We investigated the requirement for Syk activation to initiate downstream signaling events during polymorphonuclear leukocyte (PMN) phagocytosis of Ab-coated erythrocytes (EIgG). When PMN were challenged with EIgG, Syk phosphorylation increased in a time-dependent manner, paralleling the response of PMN phagocytosis. Pretreatment of PMN with piceatannol, a Syk-selective inhibitor, blocked EIgG phagocytosis and Syk phosphorylation. We found that piceatannol inhibited protein kinase Cδ (PKCδ) and Raf-1 translocation from cytosol to plasma membrane by >90%. Extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2) phosphorylation was similarly blocked. We also investigated phosphatidylinositide 3-kinase (PI 3-kinase) activity and Syk phosphorylation using piceatannol, wortmannin, and LY294002, inhibitors of PI 3-kinase. The phosphorylation of Syk preceded the activation of PI 3-kinase. Both wortmannin and piceatannol inhibited PI 3-kinase, but only piceatannol inhibited Syk. In contrast to piceatannol, wortmannin did not inhibit PKCδ and Raf-1 translocation. To elucidate signaling downstream of Syk activation, we assessed whether the cell-permeable diacylglycerol analogue didecanoylglycerol could normalize PMN phagocytosis, PKCδ and Raf-1 translocation, and ERK1 and ERK2 phosphorylation inhibited by piceatannol. The addition of didecanoylglycerol to the Syk-inhibited phagocytosing PMN normalized all three without a concomitant effect on PI 3-kinase activity and Syk phosphorylation. We conclude that Syk activation following Fcγ receptor engagement initiates downstream signaling events leading to mitogen-activated protein kinase activation independent of PI 3-kinase activation.

Polymorphonuclear leukocytes (PMN)4 play an important role in host defense through their ability to phagocytose particles and to initiate the respiratory burst. These responses are initiated through several possible stimuli, including binding of complement-opsonized particles to the complement receptor or the Fc domain of an IgG-Ag complex to an Fcγ receptor on the cell (1, 2, 3). PMNs possess three classes of Fcγ receptors, inducible and constitutive, FcγRI, FcγRIIA, and FcγRIIIB, all of which are involved in phagocytosis (1, 4). Protein tyrosine kinases have been implicated in signaling events initiated by Fcγ receptors (5, 6). FcγIIA receptors do not possess intrinsic tyrosine kinase activity, but their cytoplasmic domains possess sequences that facilitate interactions with protein tyrosine kinases. Recently, the subunits of the cytoplasmic domain of the FcγIIA receptor have been implicated in signal transduction because they include conserved tyrosine-containing sequences, which are thought to bind to the SH2 (SRC homology 2) domain of protein tyrosine kinases (7, 8).

Syk possesses two N-terminal SH2 domains that bind in tandem to sites located within the immunoreceptor tyrosine-based activation motifs (ITAMs) of Ag receptor subunit (9). Following Fcγ receptor engagement in monocytes and macrophages, Syk is associated with the γ-chain of FcγRI and FcγRIIIA and with the cytoplasmic domain of FcγRIIA, whereupon it becomes phosphorylated on tyrosine, and is activated. Syk is recruited through its SH2 domains to the Fcγ receptors, subsequently undergoes autophosphorylation, and induces the phosphorylation of multiple substrates, including other Fcγ receptor ITAMs and downstream effectors (10, 11). Upon transfection with human Fcγ receptors, COS-1 cells acquire phagocytotic properties, which in the case of FcγRI and Fcγ RIIIA isoforms are dependent on an ITAM within the γ-chain of the receptor (12, 13, 14). However, reconstitution of the receptor complex results in only marginal phagocytic activity, which can be significantly increased by cotransfection with Syk (1). Syk plays a major role in phagocytosis, because a chimeric transmembrane protein containing only the Syk catalytic domain is capable of triggering phagocytosis in COS cells (15). Clustering of the FcγRIII-Syk fusion in COS-1 cells results in a phagocytic signal that is dependent on an intact Syk kinase domain (15). Treatment of monocytes with Syk antisense oligodeoxynucleotides has been reported to abrogate phagocytosis (16). Although it is known that Syk phosphorylation is involved in Fcγ receptor engagement, little is known concerning either the mechanism or the identity of many of the downstream tyrosine-phosphorylated substrates in EIgG-stimulated PMN undergoing phagocytosis.

We have shown that the MAP kinase pathway is another key component in the transduction of signals leading to Fcγ receptor-mediated phagocytosis in PMN (17). This pathway consists of a linear cascade of the protein kinases Raf-1, MEK, ERK1, and ERK2 (18, 19, 20). The ERK proteins are phosphorylated and activated by the dual specificity kinase MEK, which is phosphorylated and activated by the serine/threonine kinase Raf. We previously reported that PKCδ is a key component of the phagocytic pathway and is translocated from cytosol to plasma membrane in phagocytosing PMN. This, in turn, leads to translocation of Raf-1 to the plasma membrane followed by phosphorylation of ERK1 and ERK2 (21).

Phosphatidylinositide 3-kinase (PI 3-kinase) has also been implicated previously in signaling by all three classes of Fcγ receptor (1, 22). Activation of PI 3-kinase results in the appearance of the lipid products of this enzyme, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (23, 24). The PI 3-kinase is composed of a catalytic subunit p110 and an associated regulatory subunit, p85 (25, 26). Wortmannin, an inhibitor of PI 3-kinase, binds to the catalytic subunit of PI 3-kinase in PMN (27).

The purpose of the present study was to examine the requirement of PMN Syk activation to initiate downstream signaling events during Fcγ receptor-mediated phagocytosis. We evaluated whether Syk activation would lead to PI 3-kinase activation and PKCδ and Raf-1 translocation, the latter translocations being associated with ERK activation.

