensitized mice (Fig. 3) .



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  		Figure 3. Effects of the pretreatment with the PI3K inhibitors wortmannin and LY294002 on the recruitment of eosinophils induced by antigen challenge of sensitized mice. Wortmannin or LY294002 was administered (A) systemically (i.p.) or (B) locally (i.pl.) at the dose of 30 µg/mouse (1.0 mg/kg), 60 min prior to the challenge with OVA (1 µg/cavity) or PBS. Eosinophil recruitment was assessed 48 h after antigen challenge. Results are expressed as the means ± SEM of five mice in each group. **, P < 0.01, when compared with PBS-injected mice and #, P < 0.01, when compared with vehicle-treated, OVA-challenged mice.

 

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  		Table 3. Effects of the Systemic Pretreatment with the PI3K Inhibitor, Wortmannin, on the Recruitment of Leukocytes Induced by Antigen Challenge of Sensitized Mice

 
As eosinophil accumulation in PI3K{gamma}–/– mice was inhibited at 48 h as compared with WT mice but not at earlier time-points (6 and 24 h), we evaluated whether administration of wortmannin 1 h before antigen challenge could also affect responses in PI3K{gamma}–/– mice. The recruitment of eosinophils into the pleural cavity 24 h after antigen challenge was reduced significantly in sensitized PI3K{gamma}–/– mice that had been pretreated systemically with the PI3K inhibitor wortmannin (vehicle+OVA: 2.5±0.4x105 eosinophil/cavity; wortamannin+OVA: 0.2±0.2x105 eosinophil/cavity; n=4).

The serine-threonine PKB/Akt is a major target of PI3K activation [2 , 11 , 17 , 18 ]. Thus, a series of experiments analyzing Akt phosphorylation was carried out to verify whether OVA challenge was accompanied by PI3K activation and whether the dose of wortmannin had been effective for enzyme inhibition. When compared with PBS controls, OVA challenge of sensitized mice triggered significant Akt phosphorylation in pleural cell extracts at 2 h after challenge (Fig. 4 ). Systemic treatment with wortmannin 1 h before OVA challenge inhibited Akt phosphorylation to baseline levels (Fig. 4) .



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  		Figure 4. Effects of pre- and post-treatment with wortmannin on OVA-induced Akt phosphorylation. Wortmannin or drug vehicle was administered systemically (i.p.), 60 min prior to or locally (i.pl.), 24 h after the challenge with OVA (1 µg/cavity) in sensitized mice. Controls received PBS in challenge. Cell lysates harvested from the pleural cavity 2 or 26 h after the challenge were then immunoprecipitated with anti-Akt antibody and immunoblotted with anti-Akt and antiphosphoserine antibodies. The bands were quantified by densitometry, using Scion image software. Negative control is shown by using blocking peptide. The position to which the molecular mass standards migrated is marked: BSA (66 kDa); OVA (45 kDa). Results are expressed as the means ± SEM of three mice in each group.

 
We then evaluated whether systemic treatment with wortmannin 1 h before OVA challenge could affect the antigen-associated increase of eosinophils in the bone marrow. Sensitized mice were challenged with OVA, and the number of eosinophils in bone marrow was examined. Antigen challenge resulted in a decrease in the number of eosinophils in the bone marrow that was first noticed at 6 h and was marked at 24 h after challenge when compared with sensitized mice challenged with PBS (Fig. 5 ). Systemic treatment with wortmannin prevented the fall in bone marrow eosinophil numbers at 6 h after antigen challenge but had no significant effects at 24 (Fig. 5) or 48 h (data not shown). Consistently, a lower number of circulating eosinophils was detected 24 h after antigen challenge in wortmannin-treated mice (OVA, 2.1±0.3x105 eosinophils/ml blood; wortmannin+OVA, 1.2±0.2x105 eosinophils/ml blood; n=5, P<0.001). It is interesting that the antigen-induced increase in concentrations of IL-5 in the pleural cavity at 6 h after challenge was prevented by systemic treatment with wortmannin (Fig. 6 ).



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  		Figure 5. Effects of the pretreatment with the PI3K inhibitor wortmannin on the number of eosinophils in the bone marrow after antigen challenge of sensitized mice. Wortmannin was administered systemically (i.p.) at the dose of 30 µg/mouse (1.0 mg/kg), 60 min prior to the challenge with OVA (1 µg/cavity) or PBS. Eosinophil number in the bone marrow was assessed at 6 and 24 h after antigen injection. Results are expressed as the means ± SEM of five mice in each group. *, P < 0.01, when compared with PBS-injected mice and #, P < 0.01, when compared with vehicle-treated, OVA-challenged mice.

