Class I Phosphoinositide 3-Kinase p110β Is Required for Apoptotic Cell and Fcγ Receptor-mediated Phagocytosis by Macrophages*

Phosphoinositide 3-kinases (PI3Ks) play an important role in a variety of cellular functions, including phagocytosis. PI3Ks are activated during phagocytosis induced by several receptors and have been shown to be required for phagocytosis through the use of inhibitors such as wortmannin and LY294002. Mammalian cells have multiple isoforms of PI3K, and the role of the individual isoforms during phagocytosis has not been addressed. The class I PI3Ks consist of a catalytic p110 isoform associated with a regulatory subunit. Mammals have three genes for the class IA p110 subunits encoding p110α, p110β, and p110δ and one gene for the class IB p110 subunit encoding p110γ. Here we report a specific recruitment of p110β and p110δ (but not p110α) isoforms to the nascent phagosome during apoptotic cell phagocytosis by fibroblasts. By microinjecting inhibitory antibodies specific to class IA p110 subunits, we have shown that p110β is the major isoform required for apoptotic cell and Fcγ receptor-mediated phagocytosis by primary mouse macrophages. Macrophages from mice expressing a catalytically inactive form of p110δ showed no defect in the phagocytosis of apoptotic cells and IgG-opsonized particles, confirming the lack of a major role for p110δ in this process. Similarly, p110γ-deficient macrophages phagocytosed apoptotic cells normally. Our findings demonstrate that p110β is the major class I catalytic isoform required for apoptotic cell and Fcγ receptor-mediated phagocytosis by primary macrophages.

Phosphoinositide 3-kinases (PI3Ks) play an important role in a variety of cellular functions, including phagocytosis. PI3Ks are activated during phagocytosis induced by several receptors and have been shown to be required for phagocytosis through the use of inhibitors such as wortmannin and LY294002. Mammalian cells have multiple isoforms of PI3K, and the role of the individual isoforms during phagocytosis has not been addressed. The class I PI3Ks consist of a catalytic p110 isoform associated with a regulatory subunit. Mammals have three genes for the class IA p110 subunits encoding p110␣, p110␤, and p110␦ and one gene for the class IB p110 subunit encoding p110␥. Here we report a specific recruitment of p110␤ and p110␦ (but not p110␣) isoforms to the nascent phagosome during apoptotic cell phagocytosis by fibroblasts. By microinjecting inhibitory antibodies specific to class IA p110 subunits, we have shown that p110␤ is the major isoform required for apoptotic cell and Fc␥ receptor-mediated phagocytosis by primary mouse macrophages. Macrophages from mice expressing a catalytically inactive form of p110␦ showed no defect in the phagocytosis of apoptotic cells and IgG-opsonized particles, confirming the lack of a major role for p110␦ in this process. Similarly, p110␥-deficient macrophages phagocytosed apoptotic cells normally. Our findings demonstrate that p110␤ is the major class I catalytic isoform required for apoptotic cell and Fc␥ receptor-mediated phagocytosis by primary macrophages.
Phagocytosis is the process by which professional and nonprofessional phagocytes internalize large particles. It is a conserved mechanism that is critical for the immune system and that is implicated in host defense and the removal of dying cells (1). Uptake of pathogens via various pattern recognition receptors and uptake of IgG-opsonized foreign particles via the receptors for the Fc portion of IgG (Fc␥Rs) 1 allow their degrada-tion as well as the subsequent presentation of foreign antigens to the cells of the adaptive immune system (1). In addition, phagocytosis allows the removal of cells dying by apoptosis in vivo, a process thought to be essential for the resolution of inflammation (2) and the modulation of the immune system (3).
