Class IA Phosphatidylinositide 3-Kinases, rather than p110γ, Regulate Formyl-Methionyl-Leucyl-Phenylalanine-Stimulated Chemotaxis and Superoxide Production in Differentiated Neutrophil-Like PLB-985 Cells

Isaline Boulven, Sylvain Levasseur, Sébastien Marois, Guillaume Paré, Emmanuelle Rollet-Labelle and Paul H. Naccache
J Immunol June 15, 2006, 176 (12) 7621-7627; DOI: https://doi.org/10.4049/jimmunol.176.12.7621

Abstract

Class I PI3Ks, through the formation of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), are thought of as essential elements of the neutrophil response to chemotactic factors. Moreover, the recent development of PI3K-deficient mice and isoform-specific inhibitors enabled examinations of the contribution of the distinct PI3K isoforms in neutrophil activation. However, the results of these various studies are conflicting, and the exact role of the different PI3K isoforms is not yet clearly established, particularly in human cells. In the present study, we used a different approach to assess the role of the distinct PI3K isoforms in response to the chemotactic agent fMLP. We inhibited PI3K activities by the transient expression following nucleofection of dominant negative mutants of either p85α or p110γ in the human myeloid cell line PLB-985, which can be induced to express a neutrophil-like phenotype. The data obtained with this approach showed that the production of PI(3,4,5)P3 triggered by fMLP is biphasic, with a peak of production observed in a short time period that entirely depends on p110γ activity, and a delayed phase that is mediated by class IA PI3K. We also provide evidence that the PI3K-dependent functional responses (i.e., superoxide production and chemotaxis) induced by the chemotactic factor mainly involve PI3K IA and, by implication, the delayed phase of PI(3,4,5)P3 production, whereas p110γ and the early peak of PI(3,4,5)P3 do not play major roles in the initiation or the control of these responses.

The activation of human polymorphonuclear neutrophils (PMNs)3 by chemotactic factors leads to the stimulation of multiple signal transduction pathways and is associated with the production of a variety of second messengers, among which are polyphosphoinositides. Of the enzymes involved in generating distinct phosphoinositides, members of a family of lipid kinases known as PI3Ks are thought to play critical although incompletely characterized roles in the initiation and control of various neutrophil functions, including chemotaxis, adhesion, phagocytosis, respiratory burst, and apoptosis (1, 2, 3, 4).

The PI3K family is a group of intracellular proteins that catalyze the phosphorylation of the D3 position of phosphoinositides. Based on their substrate specificity, three classes of PI3Ks can be defined. Of particular interest to neutrophil physiology are class I PI3Ks, which are further divided into class IA and class IB. Class IA enzymes comprise three types of p110 catalytic subunits (p110α, p110β, and p110δ), which are activated primarily by tyrosine kinases and are associated predominantly with a p85α regulatory subunit (5, 6). In contrast to class IA PI3Ks, one member of the class IB PI3K family, p110γ, acts downstream of G protein-coupled receptors. It does not associate with p85 family proteins but interacts rather with p101 (7) and/or p84 (8) regulatory proteins. All of the class I PI3Ks phosphorylate phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate in vitro, but phosphatidylinositol 4,5-bisphosphate is the predominant substrate in cells, and the major product of these kinases is phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). The latter is a key lipid second messenger that controls a wide range of cellular responses via downstream effectors that include, among others, adaptor proteins, protein kinases, nucleotide exchange factors, and GTPase-activating proteins (5, 6, 9).

Using PI3K inhibitors (wortmannin and LY294002), a number of effects of neutrophil activation have been shown to be PI3K dependent (10, 11, 12, 13). However, the specific roles of the class I PI3K isoforms in neutrophil responses have long been difficult to evaluate because of the absence (until very recently) of PI3K inhibitors specific for individual family members. The development of PI3K-deficient mice enabled key roles for specific PI3K isoforms to be uncovered, although certain results are conflicting. A critical role of p110γ in neutrophil functions was suggested by different studies conducted in p110γ-null mice. Indeed, analysis performed with p110γ knockout mice showed decreased migration (14, 15, 16, 17, 18) and reduced respiratory burst (16, 17, 18) in neutrophils stimulated by chemotactic agents. These studies reached the conclusion that p110γ was the sole PI3K isoform coupled to the activation of neutrophil by chemoattractants. However, others have shown that tyrosine phosphorylation events and, by implication, class IA PI3Ks are involved in PI(3,4,5)P3 formation induced by chemotactic factors (11, 19, 20). Moreover, a recent study based on the use of a specific inhibitor of p110δ indicated that this isoform was implicated in neutrophil migration (21). A possible role of p110δ in PI(3,4,5)P3 formation and in the chemotactic behavior of neutrophils stimulated by G protein-coupled receptor was also shown by using p110δ-null mice (22).