 


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  		Figure 6. Effects of the pretreatment with the PI3K inhibitor wortmannin on the production of IL-5 in the pleural cavity after antigen challenge of sensitized mice. Wortmannin was administered systemically (i.p.) at the dose of 30 µg/mouse (1.0 mg/kg), 60 min prior to the challenge with OVA (1 µg/cavity) or PBS. IL-5 concentrations were assessed 6 h after antigen challenge. Results are expressed as the means ± SEM of five mice in each group. *, P < 0.01, when compared with PBS-injected mice and #, P < 0.01, when compared with vehicle-treated, OVA-challenged mice.

 
Effects of treatment with PI3K inhibitors (wortmannin and LY294002) on survival of eosinophils
Next, we examined whether in addition to suppressing eosinophil recruitment, wortmannin could affect eosinophil survival in the allergic pleurisy model. As seen in Figure 4 , there was significant Akt phosphorylation (and hence, PI3K activation) at 26 h after challenge. To this end, wortmannin or drug vehicle was administered 24 h after challenge, and the number of eosinophils accumulated in the cavity was examined after a further 24 h. Our previous studies have shown that the number of eosinophils in the pleural cavity of allergen-challenged, sensitized mice is stable between 24 and 48 h after challenge [26]. As seen in Figure 7 , systemic treatment with wortmannin 24 h after OVA challenge failed to affect the accumulation of eosinophils in the pleural cavity observed after a further 24 h (Fig. 7A) . In contrast, local treatment with the drug markedly diminished the accumulation of these cells (Fig. 7B) . Again, the reduction in the number of eosinophils was also seen when LY294002 (1.0 mg/kg) was injected only locally 24 h after challenge in sensitized mice (OVA, 6.9±1.1x105 eosinophils/cavity; OVA+LY294002, 2.8±0.3x105 eosinophils/cavity; n=5, P<0.05). In addition, local treatment with wortmannin 24 h after the challenge inhibited the late antigen-associated Akt phosphorylation to background levels (Fig. 4) .



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  		Figure 7. Effects of therapeutic treatment with the PI3K inhibitor wortmannin on the accumulation of eosinophils in the pleural cavity of allergic mice. Immunized animals were challenged with OVA (1 µg/cavity) or PBS and 24 h later, received a (A) systemic (i.p.) or (B) local (i.pl.) injection of wortmannin at the dose of 30 µg/mouse (1.0 mg/kg). The number of eosinophils accumulating in the pleural cavity was assessed after a further 24 h. Results are expressed as the means ± SEM of five mice in each group. *, P < 0.01, when compared with PBS-injected mice and #, P < 0.01, when compared with vehicle-treated, OVA-challenged mice.

 
As few eosinophils were recruited from 24 to 48 h after antigen challenge, and local wortmannin treatment markedly inhibited the accumulation of these cells and Akt phosphorylation in the pleural cavity, we tested whether wortmannin-induced apoptosis could account for the observed results. The number of apoptotic eosinophils, as assessed by morphologic criteria, was markedly elevated in the pleural cavity of antigen-challenged and then wortmannin-treated mice (Fig. 8A ). Similarly, local administration of LY294002 24 h after antigen challenge significantly increased the number of apoptotic cells (Fig. 8A) . In both cases, the majority of apoptotic cells had typical eosinophil granules and was located within macrophages, presumably having been engulfed by the latter cells (data not shown). To confirm the morphological findings, we performed additional experiments in which surface changes associated with apoptosis were assessed at earlier time-points, as the exposure of phosphatidylserine precedes the recognition of the apoptotic cells by phagocytes [27 ]. To this end, we used FITC-labeled annexin-V binding in the presence of Ca+2 to label phosphatidylserine molecules exposed on the outer membrane of apoptotic cells [27 , 28 ]. In agreement with the morphological assessment, there was a rapid increase in the number of apoptotic events 2 h after treatment with wortmannin when compared with untreated mice (Fig. 8B) .