A variety of cells, including fibroblasts, epithelial cells, endothelial cells, and Sertoli cells, are capable of engulfing their dying neighbors. However, professional phagocytes such as macrophages are thought to be responsible for uptake of the majority of dying cells (4). An important feature of the apoptosis program is to make the apoptotic cells recognizable as targets for phagocytosis. Rapid engulfment is thought to avoid injury to surrounding tissues by preventing the dying cells from releasing their intracellular contents. Several receptors have been shown to play a role in apoptotic cell recognition, including the vitronectin receptor (an ␣ V ␤ 3 integrin), scavenger receptors, CD14, and the phosphatidylserine receptor (5). Recent studies in the worm Caenorhabditis elegans and in mammalian systems have identified some of the signaling pathways required for apoptotic cell uptake (5). An early step is the polymerization of actin to form plasma membrane extensions called pseudopods (6). The pseudopods extend over the particle and close to form a phagosome. The process of actin cup formation requires the activation of tyrosine kinases (7) and the participation of members of the Rho GTPase family (6). The closure of the actin cup is inhibited by LY294002 and wortmannin, inhibitors of phosphoinositide 3-kinases (PI3Ks) (6). The completion of other forms of phagocytosis such as Fc␥R-and complement receptor-3-mediated phagocytosis is also dependent on PI3K activity (8 -10). Therefore, one or more isoforms of PI3K are required for the phagocytic process.
PI3Ks are activated by a variety of signals and control cellular functions such as cytoskeleton reorganization, migration, vesicular trafficking, metabolic control, and cell survival and proliferation (11,12). PI3Ks form a family of three classes of enzymes that phosphorylate phosphatidylinositol (PI) lipids at the 3Ј-position (11,12). Among the PI3Ks, class I (divided into two subclasses, IA and IB) has been shown to transmit signals from tyrosine kinase-coupled receptors and G protein-coupled receptors. In vivo, class I PI3Ks catalyze mainly the conversion of PI(4,5)P 2 to PI(3,4,5)P 3 . Class IA PI3Ks are heterodimeric proteins consisting of a p110 catalytic subunit associated with a p85, p55, or p50 regulatory subunit (12). Mammals have three forms of the class IA p110 subunit: p110␣, p110␤, and p110␦. Class IB consists of a p110␥ catalytic subunit and a p101 regulatory protein. The main function of the class IA regulatory subunit is to recruit the p110 subunit from the cytoplasm to activated receptors at the plasma membrane, where the catalytic subunit phosphorylates its lipid substrate. The role of the p101 regulatory subunit in the regulation of p110␥ activity is less clear at present. The 3-phosphorylated PI lipids are second messengers that interact with the lipid-binding domains of a variety of cellular proteins (13). A number of PI-binding domains have been identified, and chimeras with such domains coupled to green fluorescent protein have been used to detect the levels of various PIs in live cells by fluorescence imaging during Fc␥R-mediated phagocytosis. Using this approach, it has been demonstrated that PI(3,4,5)P 3 is produced at the plasma membrane in a PI3K-dependent way during phagocytosis (14). PI(3,4,5)P 3 localizes to the actin cup at an early stage, before closure of the phagosomal vacuole. This is in accord with the early recruitment of p85␣ to the Fc␥R (14). The accumulation of PI(3,4,5)P 3 is transient, followed by the accumulation of PI(3)P concomitant with the sealing of the vacuole (15,16).
The class III PI3K group comprises one enzyme (human VPS34) that produces PI(3)P from PI and that is involved in intracellular trafficking (12). During the phagocytic process, human VPS34 is not essential for engulfment, but is required for the fusion of phagosomes with endosomes (15). Accordingly, it has been shown that PI(3)P controls the early stages of phagosome maturation (16). Finally, class II PI3K enzymes (PI3KC2␣, PI3KC2␤, and PI3KC2␥) are large enzymes characterized by a C-terminal C2 domain (17). They have the potential to produce PI(3)P and PI(3,4)P 2 and possibly PI(3,4,5)P 3 . Their contribution in phagocytosis has not been examined yet.
In this study, we sought to examine the requirement for different class I PI3Ks during apoptotic cell and Fc␥R-mediated phagocytosis. Our approach has been to study the recruitment of class IA p110 isoforms to the phagosome and to inhibit their lipid kinase activity by microinjecting specific inhibitory antibodies. Moreover, we have studied the phagocytic capabilities of macrophages expressing catalytically inactive p110␦ and of macrophages lacking p110␥.