Wortmannin and LY294002 were purchased from Biomol (Plymouth Meeting, PA), and piceatannol (3,4,3′,5′-tetrahydroxy-trans-stilbene) was obtained from Alexis (San Diego, CA); FMLP, cytochalasin B, and di-isopropyl fluorophosphate were purchased from Sigma (St. Louis, MO). Polyclonal Abs against ERK1 and ERK2 (p44/42) recognizing the phosphorylated form of both p42 and p44 were obtained from New England BioLabs (Beverly, MA). Polyclonal and monoclonal Ab against Syk and polyclonal Ab against Hck and Fgr were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-phosphotyrosine Ab 4G10 and polyclonal Ab against PI 3-kinase p85 were purchased from Upstate Biotechnology (Lake Placid, NY), and monoclonal Abs against PKCδ and Raf-1 were obtained from Transduction Laboratories (Lexington, KY). The HRP-conjugated sheep anti-mouse Abs were obtained from Amersham (Arlington Heights, IL), and HRP-conjugated anti-rabbit Ab was obtained from Santa Cruz Biotechnology. sn-1,2-dioctanoylglycerol (DiC8), sn-1,2-didecanoylglycerol (DiC10), and l-α-phosphatidylinositol (liver) were purchased from Avanti Polar Lipids (Alabaster, AL). The [γ-32P]ATP was obtained from ICN Pharmaceuticals (Irvine, CA). The G-CSF was a gift from Amgen (Thousand Oaks, CA).

Human PMN were isolated from human peripheral blood as described previously (28). Briefly, fresh whole blood was obtained by venipuncture from healthy volunteers and immediately added to acid citrate dextrose. The PMN were purified by dextran sedimentation followed by hypotonic lysis to remove the majority of erythrocytes and then centrifuged through Ficoll-Paque (Pharmacia LKB Biotechnology, Piscataway, NJ) to remove contaminating mononuclear cells. Before activation of cells and subcellular fractionation, the cells were incubated for 5 min on ice with 5 mM di-isopropyl fluorophosphate, washed, and resuspended in the desired buffer.

Sheep erythrocytes were purchased from BioWhittaker (Walkersville, MD) and were opsonized with anti-sheep erythrocyte IgG (ICN Pharmaceuticals) as previously described (29).

The phagocytosis assay was conducted essentially as outlined by Pommier et al. (30). For studies with inhibitors, PMN were suspended at 2 × 106/ml in PBS containing 1 mM Ca2+ and 1 mM Mg2+ and were incubated with different concentrations of inhibitors for 30 min at 22°C. In other experiments PMN were preincubated with inhibitors and then treated with DiC10. Following the incubation, PMN underwent phagocytosis with EIgG (1 × 108/ml), and the incubation was continued for an additional 30 min at 37°C. EIgG that were not ingested were lysed with distilled water, and isotonicity was restored using KCl. Samples were fixed with glutaraldehyde and evaluated microscopically. The number of EIgG ingested per 100 PMN was determined. Inhibition of phagocytosis in the presence of inhibitors was expressed as a percentage of the control value, with control being phagocytosis by PMN in the absence of inhibitor treatment.

PMN lysates (1–2 × 106 PMN/sample in 30–40 μl of buffer) were combined with sample buffer, boiled for 5 min, and run on 10% SDS-PAGE minigels. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Schleicher & Schuell, Keene, NH) for 2 h at 100 V, and the membrane was blocked with 2% BSA in PBS containing 1 mM EDTA, 0.05% Tween 20, and 1 mM Na3VO4. The membrane was probed with Ab against phosphorylated p44/42 in blocking buffer, washed three times with 0.2% Tween 20 in 50 mM Tris (pH 8.0) and 100 mM NaCl, then incubated with a second Ab (HRP-conjugated goat anti-rabbit Ab) in wash buffer containing 5% nonfat dry milk. Phosphorylated bands were visualized using the enhanced chemiluminescence system (ECL, Amersham, Arlington Heights, IL). Immunoblotting was also conducted using anti-phosphotyrosine 4G10, anti-Raf-1, and anti-PKCδ Ab. The HRP-conjugated sheep anti-mouse Ab served as a second Ab for anti-phosphotyrosine, anti-Raf-1, and anti-PKCδ. For these experiments PMN were treated as described in the section, Cell fractionation for immunoblotting.

The PMN (4 × 106/sample) were lysed in lysis buffer containing 1% Triton X-100 along with 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM Na3VO4, 1 mM PMSF, 10 μg/ml soybean trypsin inhibitor, and 1 μg/ml each of leupeptin, aprotinin, and pepstatin. Lysates were precleared with protein A-Sepharose for 30 min and incubated overnight at 4°C with anti-Syk, anti-Hck, or anti-Fgr Ab. Protein A-Sepharose was added to each sample and incubated for 2 h with rotation at 4°C. The beads were washed briefly three times with lysis buffer and twice with buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 1 mM Na3VO4. Adsorbed proteins were solubilized in sample buffer and separated on 10% SDS-PAGE minigels. Transfer to PVDF and subsequent immunoblotting with 4G10 anti-phosphotyrosine Ab were conducted as described above. The PVDF membranes were stripped with 100 mM 2-ME, 2% SDS, and 62.5 mM Tris (pH 6.5) at 50°C and reprobed with the appropriate Ab to demonstrate equivalent amounts of immunoprecipitated protein.

Phagocytosing PMN were lysed and immunoprecipitated as described in the previous section. Immunoprecipitates were washed three times with lysis buffer, then three times with kinase buffer (50 mM HEPES (pH 7.5), 5 mM MnCl2, 2 mM MgCl2, 10 μM Na3VO4, and 1 mM 4-nitrophenyl phosphate). Beads were suspended in kinase buffer containing 2 μM ATP and 10 μCi [γ-32P]ATP, and Syk autophosphorylation was conducted for 10 min at 30°C. Samples were then combined with sample buffer, boiled 5 min, then run on 7.5% SDS-PAGE. Gels were stained, destained, and dried, and autoradiography was performed.