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  		Figure 8. Effects of therapeutic treatment with the PI3K inhibitors wortmannin or LY294002 on the number of apoptotic cells in the pleural cavity of allergic mice. Immunized animals were challenged with OVA (1 µg/cavity) or PBS and 24 h later, received a local (i.pl.) injection of wortmannin or LY294002 at the dose of 30 µg/mouse (1.0 mg/kg). The number of cells undergoing apoptosis was defined (A) morphologically or (B and C) by flow cytometry at the indicated times. (A) Cells (5x104) were collected 48 h after antigen challenge and were cytocentrifuged, fixed, and stained with May-Grunwald-Giemsa. The proportion of cells with distinctive apoptotic morphology was evaluated in 25 fields. Results are expressed as the mean ± SEM of the number of apoptotic cells in 25 fields. (B and C) Cells (2.5x105) were collected 2 h (B and C) and 6 h (B) after wortmannin or vehicle treatment and incubated with FITC-labeled annexin-V and propidium iodide. Results are expressed as the mean ± SEM of the percentage of cells staining with annexin-V-FITC alone. There were at least five mice in each group. #, P < 0.01, when compared with vehicle-treated, OVA-challenged mice.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In allergic diseases, such as asthma and atopic dermatitis, there is a characteristic, T cell-mediated recruitment of eosinophils into the affected tissues [12 ]. Although the role of eosinophils in asthma and other allergic diseases is not completely understood, it is thought that the recruitment and action of eosinophils may underlie several of the pathophysiological manifestations of allergic diseases [12 , 13 , 15 ]. Thus, an understanding of the mechanisms involved in eosinophil recruitment, activation, and survival in sites of allergic inflammation may be useful for the development of novel pharmacological therapies to control allergic diseases. More recently, there has been a great interest in the understanding of the signal transduction pathways following activation of serpentine receptors and especially following activation of chemokine receptors, which appear to play a major role in the recruitment of leukocytes in vivo [29 ]. In the present study, we have used pharmacological and genetic approaches to evaluate the role of PI3K for the migration of eosinophils in allergic reactions in the pleural cavity of mice. In addition, we investigated the importance of signaling through the PI3K pathway for eosinophil survival in these inflammatory sites.

Recent studies have demonstrated the importance of the class IB of PI3K (PI3K{gamma}) for leukocyte recruitment during acute inflammation [6 , 8 ]. Indeed, the recruitment of neutrophils and macrophages was reduced in PI3K{gamma}–/– mice after stimulation with various chemoattractants and in a septic peritonitis model [6 , 8 , 30 ]. Here, we demonstrate that eosinophil accumulation was similar in PI3K{gamma}–/– and WT mice in an allergic pleurisy model when evaluated 6 and 24 h after antigen challenge. However, at 48 h, the accumulation of eosinophils at sites of allergic inflammation was markedly reduced in PI3K{gamma}–/– mice when compared with their WT counterparts. We made chimerical mice lacking PI3K{gamma} on bone marrow-derived cells or on non-bone marrow-derived cells. Our results demonstrate that the lack of PI3K{gamma} in bone marrow-derived leukocytes but not on non-bone marrow cells is responsible for the reduction in the accumulation of eosinophils at 48 h after antigen challenge. Thus, it is clear from the data above that the expression of PI3K{gamma} by bone marrow-derived leukocytes, presumably by the eosinophil itself, is not essential for the early recruitment (arrival) of eosinophils but is necessary for their persistence in the pleural cavity 48 h after allergen challenge of sensitized mice. Although we failed to detect enhanced numbers of apoptotic figures in PI3K{gamma}–/– mice, the clearance of eosinophils observed at 48 h after challenge in PI3K{gamma}–/– mice may be related to the relevance of this enzyme in preventing eosinophil apoptosis and the resolution of the inflammatory process.