EXPERIMENTAL PROCEDURES
Primary Macrophages and Cell Lines-Baf-3 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 6% heatinactivated fetal bovine serum (Sigma) and 5% WEHI-3B cell-conditioned medium as a source of interleukin-3 (18). Apoptosis was induced by 20 h of growth factor deprivation as previously described (19). The Jurkat human leukemia T cell line was cultured in RPMI 1640 medium (Invitrogen) containing 10% fetal calf serum. Apoptosis was induced by incubation for 2 h with staurosporine (1 M; Sigma). Under these conditions, Jurkat cells were Ͻ5% trypan blue (Sigma)-positive and 50 -70% annexin V (Sigma)-positive, indicating that they were at an early stage of apoptosis. NIH 3T3 fibroblasts were maintained in RPMI 1640 medium containing 10% donor calf serum (Invitrogen) and seeded at 2 ϫ 10 4 cells/13-mm glass coverslips in 4-well multidishes 24 h before phagocytosis assay.
To purify peritoneal macrophages, mice were killed; 20 ml of phosphate-buffered saline (PBS) was injected into the peritonea; the abdomens were massaged; the PBS was withdrawn; and the cells were resuspended in macrophage medium and seeded at 2 ϫ 10 5 cells on 13-mm glass coverslips. After 2 h, non-adherent cells were removed, and the adherent cells were cultured for an additional 20 h prior to phagocytosis assay.
Antibodies and Microinjections-Rabbit polyclonal antibodies against synthetic peptides corresponding to the carboxyl termini of p110␣, p110␤, and p110␦ have been previously described (see supplementary information in Ref. 23). Preimmune IgGs were purified over protein A. For pre-blocking experiments, anti-p110␤ and anti-p110␦ antibodies were preincubated for 15 min with a 15-fold molar excess of their cognate synthetic peptides (KVNWMAHTVRKDYRS and (C)K-VNWLAHNVSKDNRQ, respectively; Alta Bioscience). Commercial rabbit IgGs (Sigma) were used as controls.
Wild-type macrophages seeded on 13-mm glass coverslips were injected into the cytoplasm with 1 mg/ml rabbit polyclonal antibodies using an Eppendorf Transjector. After microinjection, cells were subjected to phagocytosis assays.
Phagocytosis Assays and Quantification-Sheep red blood cells (ICN) were opsonized with a subagglutining concentration of rabbit antisheep red blood cell IgG (ICN), fluorescently labeled in the presence of 10 M 5(6)-carboxyfluorescein succinimidyl ester (Molecular Probes, Inc.) for 10 min at 37°C, washed with PBS, and resuspended in macrophage medium. 5 ϫ 10 5 red blood cells were added to 4 ϫ 10 4 macrophages for the indicated times.
Prior to the induction of apoptosis, Baf-3 and Jurkat cells were fluorescently labeled by incubation for 10 min with 10 M 5(6)-carboxyfluorescein succinimidyl ester. Cells were washed with PBS containing 1 mM MgCl 2 (PBS/MgCl 2 ) and then incubated at 37°C for 10 min in PBS/MgCl 2 containing 1 mg/ml biotinamidocaproate N-hydroxysuccimide ester (Sigma) to biotinylate membrane proteins. Cells were washed with PBS/MgCl 2 and then resuspended at 5 ϫ 10 5 cells/ml in culture medium. After apoptosis induction (see above), the cells were washed, and 5 ϫ 10 5 cells were added to 4 ϫ 10 4 phagocytes for the indicated times. To remove unbound cells but to preserve bound particles, coverslips were gently washed with PBS and then fixed with 4% paraformaldehyde (BDH) in PBS for 10 min.
To quantify apoptotic cell phagocytosis by macrophages, specimens were stained for 1 h at room temperature with rhodamine-conjugated streptavidin (10 g/ml; Sigma) and a fluorescein-conjugated rat antibody against the macrophage cell-surface marker F4/80 (2 g/ml; Serotec Ltd.) in PBS and then washed twice with PBS for 5 min. Coverslips were mounted with Mowiol (Calbiochem) and examined by epifluorescence microscopy (Zeiss). This procedure has been described in detail previously (6). Briefly, the macrophages were immunodetected by the antibody against F4/80 and fluoresced green. The apoptotic cells fluoresced intensely green due to the 5(6)-carboxyfluorescein succinimidyl ester staining. The non-internalized apoptotic cells also fluoresced red, as their plasma membrane was biotinylated and remained accessible to the rhodamine-conjugated streptavidin.