The PMN (8 × 106/sample) were lysed in 800 μl of a buffer containing 25 mM HEPES (pH 7.5), 50 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 1 mM Na3VO4, and 40 mM 4-nitrophenyl phosphate. Lysates were precleared with protein A-Sepharose for 30 min, and lysates were incubated with anti-PI 3-kinase Ab overnight with rotation at 4°C. Protein A-Sepharose was added to each sample and incubated for 2 h with rotation at 4°C. The beads were washed three times with the lysis buffer and three times with kinase buffer containing 100 mM NaCl, 20 mM Tris (pH 7.5), and 1 mM EDTA. Beads were incubated in kinase buffer containing 200 μg/ml of sonicated phosphatidylinositol substrate for 10 min at 20°C followed by a 30-min incubation at 20°C with the addition of 2 mM MgCl2, 2 μM ATP and 5 μCi/sample [γ-32P]ATP in a total volume of 52 μl. The reaction was terminated by adding HCl (1 M), and each sample was mixed with chloroform and methanol to obtain a chloroform/methanol/HCl ratio of 100/200/2. The samples were transferred to clean glass tubes and washed with equal volumes of 1 M NaCl/chloroform/methanol (1/4/4). After vortexing and centrifugation, 2 ml of 1% HCl was added to the lower phase, vortexed, and centrifuged again. The lower phase was transferred to a clean tube and dried under N2. Lipids were spotted in a thin line on silica plates and separated using a solvent system of chloroform/methanol/water/ammonium hydroxide (43/38/7/5, v/v). Phosphatidylinositide 4-kinase, whose Rf value is the same as PI-3P in this system, was spotted as a standard and visualized using primulin. Phosphorylated substrate was visualized by autoradiography, and the bands scraped and counted in a liquid scintillation counter (Wallac, Gaithersburg, MD). Activity was expressed as a percentage of the control value.

The PMN were resuspended at 1 × 107/ml in PBS and labeled with 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine (10−8 M; Amersham) for 30 min at 37°C. The labeled cells were washed with PBS and resuspended at 2 × 106 cells/ml in PBS containing 1 mM Ca2+ and 1 mM Mg2+. Cells were incubated with piceatannol for 30 min at 22°C, then with 200 mM ethanol for 5 min at 37°C. the PMN were then stimulated at 37°C with EIgG for 15 min or with 5 μg/ml cytochalasin B for 5 min, followed by 100 nM FMLP for 30 s. Lipids were extracted according to the method of Van Veldhoven and Bell (31), and assays for 3H-labeled phosphatidylethanol and phosphatidic acid were performed as previously described (32).

For fractionation studies phagocytosis was stopped as described previously at 3 min after initiating the ingestion of EIgG (21). The PMN were resuspended at 1 × 108/ml in extraction buffer (50 mM Tris (pH 7.5), 2 mM EGTA, 1 mM PMSF, leupeptin (1 μg/ml), 10 μM benzamidine, 10 μM pepstatin, and aprotinin (0.2 μg/ml)). The cells were disrupted by sonication on ice, and the resulting homogenate was centrifuged (400 × g, 10 min, 4°C) to remove unbroken cells and nuclei. The supernatant of each sample was applied to a 15–40% discontinuous sucrose gradient and centrifuged for 30 min at 150,000 × g at 4°C to obtain cytosolic, membrane, and granule fractions (21). The cytosol was removed from the top of the gradient, and the membrane fraction was collected at the 15–40% interface. The granule fraction was seen as the pellet. Protein was measured by the bicinchoninic acid method (Pierce, Rockford, IL), using BSA as a standard. The cytosol and the membrane fraction were combined with sample buffer and boiled for 5 min.

Two-tailed Student’s t tests were used to assess statistical significance.

Both Syk and PI 3-kinase are implicated in mediating FcγR-mediated cell activation (9, 33, 34, 35). To determine whether these proteins are involved in FcγR-mediated phagocytosis in PMN, we employed selective inhibitors against Syk (piceatannol) and PI 3-kinase (wortmannin) and evaluated EIgG-mediated phagocytosis by PMN. Both piceatannol (Fig. 1,A) and wortmannin (Fig. 1,B) incubated with PMN inhibited phagocytosis in a concentration-dependent manner. At 100 μM piceatannol and 100 nM wortmannin, EIgG-mediated phagocytosis was inhibited by 98 and 95%, respectively (Fig. 1, A and B). At 100 μM, LY294002, another inhibitor of PI 3-kinase, also inhibited phagocytosis by 86.7 ± 3.0%. The ID50 of piceatannol and wortmannin were 1 μM and 1 nM, respectively.

FIGURE 1.

Effect of piceatannol (A), an inhibitor of Syk and wortmannin (B), an inhibitor of PI 3-kinase, on Fcγ receptor-mediated phagocytosis in PMN in the absence (A and B) and presence (C) of DAG. The PMN (2 × 106/ml) were preincubated with different concentrations of inhibitor for 30 min at 22°C. Following the incubation PMN underwent phagocytosis with EIgG at once (A and B) or were washed twice and incubated with DiC10 at different concentrations at 22°C for 30 min (C), and then EIgG were added. Phagocytosis in cells without inhibitor treatment was 18.4 ± 2.4 EIgG ingested/100 PMN (100% control). The values represent the mean ± SD for three experiments. Comparisons were made between control PMN vs PMN with inhibitor treatment (A and B; ∗∗, p < 0.005; ∗, p < 0.05) and between DiC10 vs cells not treated with DiC10 (C; ∗∗, p < 0.0001; ∗, p < 0.005).

FIGURE 1.

Effect of piceatannol (A), an inhibitor of Syk and wortmannin (B), an inhibitor of PI 3-kinase, on Fcγ receptor-mediated phagocytosis in PMN in the absence (A and B) and presence (C) of DAG. The PMN (2 × 106/ml) were preincubated with different concentrations of inhibitor for 30 min at 22°C. Following the incubation PMN underwent phagocytosis with EIgG at once (A and B) or were washed twice and incubated with DiC10 at different concentrations at 22°C for 30 min (C), and then EIgG were added. Phagocytosis in cells without inhibitor treatment was 18.4 ± 2.4 EIgG ingested/100 PMN (100% control). The values represent the mean ± SD for three experiments. Comparisons were made between control PMN vs PMN with inhibitor treatment (A and B; ∗∗, p < 0.005; ∗, p < 0.05) and between DiC10 vs cells not treated with DiC10 (C; ∗∗, p < 0.0001; ∗, p < 0.005).