In the present study, we aimed to further characterize the role of the different isoforms of PI3Ks in PI(3,4,5)P3 formation and in neutrophil responses to the chemotactic factor fMLP. We examined the roles of PI3K IA and IB using differentiated neutrophil-like PLB-985 cells (dPLB-985) in which PI3K activity was inhibited by the transient expression of dominant negative mutants of either p85α or p110γ. Our data confirmed that p110γ represents the major PI3K isoform activated in response to fMLP in short time periods. However, our results also indicated that class IA PI3Ks are implicated in the formation of PI(3,4,5)P3 at longer times of fMLP stimulation and play a major role in the responses triggered by the chemotactic factor.

Materials and Methods

Reagents and antibodies

Wortmannin, sodium orthovanadate, DMSO, and fMLP were purchased from Sigma-Aldrich. Aprotinin and leupeptin were bought from Roche Diagnostic Systems. [32P]Orthophosphate (1000 Ci/mmol) and the ECL reagents used for immunoblotting were obtained from PerkinElmer. Calcein-acetoxymethyl ester was obtained from Molecular Probes. Cytochrome c was purchased from MP Biomedicals. RPMI 1640 was obtained from Invitrogen Life technologies. Penicillin/streptomycin and FBS were purchased from Wisent. Anti-p110γ antisera were raised in rabbits as described previously (23). Anti-p85α was obtained from Upstate Biotechnology (catalog number 06-195). HRP-labeled donkey anti-rabbit IgG was purchased from Jackson ImmunoResearch Laboratories (catalog number 711-035-152).

Vectors

The pcDNA3.1 vector was obtained from Invitrogen Life Technologies. The cDNA coding for kinase-dead (KD) mutant (K832R) p110γ (designated as p110γ KD) (24) was provided by Dr. M. P. Wymann (University of Basel, Basel, Switzerland) and subcloned in the pcDNA3.1 vector. The cDNA coding for deleted mutant p85α (designated as p85Δ) (25) was provided by Dr. B. Barbeau (Centre de Recherche en Infectiologie, Université Laval, Sainte-Foy, Quebec, Canada) and subcloned in the pcDNA3.1 vector. The pEGFP-C1 vector was purchased from Clontech Laboratories.

Cells

PLB-985 cells (from the German Collection of Microorganisms and Cell Cultures (DSMZ)) were grown in RPMI 1640 medium containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. The cells were maintained in culture for 12 passages before new batches were thawed. To induce a differentiation to a neutrophil-like phenotype, PLB-985 cells were cultured in medium supplemented with 1.25% DMSO for 5 days before each experiment.

Peripheral blood PMNs were obtained from healthy adult volunteers and sterilely isolated as described previously (26).

Transfection

Differentiated PLB-985 cells were transiently transfected using the Nucleofector system from Amaxa Biosystems. After centrifugation, 10 × 106 cells were suspended in 100 μl of prewarmed Nucleofector solution (Lymphocyte B kit) containing 8 μg of vector pEGFP-C1 (encoding for the enhanced GFP), pcDNA3.1, p110γ KD, or p85Δ. The samples were transferred into an electroporation cuvette, and transfections were performed with the electrical setting U-15. After nucleofection, the cells were immediately transferred into prewarmed complete RPMI 1640 medium containing 1.25% DMSO and cultured at 37°C in a humidified atmosphere of 5% CO2. At specific times after nucleofection, cells were harvested and resuspended in Mg2+-free HBSS containing 1.6 mM CaCl2 for analysis. Similar transfection efficiencies and functional preservation were obtained using the kits for lymphocyte B, lymphocyte T, dendritic cells, and PLB-985 cells (data not shown).