To quantify apoptotic cell phagocytosis by NIH 3T3 cells, specimens were stained with rhodamine-conjugated streptavidin, permeabilized with 0.2% Triton X-100 (BDH) in PBS for 5 min, and stained with fluorescein-conjugated phalloidin (Sigma) to visualize the fibroblasts. To quantify opsonized red blood cell phagocytosis by macrophages, specimens were stained with fluorescein-conjugated antibody against F4/80 and rhodamine-conjugated anti-rabbit IgG (5 g/ml; Southern Biotechnology) to detect non-internalized red blood cells.
Phagocytosis was quantified by counting ϳ100 phagocytes from 5 to 10 randomly selected fields. Apoptotic cells or red blood cells were counted as phagocytosed if they fluoresced only green and as bound if they fluoresced green and red. The phagocytic index (number of particles internalized per 100 macrophages) and binding index (number of particles bound per 100 macrophages) were determined. When required, microinjected macrophages were detected by a final step of permeabilization with 0.2% Triton X-100 and staining with aminomethylcoumarin-conjugated anti-rabbit IgG (50 g/ml; Jackson Immu-noResearch Laboratories, Inc.).

Recruitment of p110␤ and p110␦ to Phagocytic Cups-To
address the role of individual p110 isoforms during apoptotic cell phagocytosis, we first investigated their recruitment to the nascent phagosome. In the absence of antibodies sensitive enough to detect endogenous class IA p110 isoforms, we increased their expression levels in NIH 3T3 fibroblasts by transfection. Although these cells are not professional phagocytes, they are able to phagocytose apoptotic cells, albeit at low levels, using the phosphatidylserine receptor (24). The engulfment (but not the binding) of apoptotic cells by NIH 3T3 cells was inhibited by the PI3K inhibitor LY294002 (Fig. 1), showing that the phagocytosis of apoptotic cells by NIH 3T3 cells is PI3K-dependent. NIH 3T3 cells were transfected with constructs coding for Myc-tagged p110 isoforms and then incubated with apoptotic cells. In the absence of bound particles, Myc-tagged p110␣, p110␤, and p110␦ were detected mainly in the cytoplasm (data not shown). When NIH 3T3 cells engulfed apoptotic cells, F-actin accumulated to form an actin cup at the site of binding with the apoptotic cell (Fig. 2). Localized accumulation of p110␤ and p110␦ (but not p110␣) was observed in the actin cup (Fig. 2). When apoptotic cells were bound only to the NIH 3T3 cells without inducing the formation of an actin cup, p110␤ and p110␦ did not localize to the site of binding (data not shown). This result indicates that p110␤ and p110␦ can be specifically recruited at the site of apoptotic cell phagocytosis in transfected NIH 3T3 fibroblasts.
p110␤ Is Required for Apoptotic Cell and Fc␥R-mediated Phagocytosis-Several studies have used the microinjection of isoform-specific inhibitory antibodies to evaluate the role of class IA p110 subunits in growth factor signaling (23,(25)(26)(27). Rabbit polyclonal antibodies against the carboxyl termini of p110␣, p110␤, and p110␦ were produced and characterized previously (see supplementary information in Ref. 23). Each antibody (but not preimmune IgG) selectively neutralized the lipid kinase activity of its specific p110 in vitro. In the BAC1.2F5 macrophage cell line, antibodies to p110␤ and p110␦ were shown to inhibit CSF-1-induced chemotaxis, whereas antibody to p110␣ was shown to inhibit CSF-1-induced proliferation (23).