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We have previously reported that maximal levels of diacylglycerol (DAG) were generated endogenously during the peak of IgG-dependent phagocytosis in PMN and that DAG is required for Fc receptor-mediated phagocytosis to occur (21). As previously observed, the addition of 50, 100, and 200 μM of a cell-permeable DAG analogue, sn-1,2-didecanoylglycerol (DiC10), increased phagocytosis in a concentration-dependent manner by 9, 42, and 46%, respectively (21) (Fig. 1,C). To ascertain whether the addition of DiC10 could abrogate the inhibition by piceatannol and wortmannin of EIgG-mediated phagocytosis, PMN were preincubated with each of the different inhibitors, and then various concentrations of DiC10 were added. Phagocytosis was normalized in the presence of 100 μM piceatannol after the addition of 50 μM DiC10 (Fig. 1,C). At higher concentrations of DiC10 (100 and 200 μM), phagocytosis remained similar to that in untreated controls in the presence of 100 μM piceatannol. In contrast, when PMN were incubated with 100 nM wortmannin, the addition of all concentrations of DiC10 was able to augment phagocytosis of EIgG, but failed to completely normalize phagocytosis (Fig. 1 C). The maximal increase was seen at 200 μM DiC10 addition, which led to restoration of phagocytosis from 5–46% of the control value. Similar results were obtained with 100 μM of the PI 3-kinase inhibitor LY294002. LY294002 inhibited EIgG phagocytosis to 13.2 ± 3.0% of control value, and 200 μM DiC10 increased the phagocytic response to 67.9 ± 10% of the control value. Similar studies were conducted using DiC8 in lieu of DiC10 to determine whether DiC8 could reverse the inhibitory effects of piceatannol and wortmannin on phagocytosis. Using 10 μM DiC8, phagocytosis was restored to 77.7 ± 8.2 and 33.7 ± 1.3% of control values in the presence of 100 μM piceatannol and 100 nM wortmannin, respectively. These results indicate that DAG analogues were able to normalize piceatannol and augment wortmannin inhibition of phagocytosis in a concentration-dependent manner.

Since, following EIgG stimulation, DAG can be generated from phosphatidic acid in a pathway activated initially by phospholipase D (PLD), PLD activity was measured in PMN that were preincubated with piceatannol as another means of determining the sequence of signaling events. The 3- to 4-fold increase in PLD stimulated by phagocytosis was inhibited by piceatannol in a dose-dependent manner: 30, 61, and 99% inhibition for 1, 10, and 100 μM, respectively. In contrast, PLD stimulated by FMLP in cytochalasin B-treated PMN increased by 2-fold at 30 s and was not inhibited by 100 μM piceatannol. This supports the idea that PLD is activated by Syk during phagocytic signaling.

Tyrosine phosphorylation is one of the earliest responses in PMN activation and is required for FcγR-mediated phagocytosis by macrophages (6, 11). To evaluate whether enhanced Syk phosphorylation occurred during ingestion of EIgG in PMN, we performed anti-phosphotyrosine immunoblotting on lysates of phagocytosing PMN. There was no evidence of tyrosine phosphorylation of Syk at time zero, which was obtained by lysing PMN just before EIgG were added. Thirty percent of maximal Syk phosphorylation was apparent within 15–30 s after initiating phagocytosis of EIgG (Fig. 2,A). Phosphorylation of Syk peaked (arbitrarily assigned 100% on autoradiographs by densitometry) at 3–5 min, corresponding to the maximal rate of ingestion of EIgG by PMN. Syk phosphorylation decreased by 10 min (Fig. 2,A). Blots were reprobed with mAb against Syk to demonstrate equal amounts of Syk in immunoprecipitated samples (Fig. 2,B). The phosphorylation of Syk was an EIgG-dependent event; in contrast to phagocytosis, activation of PMN with FMLP did not lead to phosphorylation of Syk (data not shown). We also tested Syk kinase activity by monitoring autophosphorylation. Syk autophosphorylation increased to a maximum in the first few minutes of phagocytosis and decreased by 10 min (Fig. 2 C).

FIGURE 2.

A, Kinetics of Syk phosphorylation in PMN during phagocytosis of EIgG. The PMN (2 × 106/ml, 4 × 106/sample) were activated with EIgG, and at the indicated times phagocytosis was terminated. The samples were immunoprecipitated with anti-Syk Ab and were run on 10% SDS-PAGE followed by protein transfer to PVDF membranes. The membranes were probed with anti-phosphotyrosine Ab. The figure is representative of three experiments. B, Equal immunoprecipitation of Syk. Membranes used for evaluation of tyrosine phosphorylation were stripped and reprobed with mAb against Syk. C, Kinetics of Syk activity in PMN during phagocytosis of EIgG. Samples were activated with EIgG and immunoprecipitated as described for A. Immunoprecipitates were suspended in kinase buffer containing [α-32P]ATP and incubated at 30°AC for 10 min to allow autophosphorylation of Syk. Samples were then run on 7.5% SDS-PAGE, and autoradiography was performed.

FIGURE 2.

A, Kinetics of Syk phosphorylation in PMN during phagocytosis of EIgG. The PMN (2 × 106/ml, 4 × 106/sample) were activated with EIgG, and at the indicated times phagocytosis was terminated. The samples were immunoprecipitated with anti-Syk Ab and were run on 10% SDS-PAGE followed by protein transfer to PVDF membranes. The membranes were probed with anti-phosphotyrosine Ab. The figure is representative of three experiments. B, Equal immunoprecipitation of Syk. Membranes used for evaluation of tyrosine phosphorylation were stripped and reprobed with mAb against Syk. C, Kinetics of Syk activity in PMN during phagocytosis of EIgG. Samples were activated with EIgG and immunoprecipitated as described for A. Immunoprecipitates were suspended in kinase buffer containing [α-32P]ATP and incubated at 30°AC for 10 min to allow autophosphorylation of Syk. Samples were then run on 7.5% SDS-PAGE, and autoradiography was performed.

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Pretreating cells with piceatannol blocked Syk phosphorylation in a concentration-dependent manner (>90% inhibition at 100 μM and >50% inhibition at 10 μM; Fig. 3,A). Syk phosphorylation was not quite as sensitive to piceatannol as was phagocytosis, which was inhibited 80% at 10 μM (Fig. 1,A). Fig. 3,B shows that the amount of Syk was approximately equal among the samples shown in Fig. 3,A. To demonstrate the specificity of piceatannol, we tested its effect on other kinases. The protein tyrosine kinases Hck and Fgr have also been shown to activate upon PMN FcR ligation (36). The PMN were preincubated with 10 or 100 μM piceatannol, as in Syk analyses, then stimulated with 100 nM FMLP and lysed. Hck and Fgr were immunoprecipitated, and samples were subjected to Western blotting for phosphotyrosine. Piceatannol up to 100 μM did not inhibit the FMLP-stimulated increase in phosphorylation of Hck or Fgr (Fig. 3, C and D), demonstrating that piceatannol is selective for Syk among these tyrosine kinases implicated in phagocytosis. These data suggest a direct relationship between the extent of phosphorylation of Syk and phagocytosis of EIgG in PMN.