Immunoblotting of PI3Ks

Transfected PLB-985 cell suspensions (20 × 106 cells/ml) were added to an equal volume of boiling 2× Laemmli’s sample buffer (composition of 1× 62.5 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 5% (v/v) 2-ME, 8.5% (v/v) glycerol, 2.5 mM orthovanadate, 10 mM para-nitrophenylphosphate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 0.025% bromophenol blue) and boiled for 10 min. Samples were then subjected to electrophoresis on 7.5–20% SDS-polyacrylamide gels and transferred to Immobilon polyvinylidene difluoride membranes (Millipore). Immunoblotting was performed as follows. Nonspecific sites were blocked using 2% gelatin in TBS-Tween 20 (25 mM Tris-HCl (pH 7.8), 190 mM NaCl, and 0.15% Tween 20) for 20 min at 37°C. Anti-p110γ (final dilution of 1/1000) or anti-p85 (final dilution of 1/3000) Abs were incubated with the membranes for 1 h at 37°C in TBS-Tween 20. The membranes were then washed three times at room temperature in TBS-Tween 20 for a total duration of 30 min and incubated with HRP-labeled donkey anti-rabbit IgG for 30 min at 37°C at a final dilution of 1/20,000 in TBS-Tween 20. The membranes were washed three times with TBS-Tween 20 at room temperature, and the protein bands were revealed using the ECL Western blotting detection system following the manufacturer’s directions.

PI(3,4,5)P3 formation

Cells (50 × 106 cells/ml) were incubated with 0.5 mCi/ml [32P]orthophosphate at 37°C in a labeling buffer (110 mM NaCl, 10 mM KCl, 1 mM MgCl2, 10 mM glucose, 1.6 mM CaCl2, and 30 mM HEPES (pH 7,4)) for 90 min for differentiated PLB-985 cells and 60 min for PMNs. Unincorporated radioactivity was discarded, and the cells were washed twice in HBSS. The cells were resuspended at 10 × 106 cells/ml in HBSS for differentiated PLB-985 cells and 50 × 106 cells/ml for PMNs and then stimulated with fMLP (10−7 M) for the indicated time. The reactions were stopped by the transfer of 100 μl of the cells into 400 μl of CHCl3-MeOH-HCl (1:1:0.1; v/v/v), followed by rapid vortexing (15–30 s). The samples were centrifuged for 5 min at 13,000 × g, and the organic phase (lower phase) was transferred into borosilicate tubes. The samples were applied to oxalate-treated silica gel 60 plates (EMD Chemicals), which were developed in chloroform-acetone-methanol-acetic acid-H2O (80:30:26:24:14; v/v/v/v/v) for 3h. The plates were dried, and the products were visualized with a bioimaging analyzer (Fuji Film BAS-1800).

Measurement of PLB-985 chemotactic responses

Differentiated PLB-985 cells, transfected with pcDNA3.1, p110γ KD, or p85Δ, were resuspended in HBSS containing 10% FBS (HBSS/FBS) at 107 cells/ml. The cells were preincubated with 5 μg/ml calcein-acetoxymethyl ester at 37°C for 30 min in the dark with constant agitation. The cells were then washed and resuspended in HBSS/FBS at 2 × 106 cells/ml. For assays in the presence of wortmannin, untransfected dPLB-985 cells were incubated with 5 μg/ml calcein-acetoxymethyl ester and then washed and incubated at 37°C for 10 min with either wortmannin (200 nM in 0.01% DMSO final concentration) or DMSO (0.01%). Cell migration was monitored using a 96-well ChemoTX disposable chemotaxis system (Neuro Probe). The wells of the lower chamber of the plate were filled with 31 μl of fMLP at the indicated concentrations. The polycarbonate filters (3 μM) were positioned on the plate, and transfected dPLB-985 cells (30 μl; 60,000 cells/well) were placed on the filter and allowed to migrate for 120 min at 37°C in the presence of 5% CO2 in the dark. The cells that had not migrated were removed by gently wiping the filters with a tissue. The fluorescence of the cells in the filters was measured with a microplate fluorescence reader (FL600 from Bio-Tek Instruments; excitation and emission wavelengths were 485 and 530 nm, respectively). The fluorescence from known numbers of differentiated PLB-985 was obtained by placing them into the bottom chamber. The results are expressed as the percentage of the maximal response.

Superoxide measurements

Superoxide production was measured using the reduction of cytochrome c assay. Differentiated PLB-985 cells transfected with pcDNA3.1, p85Δ, or p110γ KD (2 × 107 cells/ml) were preincubated for 10 min at 37°C in the presence of 125 μM cytochrome c. Cells were then stimulated at room temperature by adding fMLP (10−7 M), and the differences between the OD readings at 550 nm and 540 nm were monitored for 5 min. For assays in the presence of wortmannin, untransfected dPLB-985 cells were preincubated for 10 min at 37°C with wortmannin (200 nM in 0.01% DMSO final concentration) or DMSO (0.01%) in the presence of 125 μM cytochrome c before being stimulated. The results are expressed as the percentage of the maximal response.