To evaluate the requirement for the distinct class IA p110 isoforms during apoptotic cell phagocytosis, bone marrow-derived macrophages were injected intracytoplasmically with each antibody, and apoptotic cell phagocytosis was quantified. Bone marrow-derived macrophages use the ␣ V ␤ 3 integrin for the uptake of apoptotic cells (6,28). Microinjection of control IgG or preimmune IgG did not affect apoptotic cell binding or apoptotic cell internalization by the macrophages (Fig. 3), indicating that the microinjection process did not affect the phagocytosis process. Macrophages microinjected with anti-p110␣ antibody phagocytosed apoptotic cells at levels similar to control macrophages, whereas macrophages microinjected with anti-p110␦ antibody showed a small but significant reduction in the phagocytosis of apoptotic cells. Macrophages injected with anti-p110␤ antibody were most severely blocked in the phagocytosis of apoptotic cells (70% inhibition). The effects observed were not due to impaired binding, as none of the antibodies blocked apoptotic cell binding significantly (Fig. 3B). Moreover, pre-blocking the anti-p110␤ and anti-p110␦ antibodies with their cognate peptides fully reversed the phagocytosis inhibition (Fig. 3A).
We subsequently investigated the contribution of p110␤ to the phagocytosis of other particles or by other types of macrophages. Phagocytosis of apoptotic Baf-3 cells by peritoneumderived macrophages (Fig. 4A) as well as phagocytosis of apoptotic Jurkat cells by bone marrow-derived macrophages (Fig.  4B) were significantly inhibited by injection of anti-p110␤ antibody. Moreover, phagocytosis of IgG-opsonized particles mediated by Fc␥R was also very significantly inhibited by injection of anti-p110␤ antibody (Fig. 4C). Fc␥R-mediated  2. p110␤ and p110␦ are recruited to the actin cup during apoptotic cell phagocytosis by fibroblasts. NIH 3T3 fibroblasts were transfected with cDNAs encoding 5ЈMyc-p110␣KD (A), 5ЈMyc-p110␤ (B), and p110␦-3ЈMyc (C). Transfected cells were incubated for 24 -32 h to allow expression and then incubated for an additional 1.5 h with biotinylated apoptotic Baf-3 cells. F-actin was detected using rhodamine-conjugated phalloidin (left panels). Apoptotic cells were detected using Cy5-conjugated streptavidin (middle panels). p110 isoforms were detected using mouse anti-Myc antibody, followed by fluorescein-conjugated anti-mouse antibody (right panels). Arrows indicate colocalization of the actin cup, the p110 isoform, and an apoptotic cell. Arrowheads indicate an actin cup surrounding an apoptotic cell, but devoid of p110␣ staining. Similar results were obtained with wildtype p110␣-3ЈMyc (data not shown). Bar ϭ 10 m. phagocytosis was not inhibited or was only slightly inhibited following microinjection of anti-p110␣ or anti-p110␦ antibody, respectively. These results indicate that phagocytosis of both apoptotic cells and immunoglobulin-coated particles is dependent on p110␤.
Phagocytosis Is Not Impaired in Macrophages from Mice Lacking Functional p110␦ or p110␥-Given the structural and functional similarities between p110␤ and p110␦ (12,29) and the strong effect of anti-p110␤ antibody on phagocytosis, the modest effect of anti-p110␦ antibody on apoptotic cell and Fc␥R-mediated phagocytosis was unexpected. As an alternative approach to evaluate the p110␦ requirement for phagocytosis, we examined the phagocytic capabilities of macrophages derived from mice expressing a catalytically inactive form of p110␦ (p110␦ D910A/D910A ) (20). Inactivation of p110␦ did not affect the expression levels of the p110 isoforms in bone marrow-derived macrophages. 2 Macrophages in similar numbers and expressing equivalent levels of the macrophage cell-surface marker F4/80 were generated from the bone marrow or peritoneal cavities of wild-type and p110␦ D910A/D910A mice, indicating that there is no apparent defect in macrophage differentiation (data not shown). Macrophages were derived from the bone marrow of wild-type and p110␦ D910A/D910A mice (matched for sex and age) and cultured for different times with apoptotic Baf-3 cells. There was no discernible difference in the ingestion of apoptotic cells between wild-type and p110␦ D910A/D910A macrophages (Fig. 5A). Moreover, phagocytosis by p110␦ D910A/D910A macrophages was still inhibited by the PI3K inhibitor LY294002, indicating that another PI3K, distinct from p110␦, is critical for phagocytosis. Phagocytosis of apoptotic Baf-3 or Jurkat T cells by peritoneal macrophages was also not affected by p110␦ inactivation (Fig. 5B). Similarly, p110␦ D910A/D910A and wild-type macrophages ingested similar numbers of IgG-opsonized red blood cells, indicating that Fc␥Rmediated phagocytosis was not affected (Fig. 5C).