FIGURE 3.

A, Piceatannol inhibits Syk activation in PMN during EIgG-mediated phagocytosis. The PMN (2 × 106/ml) were incubated with different concentrations of piceatannol for 30 min at 22°C, then activated by the addition of EIgG (1 × 108/ml) for 3 min at 37°C. Unstimulated controls (UC) were incubated in parallel for equal times with no additions. The PMN were treated as described in Fig. 2 A. B, Equal immunoprecipitation of Syk. Membranes used for evaluation of tyrosine phosphorylation were stripped and reprobed with mAb against Syk. C, Piceatannol does not inhibit phosphorylation of Hck. The PMN were incubated with piceatannol as described in A, then activated with 100 nM FMLP for 5 min at 37°C. Cells were lysed and immunoprecipitated with Ab against Hck. Samples were subjected to SDS-PAGE (7.5%), transferred to PVDF membranes, and probed with anti-phosphotyrosine. D, Piceatannol does not inhibit phosphorylation of Fgr. Samples were prepared as described in C, except for immunoprecipitation with anti-Fgr.

FIGURE 3.

A, Piceatannol inhibits Syk activation in PMN during EIgG-mediated phagocytosis. The PMN (2 × 106/ml) were incubated with different concentrations of piceatannol for 30 min at 22°C, then activated by the addition of EIgG (1 × 108/ml) for 3 min at 37°C. Unstimulated controls (UC) were incubated in parallel for equal times with no additions. The PMN were treated as described in Fig. 2 A. B, Equal immunoprecipitation of Syk. Membranes used for evaluation of tyrosine phosphorylation were stripped and reprobed with mAb against Syk. C, Piceatannol does not inhibit phosphorylation of Hck. The PMN were incubated with piceatannol as described in A, then activated with 100 nM FMLP for 5 min at 37°C. Cells were lysed and immunoprecipitated with Ab against Hck. Samples were subjected to SDS-PAGE (7.5%), transferred to PVDF membranes, and probed with anti-phosphotyrosine. D, Piceatannol does not inhibit phosphorylation of Fgr. Samples were prepared as described in C, except for immunoprecipitation with anti-Fgr.

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To determine whether PI 3-kinase activity could be demonstrated in PMN undergoing phagocytosis, we monitored the kinetics of PI 3-kinase in PMN challenged with EIgG. For these studies PMN were incubated with EIgG, the incubation was terminated at different time points, and the cells were lysed. The lysate was incubated with an Ab against the 85-kDa regulatory subunit of PI 3-kinase, and the resulting immunoprecipitates were tested for PI 3-kinase activity using phosphatidylinositol as the substrate, yielding PI-3P as the product.

The PI 3-kinase activity was detectable at 30 s (Fig. 4 A), which correlated with the onset of Syk phosphorylation. On the other hand, the peak of Syk phosphorylation correlated with the maximal rate of phagocytosis, whereas PI 3-kinase activity occurred earlier, before the maximal rate of phagocytosis. At 30 s we observed an activity of 337 ± 47% (mean ± SEM; n = 3; kinase activity in unstimulated PMN, 100%). Deactivation occurred at 1 min (159 ± 79%) and reached basal levels at 3 min (122 ± 13%).

FIGURE 4.

A, Kinetics of PI 3-kinase activity during phagocytosis in PMN. The PMN (2 × 106/ml) were stimulated with EIgG (1 × 108/ml) for the times indicated at 37°C. After lysis of EIgG, PMN were suspended in lysis buffer for 30 min at 4°C and subsequently immunoprecipitated with anti-p85, and the kinase assay was performed as described in Materials and Methods. B, DiC10 is not able to restore piceatannol-mediated inhibition of PI 3-kinase activity during phagocytosis in PMN. The PMN (2 × 106/ml) were incubated with 100 μM piceatannol or 200 μM DiC10 alone or were pretreated for 30 min with piceatannol followed by incubation for 30 min with 200 μM DiC10. PMN were then challenged with EIgG for 30 s at 37°C and treated as described above. Unstimulated controls were incubated in parallel for equal times with no additions or with DiC10 only. C, Immunoprecipitation of PI 3-kinase under different conditions and incubations. Samples from PI 3-kinase assays (A and B, and Fig. 5 A) were used to demonstrate equal immunoprecipitation. The PI 3-kinase was solubilized from protein A-Sepharose with sample buffer, run on 7.5% SDS-PAGE, transferred to PVDF, and immunoblotted with Ab against p85 PI 3-kinase.

FIGURE 4.

A, Kinetics of PI 3-kinase activity during phagocytosis in PMN. The PMN (2 × 106/ml) were stimulated with EIgG (1 × 108/ml) for the times indicated at 37°C. After lysis of EIgG, PMN were suspended in lysis buffer for 30 min at 4°C and subsequently immunoprecipitated with anti-p85, and the kinase assay was performed as described in Materials and Methods. B, DiC10 is not able to restore piceatannol-mediated inhibition of PI 3-kinase activity during phagocytosis in PMN. The PMN (2 × 106/ml) were incubated with 100 μM piceatannol or 200 μM DiC10 alone or were pretreated for 30 min with piceatannol followed by incubation for 30 min with 200 μM DiC10. PMN were then challenged with EIgG for 30 s at 37°C and treated as described above. Unstimulated controls were incubated in parallel for equal times with no additions or with DiC10 only. C, Immunoprecipitation of PI 3-kinase under different conditions and incubations. Samples from PI 3-kinase assays (A and B, and Fig. 5 A) were used to demonstrate equal immunoprecipitation. The PI 3-kinase was solubilized from protein A-Sepharose with sample buffer, run on 7.5% SDS-PAGE, transferred to PVDF, and immunoblotted with Ab against p85 PI 3-kinase.