Statistical analysis

The data were analyzed using the Student paired t test (two-tailed). The levels of significance (∗, p < 0.05; ∗∗, p < 0.01; Figs. 4⇓ and 8⇓) were determined between the cells transfected with p110γKD or p85Δ, and the cells were transfected with the empty vector pcDNA3.1.

Results

Production of PI(3,4,5)P3 in differentiated neutrophil-like PLB-985 stimulated by fMLP

In this study we used a human neutrophil-like cellular model, the human myeloid cell line PLB-985, to examine the role of class IA and class IB PI3Ks in the responses to the chemoattractant fMLP. These cells can be differentiated with DMSO to express a neutrophil-like phenotype (27).

The experiments whose results are summarized in Fig. 1⇓A showed that the stimulation of dPLB-985 cells by fMLP resulted in rapid increases of the levels of PI(3,4,5)P3. These effects were time dependent and peaked within 30 to 60 s of the addition of the chemoattractant. PI(3,4,5)P3 formation then gradually declined, with a sustained phase that persisted for at least 10 min. The kinetics of PI(3,4,5)P3 accumulation in fMLP-stimulated PMNs were similar (Fig. 1⇓B) to those observed in dPLB-985, indicating that the latter cells represent a useful model system in which to study the activation of PI3Ks in response to this chemotactic factor.

FIGURE 1.
FIGURE 1.

Accumulation of PI(3,4,5)P3 in response to fMLP in dPLB-985 and PMNs. 32P-labeled dPLB-985 (A) and PMNs (B) were stimulated with fMLP (10−7 M) for the indicated times. PI(3,4,5)P3 formation was visualized as described in Materials and Methods. The results of the densitometric analysis are expressed as the percentage of the unstimulated cells. The values represent the means of at least five separate experiments.

Transfection of dPLB-985 cells by nucleofection

To overcome the low transfection efficiency of dPLB-985 cells previously reported (28), we used the Nucleofector (Amaxa Biosystems) technology that is a novel gene transfer method designated for hard-to-transfect cell lines. To examine the efficiency of this technique, we first nucleofected the pEGFP-C1 vector encoding for enhanced GFP into dPLB-985 as described in Materials and Methods. Differentiated PLB-985 cells transfected with pEGFP-C1 were analyzed by flow cytometry at different time points after nucleofection. As depicted in Fig. 2⇓, enhanced GFP (EGFP) expression was detectable within 2 h postnucleofection. Transfection efficiencies of >60% were reached as early as 4 h postnucleofection, and EGFP expression continued to increase slowly up to 24 h. The proportion of dead cells was determined by parallel trypan blue staining. The proportion of trypan blue-positive cells increased progressively to 65% by 24 h postnucleofection. In contrast, >60% of the cells excluded trypan blue at a time when EGFP expression was near maximal (4 h), indicating that this novel technology represents a feasible approach to introduce plasmids in differentiated neutrophil-like PLB-985 cells and to specifically target intracellular proteins. Based on these data, all of the subsequent analyses were performed 4 h after nucleofection.

FIGURE 2.
FIGURE 2.

Time course of EGFP expression after nucleofection of dPLB-985. dPLB-985 were nucleofected with pEGFP-C1 and incubated as described in Materials and Methods. Cells were removed at the indicated times posttreatment, and EGFP expression was determined by FACS analysis. The proportion of dead cells was determined by trypan blue staining. The results shown represent the means of three separate experiments.

Effect of p110γ KD and p85Δ transfection on the accumulation of PI(3,4,5)P3 induced by fMLP

In an effort to identify the PI3K isoform activated by fMLP, we transiently transfected differentiated neutrophil-like PLB-985 cells with an empty vector (pcDNA3.1) or dominant negative mutants of PI3Kγ (p110γ KD) or p85α (p85Δ). Transfected dPLB-985 cells were analyzed by immunoblots using Abs against either p110γ or p85α. The results of these experiments are depicted in Fig. 3⇓, which shows the presence of bands corresponding to p110γ KD (Fig. 3⇓A) and p85Δ (Fig. 3⇓B) in the appropriate cells, indicating that both mutant proteins were properly expressed 4 h after nucleofection.

FIGURE 3.
FIGURE 3.