Next, to determine whether the class IB catalytic p110␥ isoform plays a role in apoptotic cell phagocytosis, we compared the phagocytic capabilities of bone marrow-derived macrophages from wild-type and p110␥-deficient mice. The generation of p110␥ mice has been described previously (21). Deficiency in p110␥ did not impair apoptotic Baf-3 cell phagocytosis (Fig. 6). This result obtained with apoptotic cell phagocytosis is consistent with a lack of a role for p110␥ in phagocytosis mediated by other receptors such as Fc␥R-mediated phagocytosis by neutrophils (30) and bacterial phagocytosis by peritoneal macrophages (21). DISCUSSION PI3K activity has previously been shown to be stimulated and/or required for phagocytosis by professional phagocytes (6, 8 -10). In this study, we have investigated the involvement of each of the four class I PI3K catalytic subunits in phagocytosis. By studying the recruitment and requirement of class IA p110 isoforms (p110␣, p110␤, and p110␦) and by studying macrophages either expressing inactive p110␦ or deficient in class IB p110␥, we have shown that p110␤ is the major class I catalytic isoform involved in phagocytosis. 2 C. Sawyer and B. Vanhaesebroeck, unpublished data.

FIG. 4. p110␤ is required for various forms of phagocytosis by macrophages.
Macrophages were microinjected with control IgG or anti-p110 antibodies as indicated in the legend to Fig. 3 and subsequently assayed for phagocytosis. A, peritoneal macrophages incubated for 90 min with apoptotic Baf-3 cells; B, bone marrow-derived macrophages incubated for 90 min with apoptotic Jurkat cells; C, bone marrow-derived macrophages incubated for 10 min with IgG-opsonized red blood cells. None of the antibodies blocked binding of IgG-opsonized red blood cells significantly (data not shown). The phagocytic index of injected macrophages was compared with that of non-injected neighboring macrophages. Values are means of two to six independent experiments. Results obtained with anti-p110 antibodies were compared with results obtained with control IgG using Student's paired t test, *, p Ͻ 0.001 (very significant); **, p Ͻ 0.05 (significant).
There is increasing evidence that different p110 isoforms of the class IA PI3K have distinct functions depending on the stimulus and/or cell type involved. These results have been obtained by microinjection of inhibitory antibodies specific for the p110 subunits in a variety of cell lines. For instance, in the murine BAC1.2F5 macrophage cell line, p110␣ is important for CSF-1-induced proliferation, whereas p110␤ and p110␦ are involved in CSF-1-induced actin reorganization and cell migration (23). p110␦ can also regulate cell migration in breast cancer lines (31). In porcine aortic endothelial cells, plateletderived growth factor-induced actin reorganization depends on p110␣, whereas insulin-induced actin reorganization is p110␤dependent (26). In human colon carcinoma cells, p110␣ has been implicated in survival and p110␤ in proliferation (32). Our study confirms that different PI3K catalytic subunits have non-redundant functions and identifies p110␤ as the major isoform required for Fc␥R-mediated and apoptotic cell phagocytosis. PI3Ks are also involved in other types of phagocytosis such as complement receptor-3-mediated phagocytosis (10), although the mechanism of complement-mediated phagocytosis is clearly distinct from that of Fc␥R-mediated or apoptotic cell phagocytosis (33). It will therefore be interesting to investigate which PI3K isoforms contribute to other types of phagocytosis.
We have found that, in NIH 3T3 fibroblasts, p110␤ and p110␦ (but not p110␣) can be recruited to the nascent phagosome during apoptotic cell phagocytosis. A similar selective association of p110 isoforms has been observed in Jurkat cells, where p110␤ and p110␦ (but not p110␣) associate with Ras upon redox stimulation (12). Ras effectors have been shown to be activated during Fc␥R-mediated phagocytosis and during apoptotic cell phagocytosis (7). Ras may therefore provide a mechanism for specific PI3K activation/recruitment during phagocytosis.