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To determine whether PI 3-kinase was downstream of Syk, we monitored PI 3-kinase activity after cells were incubated with 100 μM piceatannol. Samples were taken for PI 3-kinase activity determination at the 30 s point following the addition of EIgG (Fig. 4,B). Piceatannol (condition E) inhibited PI 3-kinase activity to 10 ± 8% of the control value (condition C, Fig. 4,B). In contrast, PI 3-kinase activity of G-CSF-primed PMN stimulated by FMLP was not inhibited by piceatannol (data not shown), indicating that PI 3-kinase itself was not directly affected by piceatannol. To demonstrate whether PI 3-kinase was downstream from Syk activation and to determine whether DAG could restore phagocytosis by bypassing the inhibition of Syk and PI 3-kinase, PMN were first incubated with 100 μM piceatannol. Following the addition of 200 μM DiC10, PI 3-kinase activity was evaluated (Fig. 4,B). In the presence of 100 μM piceatannol and DiC10, PI 3-kinase activity was 16% ± 9 (Fig. 4,B, condition F). Therefore, DiC10 was not able to abrogate the piceatannol-mediated inhibition of PI 3-kinase activity, suggesting that PLD is in a signaling pathway separate from PI 3-kinase. DiC10 did not significantly affect PI 3-kinase activity in phagocytosing PMN (Fig. 4,B, condition D) or unstimulated PMN (condition B); the latter condition is comparable to that in unstimulated controls (condition A). The amount of PI 3-kinase immunoprecipitated was unaffected by DiC10 or inhibitors and was similar between treatments (Fig. 4 C).

Phagocytosis-stimulated PI 3-kinase activity and phosphorylation of Syk were evaluated in the presence of wortmannin. The increased activity of PI 3-kinase following the addition of EIgG by 30 s was inhibited by 100 nM wortmannin to 12 ± 6% (Fig. 5,A). As shown in Fig. 5,A, wortmannin-mediated inhibition of PI 3-kinase activity during phagocytosis of EIgG in PMN was similar to the inhibition mediated by piceatannol. The PI 3-kinase activity of PMN treated with piceatannol or wortmannin was negligible and similar regardless of whether EIgG were present (data not shown). Phosphorylation of Syk appeared to precede the activation of PI 3-kinase, because both wortmannin and piceatannol inhibited PI 3-kinase, but only piceatannol, and not wortmannin, inhibited Syk (Fig. 5,B). Fig. 5 C shows equal amounts of immunoprecipitated Syk in all samples. Similar to the wortmannin results, the PI 3-kinase inhibitor LY294002 inhibited PI 3-kinase activity (by about 90%), but not phosphorylation of Syk (data not shown). This observation suggests that PI 3-kinase activation lies downstream of Syk in the signaling cascade and that the signaling pathway is disrupted in piceatannol-incubated PMN at the level of Syk activation.

FIGURE 5.

Wortmannin inhibits PI 3-kinase activity (A), but not Syk activation (B), during phagocytosis of EIgG. The PMN (2 × 106/ml) were incubated with 100 nM wortmannin and, for comparison, with 100 μM piceatannol for 30 min at 22°C followed by the addition of EIgG (1 × 108/ml) for 30 s (A) or 3 min (B) at 37°C. Unstimulated controls were incubated in parallel for equal times with no additions. PMN were investigated as outlined in Figs. 2 and 4. C, Equal immunoprecipitation of Syk. Membranes used for evaluation of tyrosine phosphorylation (B) were stripped and reprobed with mAb against Syk.

FIGURE 5.

Wortmannin inhibits PI 3-kinase activity (A), but not Syk activation (B), during phagocytosis of EIgG. The PMN (2 × 106/ml) were incubated with 100 nM wortmannin and, for comparison, with 100 μM piceatannol for 30 min at 22°C followed by the addition of EIgG (1 × 108/ml) for 30 s (A) or 3 min (B) at 37°C. Unstimulated controls were incubated in parallel for equal times with no additions. PMN were investigated as outlined in Figs. 2 and 4. C, Equal immunoprecipitation of Syk. Membranes used for evaluation of tyrosine phosphorylation (B) were stripped and reprobed with mAb against Syk.

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Previously, we correlated ERK1 and ERK2 phosphorylation with the engagement of FcγRII (17), demonstrated the kinetics of ERK1 and ERK2 phosphorylation, and observed maximal phosphorylation occurring after EIgG challenge at 5 min in PMN. To explore the upstream signaling events leading to ERK1 and ERK2 phosphorylation during EIgG-mediated phagocytosis, we investigated the effect of piceatannol on ERK1 and ERK2 activation using an Ab that recognizes the phosphorylated forms of both ERK1 and ERK2, which have molecular masses of 44 and 42 kDa, respectively. ERK1 and ERK2 phosphorylation was suppressed by piceatannol in a concentration-dependent manner (>80% inhibition at 100 μM and 50% inhibition at 50 μM; Fig. 6,A). Fig. 6 B demonstrates equal loading of p42/44 in all lanes. ERK phosphorylation was not as sensitive to piceatannol as were phagocytosis or Syk phosphorylation. Decreased sensitivity to piceatannol may occur because ERK1 and ERK2 can be activated by reactive oxygen intermediates (37), which are generated by PMN rendered nonphagocytic but capable of generating reactive oxygen intermediates through the p38 kinase pathway following engagement of the Fc receptor (38).

FIGURE 6.

A, Inhibition of ERK1 and ERK2 phosphorylation by piceatannol during phagocytosis of EIgG in PMN. The PMN (2 × 106/ml) were incubated with different concentrations of piceatannol for 30 min at 22°C followed by the addition of EIgG (1 × 108/ml) for 5 min at 37°C. Unstimulated controls (UC) were incubated in parallel for equal times with no additions. Samples were run on SDS-PAGE and transferred to PVDF, and the membranes were probed with anti-MAP kinase Ab that recognizes both phosphorylated isoforms, ERK1 (p44) and ERK2 (p42). B, Equal loading of ERK. Membranes used for evaluation of phosphorylation were stripped and reprobed with Ab that recognizes p42/p44 that is not phosphorylated. C, Restoration of piceatannol-mediated inhibition of ERK1 and ERK2 phosphorylation by DiC10 during phagocytosis in PMN. The PMN (2 × 106/ml) were treated with 100 μM piceatannol alone or 200 μM DiC10 alone or were pretreated for 30 min with 100 μM piceatannol followed by incubation for 30 min with 200 μM DiC10. The PMN were then challenged with EIgG. ERK1 and ERK2 phosphorylation was determined by Western blotting.

FIGURE 6.