Transfection of p110γ KD and p85Δ in dPLB-985. dPLB-985 cells were nucleofected with empty pcDNA3.1, p110γ KD, or p85Δ as described in Materials and Methods. Four hours after transfection, cells were removed and total proteins were analyzed by immunoblotting with anti-p110γ (A) or anti-p85 (B). The data show the results of one determination representative of four separate experiments.

We then examined the accumulation of PI(3,4,5)P3 in response to fMLP in control or p110γ KD- and p85Δ-transfected cells. As shown in Fig. 4⇓, the peak of PI(3,4,5)P3, observed after 30 s of stimulation with fMLP, was severely inhibited in cells expressing p110γ KD. In contrast, p85Δ transfection was without effect on the formation of PI(3,4,5)P3 observed at 30 s poststimulation by fMLP in dPLB-985 cells.

FIGURE 4.
FIGURE 4.

Effect of p110γ KD and p85Δ transfection on the peak of PI(3,4,5)P3 production induced by fMLP. dPLB-985 cells were nucleofected with pcDNA3.1, p110γ KD, or p85Δ as described in Materials and Methods. Four hours after transfection, cells were removed and labeled for 90 min with [32P]orthophosphate. The cells were then stimulated with fMLP (10−7 M) for 30 s. The amounts of PI(3,4,5)P3 was measured as described in Materials and Methods. The values are expressed as the percentage of the unstimulated cells and represent the means of at least five separate experiments. ∗∗, p < 0.01.

These data indicate that p110γ is the major, if not the only, PI3K isoform responsible of the peak of production of PI(3,4,5)P3 triggered by fMLP in differentiated neutrophil-like PLB-985 at this early time point.

Effect of p110γ KD transfection on the functional responses induced by fMLP

The activation of PI3Ks has been linked to various cell functions in PMNs, including the stimulation of the oxidative burst and cell motility. As shown in Fig. 5⇓, dPLB-985 produced superoxide anions (Fig. 5⇓A) and exhibited a classical bell-shaped chemotactic concentration curve (Fig. 5⇓B) in response to fMLP. Wortmannin, a PI3K inhibitor that affects equally PI3K IA and IB, decreased by >50% the production of superoxide triggered by fMLP in dPLB-985. The chemotactic response of the cells was also dramatically inhibited by wortmannin. These data provide evidence that the functional responsiveness of dPLB-985 cells depends on PI3Ks in much the same way as PMNs.

FIGURE 5.
FIGURE 5.

Effect of wortmannin on chemotaxis and superoxide production stimulated by fMLP. A, dPLB-985 were incubated for 10 min without or with wortmannin (wort) (200 nM) before stimulation by fMLP (10−7 M). Superoxide production was monitored as described in Materials and Methods. The results, expressed as the percentage of the maximal response, represent the means of three separate experiments. B, dPLB-985 cells were preincubated 10 min without or with wortmannin (wort) (200 nM), and the chemotaxis assays were then performed as described in Materials and Methods. The results are expressed as the percentage of the maximal response and represent the means of three separate experiments, each done in duplicate.

We next took advantage of the ability to inhibit the intrinsic activity of p110γ through the transfection of p110γ KD to directly examine the functional involvement of this specific PI3K isoform. In these experiments, dPLB-985 cells were transfected either with empty vector or with p110γ KD, and their ability to produce superoxide anions or to migrate in response to fMLP was monitored. As shown in Fig. 6⇓, neither the production of superoxide nor the chemotactic response induced by fMLP were inhibited in dPLB-985 cells expressing p110γ KD.

FIGURE 6.
FIGURE 6.

Effect of p110γ KD transfection on chemotaxis and superoxide production stimulated by fMLP. dPLB-985 cells were nucleofected with either empty pcDNA3.1 or p110γ KD as described in Materials and Methods. A, Four hours after transfection, cells were removed and stimulated by fMLP (10−7 M). Superoxide production was monitored as described in Materials and Methods. The results, expressed as the percentage of the maximal response, represent the means of three separate experiments. B, Four hours after transfection the cells were removed, and the chemotaxis assays were then performed as described in Materials and Methods. The results, expressed as the percentage of the maximal response, represent the means of three separate experiments, each done in duplicate. The maximal superoxide response was 0.81 ± 0.14 nmol of O2− per 106 cells in 5 min. The maximal number of cells migrating in response to fMLP was 3307 ± 256 cells.