It is interesting that PI3K{gamma}–/– mice had lower titers of total serum Ig and IgG1. These results are consistent with studies demonstrating that PI3K{gamma}+/– mice exhibited high titers of nitroiodophenylacetic acid conjugated to OVA-specific IgG1 antibodies but lower levels of these antibodies in PI3K{gamma}–/– mice [8 ]. Initial events during an allergic reaction involve the release of proinflammatory mediators by mast cells triggered by the binding of the allergen to IgE antibodies bound to mast cells Fc{varepsilon}RI. Murine IgG1 is also capable of sensitizing murine mast cells [31 ]. In addition, it has recently been demonstrated that mast cells from PI3K{gamma}–/– mice had defective degranulation in vitro and responded poorly to adenosine or a passive systemic anaphylaxis in vivo [32 ]. Thus, although the differences in titers between WT and PI3K{gamma}–/– mice were small, the diminished eosinophil accumulation observed in our studies could be secondary not only to a deficient production of Ig and insufficient mast cell sensitization but also to deficient mast cell function. Nevertheless, it is worth noting that the production of eotaxin and IL-5 in the pleural cavity was similar in PI3K{gamma}–/– and WT mice. We have previously shown that eotaxin is released in the allergic pleurisy model and contributes markedly to eosinophil recruitment in response to antigen challenge [25 ]. Similarly, many studies have demonstrated an important role of IL-5 for the recruitment of eosinophils in vivo [33 , 34 ]. Although IL-5 and eotaxin appear to be produced by T cells [35 ] and macrophages [25 ], respectively, the results suggest that the initial mast cell-triggered events occur adequately in PI3K{gamma}–/– mice. Thus, it appears that diminished sensitization (as seen by the decrease in Ig titers) and defective mast cell function may not be functionally relevant phenomena suppressed in PI3K{gamma}–/– mice, as the most relevant mediators of eosinophil recruitment were being produced in adequate concentrations. This is consistent with the findings that the number of eosinophils that were recruited into the pleural cavity in the initial phase (6–24 h) of the allergic response was similar in PI3K{gamma}–/– and WT mice.

Next, we evaluated whether modulation of eosinophil recruitment was also observed when a pharmacological strategy was used to prevent PI3K function. As selective PI3K{gamma} inhibitors are not commercially available, we tested the effects of the isoform-nonselective PI3K inhibitors wortmannin and LY294002 in the allergic pleurisy reaction. In our experiments, no inhibition of eosinophil recruitment was observed when wortmannin or LY294002 was administrated locally 1 h before antigen challenge. This is in agreement with another study demonstrating that intranasal-administered wortmannin could inhibit allergen-induced airway hyper-responsiveness in vivo, probably by inhibiting eosinophil degranulation, but not their accumulation in bronchoalveolar lavage fluid [21 ]. However, the intrapulmonary administration of wortmannin before allergen challenge was an efficient inhibitor of the immediate-type allergic response and the late-phase pulmonary inflammation in a model of experimental asthma in Brown Norway rats [36 ]. Why shouldn’t the drug then function when given locally? Two major possibilities may be raised to explain these apparent contradictory results. First, wortmannin may have already been cleared when the relevant chemoattractant molecules are produced. For example, eotaxin production peaks 6 h after antigen challenge [25 ] and hence, at least 6 h after local administration of wortmannin. Second, the main site of action of the drug may be the eosinophil itself. The latter possibility entails that the leukocyte has to be inhibited by the drug before it is recruited to the site of allergic inflammation. However, as discussed above, the lack of PI3K{gamma} on eosinophils was not associated with defective recruitment (arrival) but only with defective late accumulation of these cells.

In contrast to its lack of effect when administered locally, wortmannin markedly prevented eosinophil migration in the pleurisy model when given systemically. These results were confirmed by use of LY294002, which is a structurally distinct PI3K inhibitor [37 ]. The inhibition of PI3K on circulating eosinophils or at a site relevant for the recruitment of eosinophils but distinct from the site of allergic inflammation could provide an alternative possibility to explain the latter results. The bone marrow is a possible site for the action of the PI3K inhibitors, as several studies have pointed to the major role of this organ for the recruitment of eosinophils in vivo [38 , 39 ]. In allergen-challenged mice, wortmannin prevented the antigen-induced fall in bone marrow eosinophils at 6 h after challenge. The effect of systemic wortmannin on bone marrow eosinophils was consistent with the finding that antigen-induced IL-5 production and a circulating number of eosinophils were also prevented by the drug. It has been shown previously that the mobilization of bone marrow eosinophils stimulated by IL-5 involves signaling through PI3K [40 ]. It is worth noting the contrasting effects of wortmannin treatment (inhibition) and the genetic strategy (no effect) on the concentrations of IL-5 after allergen challenge, suggesting that a PI3K isoform other than PI3K{gamma} is involved in the inhibitory effects of wortmannin on IL-5 production. This result supports previous reports in which production of IL-4 and IL-5 by mesenteric lymphocyte from PI3K/p85–/– mice (deficient in class IA PI3K isoforms) infected with Strongyloides venezuelensis was reduced [41 ]. Therefore, one possibility to explain the effects of systemic but not local treatment with the PI3K inhibitors on the recruitment of eosinophils in the allergic pleurisy model could be that the inhibitors are preventing eosinophil release from bone marrow when given systemically. Alternatively (or in addition to the mechanism proposed above), it is possible that the released eosinophils could have their PI3K enzymes inhibited by the systemic treatment with the inhibitors in blood, thus, prior to being stimulated to migrate to tissue. As systemic PI3K inhibitors prevented eosinophil recruitment, and no difference was found in eosinophil recruitment into the pleural cavity 6 and 24 h after antigen challenge in PI3K{gamma}–/– mice, we suggest that a PI3K isoform other than PI3K{gamma} may be contributing to eosinophil recruitment in our model. In keeping with the latter suggestion, the migration of neutrophils and macrophage in response to some chemokines was only partially reduced in PI3K{gamma}–/– mice, although these cells were unable to produce PI(3,4,5)P3 and activate Akt/PKB [6 , 8 ]. Furthermore, this hypothesis is supported by the observation that the pretreatment of PI3K{gamma}–/– mice with wortmannin, 1 h before OVA challenge, prevented the recruitment of eosinophils to the pleural cavity of drug-treated mice. Moreover, it has recently been demonstrated that antigen-challenged mice treated with a dominant-negative form of the class IA PI3K regulatory subunit had reduced airway eosinophilia and airway hyper-reactivity [42 ]. In this regard, a role for PI3K{delta} to polymorphonuclear cell polarization and directional migration has been documented previously [43 ]. Therefore, in future studies, it will be important to verify the role of other classes of PI3K in our model.