A, Inhibition of ERK1 and ERK2 phosphorylation by piceatannol during phagocytosis of EIgG in PMN. The PMN (2 × 106/ml) were incubated with different concentrations of piceatannol for 30 min at 22°C followed by the addition of EIgG (1 × 108/ml) for 5 min at 37°C. Unstimulated controls (UC) were incubated in parallel for equal times with no additions. Samples were run on SDS-PAGE and transferred to PVDF, and the membranes were probed with anti-MAP kinase Ab that recognizes both phosphorylated isoforms, ERK1 (p44) and ERK2 (p42). B, Equal loading of ERK. Membranes used for evaluation of phosphorylation were stripped and reprobed with Ab that recognizes p42/p44 that is not phosphorylated. C, Restoration of piceatannol-mediated inhibition of ERK1 and ERK2 phosphorylation by DiC10 during phagocytosis in PMN. The PMN (2 × 106/ml) were treated with 100 μM piceatannol alone or 200 μM DiC10 alone or were pretreated for 30 min with 100 μM piceatannol followed by incubation for 30 min with 200 μM DiC10. The PMN were then challenged with EIgG. ERK1 and ERK2 phosphorylation was determined by Western blotting.

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We next explored the role of DiC10 in restoring ERK2 activation in the presence of piceatannol. In our previous studies we found that DiC10 increased ERK1 and ERK2 activation during phagocytosis of EIgG in PMN, corresponding to an increase in the extent of phagocytosis (Fig. 6,C) (21). Addition of DiC10 to unstimulated PMN modestly increased ERK1/2 phosphorylation to about 25% that occurring during phagocytosis (data not shown). PMN were incubated initially with 100 μM piceatannol followed by 200 μM DiC10, then were allowed to ingest EIgG. An increase to the phagocytosis-stimulated control value in ERK1 and ERK2 phosphorylation was observed (Fig. 6 C). The difference between DiC10 samples with and without piceatannol was not significant.

By adding the synthetic inhibitor piceatannol to PMN, we have been able to demonstrate the effect of blocking ERK1 and ERK2 signaling downstream of Syk activation. Furthermore, these results indicate that DiC10 was able to restore ERK1 and ERK2 phosphorylation and to improve phagocytosis when piceatannol was present.

We recently reported that PKCδ translocation is an upstream event leading to Raf-1 translocation to the plasma membrane during phagocytosis of EIgG in PMN and that PKCδ is a key component of the phagocytic pathway (21). In turn, Raf-1 activation leads to MEK activation followed by ERK phosphorylation (21). To provide a basis for linking PKCδ to the activation of Syk, we investigated the translocation of PKCδ and Raf-1 to the plasma membrane during phagocytosis in the presence of piceatannol.

In our previous studies we found that PKCδ and Raf-1 were present in the cytosol and were translocated to the plasma membrane at 3 min following the initiation of phagocytosis (21) (Fig. 7, A and B, condition B). Similar to the findings of others, <100% of the total PKCδ and Raf-1 content of PMN was recovered from the cytosol and membrane fraction of EIgG-stimulated cells compared with that of control unstimulated PMN (condition A) (21, 39). These findings suggest that these enzymes may undergo proteolysis or translocation to other subcellular fractions (39). Piceatannol (100 μM) inhibited both PKCδ and Raf-1 translocation to the plasma membrane following 3 min of phagocytosis (Fig. 7, condition D).

FIGURE 7.

Restoration of piceatannol-mediated inhibition of PKCδ (A) and Raf-1 (B) translocation to the plasma membrane by DiC10. The PMN (1 × 108/ml) were incubated with piceatannol or 200 μM DiC10 alone or were pretreated for 30 min with 100 μM piceatannol followed by incubation for 30 min with 200 μM DiC10 and then challenged with EIgG (5 × 109/ml) at 37°C for 3 min. Unstimulated controls were incubated in parallel for equal times with no additions. The PMN were then separated into cytosolic and membrane fractions. The fractions were analyzed for the presence of PKCδ and Raf-1 by employing SDS-PAGE and by immunoblotting using anti-PKCδ and anti-Raf-1 Ab.

FIGURE 7.

Restoration of piceatannol-mediated inhibition of PKCδ (A) and Raf-1 (B) translocation to the plasma membrane by DiC10. The PMN (1 × 108/ml) were incubated with piceatannol or 200 μM DiC10 alone or were pretreated for 30 min with 100 μM piceatannol followed by incubation for 30 min with 200 μM DiC10 and then challenged with EIgG (5 × 109/ml) at 37°C for 3 min. Unstimulated controls were incubated in parallel for equal times with no additions. The PMN were then separated into cytosolic and membrane fractions. The fractions were analyzed for the presence of PKCδ and Raf-1 by employing SDS-PAGE and by immunoblotting using anti-PKCδ and anti-Raf-1 Ab.

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These experiments were also performed in PMN treated with DiC10 (Fig. 7, A and B). In our previous studies we found that DiC10 alone led to increased translocation of PKCδ to the plasma membrane during phagocytosis of EIgG (21) (Fig. 7, condition C). When PMN were preincubated with 100 μM piceatannol followed by the addition of 200 μM DiC10, an increase in translocation of PKCδ and Raf-1 to the plasma membrane occurred during phagocytosis of EIgG (Fig. 7, condition E) compared with that in PMN treated with piceatannol alone (condition D). These results demonstrate that DiC10 was able to restore PKCδ and Raf-1 translocation during phagocytosis of EIgG when piceatannol was present. The ability of DiC10 to restore PKCδ and Raf-1 translocation correlated with the restoration of phagocytosis and ERK1 and ERK2 activation in the presence of piceatannol.

In contrast to piceatannol, there was no inhibition of PKCδ translocation to the plasma membrane in the presence of wortmannin, indicating that this PI 3-kinase inhibitor blocked phagocytosis of EIgG in PMN in a PKCδ-independent process and therefore did not lead to inhibition of ERK1 and ERK2 phosphorylation (data not shown).

In this study we investigated the early events after Fcγ receptor engagement in PMN by analyzing protein kinase activation during phagocytosis of EIgG. We previously found that FcγR engagement and associated signal transduction steps lead to PKCδ and Raf-1 translocation and, in turn, this ultimately results in activation and phosphorylation of ERK1 and ERK2 (21). The upstream signaling events leading to translocation of PKCδ and Raf-1 during EIgG-mediated phagocytosis have not been completely elucidated. We evaluated the role of Syk in Fcγ receptor-mediated signaling events and the downstream events by examining translocation of PKCδ and Raf-1 to the membrane, ERK1 and ERK2 phosphorylation, and activation of PI 3-kinase during EIgG-mediated phagocytosis.