The modest inhibition of phagocytosis observed upon microinjection of anti-p110␦ antibody was not reproduced when we examined the phagocytic capabilities of macrophages from p110␦ D910A/D910A mice. A possible explanation for this discrepancy is that the role of p110␦ in phagocytosis activity could be independent of its catalytic activity. For example, p110␦ might bind to and recruit other proteins to the phagocytic cup, and this would be inhibited by anti-p110␦ antibody. Another explanation could be that the absence of p110␦ activity in p110␦ D910A/D910A macrophages has been compensated for by an increase in the level or the activity of other class IA subunits, as was seen in p85␣ and p110␣ gene-targeted mice (34 -36). However, the levels of class IA subunits in p110␦ D910A/D910A macrophages were comparable to those in wild-type macrophages. 2 Moreover, the kinase activities of p110␣ and p110␤ were not altered in macrophages from p110␦ D910A/D910A mice. 3 Finally, we can rule out a role for the class IB PI3K in phagocytosis, as p110␥ disruption did not impair apoptotic cell phagocytosis by primary bone marrow-derived macrophages, in agreement with previous studies showing that p110␥ is not required for bacterial uptake by peritoneal macrophages and Fc␥R-mediated phagocytosis by neutrophils (21,30). Altogether, these results indicate that, among the class I PI3Ks, p110␤ is the major catalytic subunit required for apoptotic cell 3 A. Bilancio, C. Sawyer, and B. Vanhaesebroeck, unpublished data. and Fc␥R-mediated phagocytosis by macrophages. The lethal phenotype of mice deficient in p110␤ (37) unfortunately precludes their use in the study of p110␤ involvement in macrophage phagocytosis.
Interestingly, although PI3Ks have been shown to act upstream of the actin cytoskeleton reorganization following growth factor stimulation (23), their role during phagocytosis seems to occur at a different level. In fact, inhibition of PI3K activity does not prevent the formation of the actin cup accompanying Fc␥R-mediated phagocytosis or apoptotic cell phagocytosis, but prevents the engulfment of the particles (6,8,9). Recently, several mechanisms have been proposed to explain the mode of action of PI3Ks during phagocytosis. First, it has been suggested that PI3Ks might allow phagosomes to form and/or close via a localized contractile activity (38). Inhibitors of PI3Ks and of myosins have similar effects on Fc␥R-mediated phagocytosis, suggesting that the two families of proteins act at similar stages. Multiple myosin isoforms are recruited to the phagosome, but their requirement has not been investigated until recently. Myosin X, an unconventional myosin that contains a PI(3,4,5)P 3 -interacting domain, is required for Fc␥Rmediated phagocytosis, and its recruitment to the actin cup is dependent on PI3K activity (39). Myosin X has therefore been suggested to be a link between PI3Ks and myosin-based contractility. Second, PI3Ks might play a role in focal exocytosis during phagocytosis (40). During the engulfment process, the delivery of membrane compensates for the loss of membrane taken up into phagosomes. It has been suggested that the accumulation of the class I PI3K product PI(3,4,5)P 3 and other PIs in the phagosomal cup might promote the recruitment of tethering proteins, leading to the docking of exocytic vesicles (40). In this respect, it is interesting that p110␤ interacts with the small GTPase Rab5, which is involved in endosomal trafficking and recruited to newly formed phagosomes (41)(42)(43). Finally, at least in macrophages, PI3Ks also regulate the contribution of the endoplasmic reticulum as a source of membrane during phagocytosis (44).
It is interesting to note that the microinjection of anti-p110␤ antibody did not completely abrogate the forms of phagocytosis we examined in primary macrophages. This suggests that a PI3K distinct from class I is also involved. Class II PI3KC2␤ is sensitive to low concentrations of the PI3K inhibitor wortmannin and is activated by membrane receptors such as integrins (45), and it will therefore be important to investigate its role in phagocytosis.