Effect of p85Δ transfection on the delayed formation of PI(3,4,5)P3 and on the functional responses induced by fMLP

The results shown in Fig. 4⇑ and 6⇑ indicate that whereas p110γ is principally responsible for the early peak of PI(3,4,5)P3 accumulation in response to fMLP, it plays little if any role in the initiation and control of the chemotactic and oxidative responses to this chemoattractant. The kinetics of accumulation of PI(3,4,5)P3 shown in Fig. 1⇑ provide evidence for a delayed phase of formation of PI(3,4,5)P3. This finding led us to examine the potential involvement of class IA PI3Ks in these responses.

We first examined the effects of p85Δ on the early and late phases of PI(3,4,5)P3 formation. To do so, we obtained complete kinetic curves of the accumulation of PI(3,4,5)P3 in the absence or presence of p110γKD or p85Δ. The results of these experiments are shown in Fig. 7⇓A. It should be noted that the data in this figure derive from two separate series of experiments. Although slight differences in the kinetics can be noted and are probably attributable to experimental variability, both kinetics show an initial peak of PI(3,4,5)P3 accumulation followed by a small but detectable sustained phase. These two phases are most evident at 30–60 s (initial phase) and at 300 s (sustained phase). The data at these two time points are summarized in the histograms shown in Fig. 7⇓B. As shown in Fig. 7⇓A, whereas p85Δ had, as previously shown in Fig. 4⇑, no effect on the peak of PI(3,4,5)P3 accumulation observed at 30 s, it dramatically reduced the levels of PI(3,4,5)P3 that were observed in control cells at 5 min. In contrast, the expression of p110γKD severely reduced the early peak of PI(3,4,5)P3 but had no effect on the accumulation of PI(3,4,5)P3 observed at 5 min poststimulation by fMLP.

FIGURE 7.
FIGURE 7.

Effect of p85Δ and p110γ KD transfection on the accumulation of PI(3,4,5)P3. dPLB-985 cells were nucleofected with pcDNA3.1 and p85Δ or p110γ KD as described in Materials and Methods. Four hours after transfection the cells were removed and labeled for 90 min with [32P]orthophosphate. The cells were then stimulated with fMLP (10−7 M) for the indicated times. The amounts of PI(3,4,5)P3 was measured as described in Materials and Methods. A, Time dependence of the accumulation of PI(3,4,5)P3 in the control and transfected cells. B, Summarization of the results at 30 s and at 5 min. The values are expressed as the percentage of the unstimulated cells and represent the means of at least four separate experiments ± SEM.

The effects of the expression of p85Δ on the production of superoxide anions and on the chemotactic responses induced by fMLP in dPLB-985 were monitored next. As shown in Fig. 8⇓, the expression of p85Δ significantly inhibited the production of superoxide anions and decreased dramatically the chemotactic response induced by fMLP.

FIGURE 8.
FIGURE 8.

Effect of p85Δ transfection on chemotaxis and superoxide production stimulated by fMLP. dPLB-985 cells were nucleofected with either empty pcDNA3.1 or p85Δ as described in Materials and Methods. A, Four hours after transfection, cells were removed and stimulated by fMLP (10−7 M). Superoxide production was monitored as described in Materials and Methods. The results, expressed as the percentage of the maximal response, represent the chemotaxis assays that were performed as described in Materials and Methods. The results, expressed as the percentage of the maximal response, represent the means of four separate experiments, each done in duplicate. The maximal superoxide response was 1.57 ± 0.20 nmol of O2− per 106 cells in 5 min. The maximal number of cells migrating in response to fMLP was 4247 ± 375 cells. ∗∗, p < 0.01.

Discussion

Class I PI3Ks and their lipid product PI(3,4,5)P3 are recognized as fundamental elements for the regulation of a vast array of cellular responses, in particular in the immune system (1, 2, 3, 29). However, it is less clear how the multiple isoforms of PI3K fit into this general scheme of PI3K signaling.

In the present study, we aimed to assess the role of the distinct PI3K isoforms in PI(3,4,5)P3 formation and in the neutrophil responses induced by the chemotactic factor fMLP. We thus inhibited PI3K activities by the transient expression of dominant negative mutants of either p85α or p110γ in the human myeloid cell line PLB-985, which can be induced to express a neutrophil-like phenotype. Nucleofection enabled us to obtain very high levels of transfection while keeping most cells alive. The data obtained with this approach show that the peak of production of PI(3,4,5)P3, triggered at short times by fMLP in differentiated neutrophil-like PLB-985 cells, mainly depends on p110γ. In addition, the lack of effect of p85Δ on the initial peak of PI(3,4,5)P3 accumulation provides evidence for the specificity of the effect of the dominant negative p110γ. These results are in agreement with our previous data obtained in PMNs that led us to conclude that p110γ represented the major PI3K activated by fMLP at very early time points (<60 s) following the stimulation of human neutrophils (23). However, in the present study we now provide evidence of the existence of a delayed phase of PI(3,4,5)P3 production that mainly involves class IA PI3K. Thus, the kinetics of fMLP-stimulated PI(3,4,5)P3 accumulation appears to be biphasic, with an early peak that depends on p110γ and a sustained phase that involves class IA PI3Ks.