Several studies have shown that cross-linking Fcγ receptors results in increased tyrosine phosphorylation and enzymatic activation of Syk (11, 40, 41). We also found that EIgG is a potent stimulus for the phosphorylation of Syk in PMN. Syk phosphorylation was maximal at 3–5 min during phagocytosis of EIgG, which corresponded to the maximal rate of ingested EIgG by PMN. Kinase activity of Syk followed similar kinetics. Because piceatannol inhibits phagocytosis and Syk activation, the mechanism by which piceatannol blocked activation of key components was studied. The translocation of PKCδ and Raf-1 during EIgG-mediated phagocytosis in PMN followed by the activation of ERK1 and ERK2 were inhibited by piceatannol, indicating that these events are downstream of Syk phosphorylation. Therefore, Syk initiates a cascade of protein phosphorylation during Fcγ receptor-mediated phagocytosis of EIgG in PMN and provides a basis linking receptor engagement to the signaling events mediated by ERK activation.

Phagocytosis of EIgG in PMN is associated with an increase in the formation of DAG mass (21), which is largely generated in a pathway initiated by the activity of PLD (42). We found that engagement of the Fcγ receptor leads to activation of PLD and the generation of phosphatidic acid. Phosphatidic acid, in turn, is metabolized by phosphatidic phosphohydrolase to diglyceride. We previously observed that inhibition of diglyceride generation from the PLD pathway inhibited phagocytosis. Therefore, in these studies we determined whether exogenous diglyceride could restore the phagocytosis mediated by the Syk inhibitor piceatannol. We observed that in the presence of piceatannol, DiC10 reconstituted the ERK phosphorylation initiated by PKCδ and Raf-1 translocation without affecting Syk phosphorylation. Also, PLD activity during PMN phagocytosis was inhibited by piceatannol. These findings support the hypothesis that the latter kinases and PLD are downstream of Syk activation. Similar to the findings of others, we also demonstrated that EIgG ingestion by PMN activated PI 3-kinase maximally at 30 s and that wortmannin and LY294002, inhibitors of PI 3-kinase, prevented EIgG-stimulated phagocytosis.

Syk activation was required for PI 3-kinase activity, because PI 3-kinase activity was inhibited by piceatannol. In contrast to the observation with piceatannol, neither wortmannin nor LY294002 inhibited Syk phosphorylation in PMN ingesting EIgG. Our findings extend the observation that FcγR engagement in murine macrophages induced an increase in PI 3-kinase phosphorylation, but not in genetically engineered Syk-deficient macrophages (34). Therefore, Syk activation is probably required to modulate PI 3-kinase activity in cells with Fcγ receptors. Chacko et al. (35) have provided evidence that Syk phosphorylation might act as an adapter to recruit PI 3-kinase to activated FcγRII in platelets. Both Syk and PI 3-kinase activities rise significantly after only 30 s of phagocytosis, indicating that their position is early in the signaling cascade. The PI 3-kinase activity is transient, returning to baseline more rapidly than that of Syk.

On the other hand, our findings do not support the hypothesis that PKCδ and Raf-1 translocation and subsequent ERK1 and ERK2 phosphorylation lie downstream of PI 3-kinase activation in mediating the PMN phagocytic response. We found that 100 nM wortmannin inhibited phagocytosis without affecting PKCδ and Raf-1 translocation or ERK1 and ERK2 phosphorylation, inconsistent with PI 3-kinase regulation. The involvement of PI 3-kinase in mediating the MAP kinase cascade is controversial (18, 43, 44). It has been suggested that wortmannin inhibition of the MAP kinase pathway is cell type and ligand specific (23). Our findings are consistent with the hypothesis that PI 3-kinase may feed into the phagocytic pathway independently of MAP kinase activation. In accordance with our data, Araki et al. (45) have shown that PI 3-kinase is necessary for phagocytosis in macrophages and that PI 3-kinase contributes to the formation of phagosomes, probably due to the closure of pseudopodia to form intracellular vesicles. Because diglyceride is known to be fusogenic (46, 47), this might be an explanation of why diglyceride augments phagocytosis in wortmannin-treated PMN.

Based on these data, we propose the following model, as illustrated in Fig. 8, for Fcγ receptor signaling during PMN phagocytosis. In summary, our results demonstrated that phagocytosis of EIgG leads to phosphorylation of Syk in PMN and that Syk activation is required to initiate a process involving PKCδ, Raf-1, ERK1, and ERK2. Furthermore, we suggest a role for PI 3-kinase in early signal transduction events after Fc receptor-mediated phagocytosis in PMN, but not direct involvement in the MAP kinase cascade. It is likely that PI 3-kinase may regulate phagocytosis by a separate and parallel pathway.

FIGURE 8.

Model of signaling cascade in PMN during FcγRII-mediated phagocytosis of EIgG. FcγRII engagement promotes Syk phosphorylation, which is required to initiate a process involving DAG generation, translocation of PKCδ and Raf-1 to the plasma membrane, and ERK1 and ERK2 phosphorylation. Syk activation was required for PI 3-kinase activity, which is downstream of Syk and is not directly involved in the MAP kinase cascade. For further details, see text.

FIGURE 8.

Model of signaling cascade in PMN during FcγRII-mediated phagocytosis of EIgG. FcγRII engagement promotes Syk phosphorylation, which is required to initiate a process involving DAG generation, translocation of PKCδ and Raf-1 to the plasma membrane, and ERK1 and ERK2 phosphorylation. Syk activation was required for PI 3-kinase activity, which is downstream of Syk and is not directly involved in the MAP kinase cascade. For further details, see text.

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1

This work was supported by Deutsche Forschungsgemeinschaft Grant Ra 789/1-1 (to E.M.B.R.) and by National Institutes of Health Grants AI20065 (to L.A.B.) and DK41487 and DK39255 (to J.A.S.).

4

Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; EIgG, IgG-opsonized erythrocytes; PKC, protein kinase C; ERK, extracellular signal-regulated protein kinase; PI 3-kinase, phosphatidylinositide 3-kinase; DAG, diacylglycerol; DiC8, sn-1,2-dioctanoylglycerol; DiC10, sn-1,2-didecanoylglycerol; SH2, SRC homology 2; ITAMs, immunoreceptor tyrosine-based activation motifs; MAP, mitogen-activated protein; PVDF, polyvinylidene difluoride.

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