The eosinophil possesses a considerable variety of histotoxic molecules, which contribute to the initiation and maintenance of the allergic inflammatory response [12 , 13 , 44 ]. Thus, the clearance of eosinophils from inflammatory sites may have therapeutic implications [45 ]. In addition to their central role in cell proliferation and migration, class I PI3K has also been implicated in the prevention of apoptotic cell death [46 , 47 ]. Thus, studies have demonstrated that the PI3K/Akt pathway is constitutively activated in the majority of human pancreatic cancer cell lines [48 ]. Moreover, use of selective inhibitors of PI3K could inhibit growth and survival of tumors [49 ]. Other studies have shown that PI3K is an important factor of survival in monocytes [50 ], neutrophils [51 , 52 ], and eosinophils [53 ] when stimulated with growth factors in vitro. In our study, we demonstrate that when wortmannin or LY294002 was injected locally 24 h after challenge, there was a decrease of the number of eosinophils in the pleural cavity. It is interesting that intratracheal wortmannin also suppressed pulmonary eosinophil influx when administered before and after antigen challenge in mice [54 ]. The inhibition of eosinophil influx after post-treatment with wortmannin correlated with inhibition of Akt phophorylation and an increase in the number of apoptotic cells. Thus, local inhibition of PI3K after antigen challenge may be blocking an important signaling pathway induced by eosinophil survival factors. The nature of these PI3K-dependent eosinophil survival factors needs to be investigated. The increase of apoptotic cells and consequent decrease in number of eosinophils in the pleural cavity were not seen when wortmannin was administrated systemically, possibly as a result of limited access of the drug into the pleural cavity. Together, these results demonstrate that wortmannin may prevent the accumulation of eosinophils in an allergic pleurisy model by inhibiting eosinophil recruitment when given systemically before antigen challenge and by inducing eosinophil apoptosis when given locally after antigen challenge. The ability of PI3K inhibitors to induce eosinophil apoptosis may provide a good explanation for the absence of eosinophils 48 h after antigen challenge of immunized PI3K{gamma}–/– mice.

In conclusion, our data demonstrate an important role of PI3K for the recruitment and survival of eosinophils in a model of allergic pleurisy. Eosinophil accumulation in the allergic pleurisy model is sensitive to wortmannin and LY294002, thus suggesting a role of PI3K enzymes. However, the PI3K{gamma} isoform appears not to be the most relevant for the recruitment process, and other PI3K isoforms could be involved. Conversely, eosinophil persistence in the pleural cavity is wortmannin- and LY294002-sensitive and depends on the expression of PI3K{gamma} by bone marrow-derived cells, presumably by eosinophils themselves. Strategies modulating the resolution of the inflammatory process may yield novel therapies for the treatment of inflammatory and autoimmune diseases [45 , 55 , 56 ]. In this regard, manipulation of PI3K activation might be useful therapeutically, not only for the prevention but also for the resolution (by influencing apoptosis of eosinophils) of allergic inflammation.


   ACKNOWLEDGEMENTS
 
We are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a Pesquisas do Estado de Minas Gerais (FAPEMIG), and the Wellcome Trust for financial support for this work.

Received July 6, 2004; accepted December 23, 2004.


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 DISCUSSION
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