The idea that both class IA and class IB PI3K isoforms are required in chemoattractant-stimulated PI(3,4,5)P3 formation is supported by earlier studies presenting apparently conflicting results. A major role of p110γ in mediating PI(3,4,5)P3 accumulation was strongly suggested by different studies (16, 17, 18) showing that neutrophils from p110γ−/− mice were unable to produce PI(3,4,5)P3 in response to chemotactic agents. However, there was also evidence for the involvement of class IA PI3Ks. Indeed, the activation of class IA PI3K downstream of chemotactic agent stimulation has been reported, and tyrosine kinase inhibitors have been shown to block chemoattractant-stimulated PI(3,4,5)P3 accumulation in neutrophils (11, 19, 20, 21, 30). Recently, the use of a p110δ-selective inhibitor and studies conducted in p110δ−/− mice have shown a crucial involvement of this PI3K IA isoform in PI(3,4,5)P3 accumulation triggered by fMLP (21, 22). These various results, which were apparently contradictory, suggest that both class IA and class IB PI3Ks are activated following fMLP stimulation. Moreover, a recent study using a series of PI3K isoform-specific inhibitors has provided evidence for a sequential activation of class IB and class IA in response to fMLP in TNF-α-primed neutrophils (31). Although the latter study did not examine the direct effects of the chemotactic factor, the data provided also support the idea that multiple isoforms of PI3Ks are required for chemoattractant-stimulated PI(3,4,5)P3 production.

The mechanisms by which tyrosine kinase dependent-PI3Ks IA are activated downstream of G protein-coupled receptor stimulation are currently unknown. Direct activation of p110β by Gβγ has been described (32, 33), and this mechanism could account in part for the activation of PI3K IA by fMLP. Different lines of evidence suggest that nonreceptor tyrosine kinases, such as the Src family tyrosine kinases, could be involved in the activation of PI3K by chemotactic factors (20, 31). We also observed that PP2, an inhibitor of the Src family tyrosine kinases, significantly reduced the accumulation of PI(3,4,5)P3 in fMLP-stimulated neutrophils (our unpublished data), suggesting that this family of enzymes is implicated in the regulation of the PI(3,4,5)P3 response to the chemotactic agent. The Tec family of tyrosine kinases, which is also activated downstream of fMLP stimulation in neutrophils (34), could also be involved in PI(3,4,5)P3 regulation, although contradictory results have been published in this respect. Indeed, Condliffe et al. (31) showed that LFM-A13, an inhibitor of the Tec family of tyrosine kinases, had no effect on the accumulation of PI(3,4,5)P3 stimulated by fMLP in TNF-α-primed neutrophils, whereas Gilbert et al. (26) observed that this compound inhibited the accumulation of PI(3,4,5)P3 triggered by the chemotactic factor in neutrophils. These differences may reflect the effect of the priming regimen used in the study by Condliffe et al. (31) or differential effects on PI3Ks and lipid phosphatases.

An interesting aspect of the stimulation of PI(3,4,5)P3 production by fMLP comes from the previous observation that PI(3,4,5)P3 may stimulate its own accumulation, suggesting the existence of a positive feedback loop (35). It was then considered that PI3K IA, activated downstream of p110γ, could mediate this positive feedback loop (1, 36). This idea is supported by the recent work of Condliffe et al. (31), which shows that class IA-driven PI(3,4,5)P3 accumulation induced by fMLP in TNF-α-primed neutrophils is significantly dependent on the activity of p110γ. However, this finding is contradictory with our present work, which shows that inhibition of the activity of p110γ through the transfection of p110γ KD is without effect on the PI3K IA-dependent accumulation of PI(3,4,5)P3 triggered by fMLP at 5 min. Although we cannot exclude the possibility that the use in our study of dPLB-985 cells rather than PMNs or the use of primed cells in the study from Condliffe et al. (31) accounts for the difference observed, it is also possible that the residual p110γ activity observed in p110γ KD-transfected cells may be sufficient to enable the activation of PI3K IA in our model. We may also consider that the PI(3,4,5)P3-dependent amplification of the signal observed by Weiner et al. (35, 36) in dPLB-985 does not imply p110γ-dependent activation of PI3K IA, but rather that it is mediated by another pathway, possibly involving Rho GTPases, as has also been suggested (35, 36).

A wide variety of neutrophil functions have been shown to be PI3K dependent, including cell motility, adhesion, phagocytosis, respiratory burst, and apoptosis (1, 2, 3, 4). In the present study, we focused on superoxide anion production and chemotaxis, whose dependence on PI3K activity is well documented (2, 4, 37, 38, 39, 40), although the role of the distinct PI3K isoforms in these responses remain unclear. We took advantage of the ability to selectively inhibit PI3K IA and PI3K IB activity through the transfection of dominant negative mutants of p85α and p110γ, respectively, to directly examine the functional involvement of these specific PI3K isoforms in fMLP responses. The results obtained clearly indicate that chemotaxis and superoxide production induced by the chemotactic factor are significantly dependent on PI3K IA, whereas inhibition of p110γ does not affect these two responses. These results are in disagreement with those obtained in p110γ−/− mice, which showed that the loss of p110γ activity in mice neutrophils dramatically reduced superoxide production (16, 18) and impaired chemotaxis (15, 16, 17, 18) in response to the chemoattractant. However, others studies support the idea that PI3K IA is required for fMLP responses in neutrophils (21, 22). These apparently contradictory results may be in part explained by the recent work of Condliffe et al. (31), which suggests that the regulation of respiratory burst involves different pathways and different PI3K isoforms in mouse and human neutrophils. It is also possible that the possible scaffolding functions of p110γ (which would be expected to be preserved in kinase-dead transfected cells or mice), as opposed to the absence of p110γ in knockout mice (41), may contribute to some of these differences.

It is interesting to note that, in our study, the production of superoxide is similarly inhibited in cells pretreated with wortmannin and in cells transfected with p85Δ, suggesting that the PI3Ks involved in the respiratory burst induced by fMLP all belong to the class IA. In contrast, wortmannin was a more potent inhibitor of fMLP-induced chemotaxis than was p85Δ transfection. Given that p110γ KD transfection was without effect on chemotaxis, the more drastic effect of wortmannin may arise from non-PI3K-dependent effects of this compound, possibly on myosin L chain kinase (42), which is important for cell migration (39). All these data indicate that the PI3K-dependent respiratory burst and the cell migration induced by fMLP mainly involve PI3K IA, whereas p110γ does not play a major role. The present results call into question the essential role that has of recent years been ascribed to p110γ in neutrophil activation (36, 40). Additional experiments need to be conducted to define more precisely the involvement of this PI3K isoform in the responses induced by the chemotactic factor and to establish the contribution of the multiple waves of PI(3,4,5)P3 production in neutrophil activation

In conclusion, the results of this study indicate that the accumulation of PI(3,4,5)P3 triggered by fMLP in dPLB-985 is biphasic, with a peak of production observed at short time periods that entirely depends on p110γ activity, and a delayed, less intense phase that is mediated by class IA PI3K. The data presented above also provide evidence that the PI3K-dependent superoxide production and chemotaxis induced by the chemotactic factor mainly involves PI3K IA and, by implication, the delayed phase of PI(3,4,5)P3 production, whereas p110γ does not play a major role. Several important questions remain to be investigated, including the elucidation of the basis for the specificity of the signals produced by these distinct PI3Ks, the definition of the roles of the subcellular localization, and the intensity or duration of the production of PI(3,4,5)P3 in the initiation and regulation of the functional responsiveness of human neutrophils.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported in part by grants from the Canadian Institutes for Health Research and by fellowships from the Association pour la Recherche sur le Cancer of France and the Arthritis Society of Canada (to I.B.). P.H.N. is the recipient of the Canada Research Chair on the Physiopathology of the Neutrophil.

  • ↵2 Address correspondence and reprint requests to Dr. Paul H. Naccache, Centre de Recherche en Rhumatologie et Immunologie du Centre Hospitalier de l’Université Laval, Room T1-49, 2705, Boulevard Laurier, Sainte-Foy, Québec Canada, G1V 4G2. E-mail address: Paul.Naccache{at}crchul.ulaval.ca

  • ↵3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; dPLB-985, differentiated neutrophil-like PLB-985 cells; EGFP, enhanced GFP; KD, kinase dead.

  • Received October 11, 2005.
  • Accepted March 29, 2006.

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