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J Biol Chem, Vol. 275, Issue 17, 12416-12423, April 28, 2000

The Requirement of Both Extracellular Regulated Kinase and p38 Mitogen-activated Protein Kinase for Stimulation of Cytosolic Phospholipase A₂ Activity by Either FcRIIA or FcRIIIB in Human Neutrophils
A POSSIBLE ROLE FOR Pyk2 BUT NOT FOR THE Grb2-Sos-Shc COMPLEX^*

Inbal Hazan-Halevy, Rony Seger^§, and Rachel Levy^¶

From the  Laboratory of Infectious Diseases, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev and Soroka Medical Center, Beer Sheva 84105, Israel and the ^§ Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100 Israel

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The signal transduction pathways initiated by opsonized zymosan (OZ) leading to activation of cytosolic phospholipase A₂ (cPLA₂) in human neutrophils remain obscure. In a previous study, we showed that the activation of cPLA₂ by OZ is tyrosine kinase-dependent. The present study demonstrates that the signals initiated by OZ involve activation of tyrosine kinase Pyk2 but not the formation of the adhesion protein complex, Shc-Grb2-Sos. Stimulation of cPLA₂ activity by OZ is mediated by Fc  receptors (FcRs) and not by complement receptors for the C3b protein. Cross-linking of FcRIIA or FcRIIIB induces p38 mitogen-activated protein (MAP) kinase and extracellular regulated kinase (ERK) phosphorylation. The kinetics of cPLA₂ activity stimulated by either of the FcRs or by both is similar to that of p38 MAP kinase and was detected as early as 15 s after stimulation, maintained a plateau for 10 min, and decreased thereafter. ERK activation was detected also within 15 s but decreased significantly 5 min after stimulation. The MEK inhibitor, PD-098059, or the p38 MAP kinase inhibitor, SB-203580, caused a partial inhibition during the time course of cPLA₂ activity, whereas their combination caused a total inhibition. Thus, although ERK activation is significantly shorter than that of p38 MAP kinase, it is equally required for activation and maintenance of cPLA₂ activity by occupancy of a single receptor, FcRIIA or FcRIIIB.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Immune complexes are recognized by specific receptors present on the plasma membrane of phagocytes: Fc  receptors (FcRs)¹ and receptors for complement protein C3b. The binding of immune complexes by polymorphonuclear neutrophil receptors induces essential host defense and inflammatory responses such as adhesion, phagocytosis of antibody-coated microorganisms, degranulation, and respiratory burst (1). Three different classes of FcRs have been identified: FcRI (CD-64), FcRII (CD-32), and FcRIII (CD-16), all of which differ in relative affinity for IgG, cellular distribution, and molecular composition. Neutrophils are unique in that they express not only the transmembrane FcRIIA but also the only known nontransmembrane FcR, the glycan phosphatidylinositol-linked FcRIIIB. FcRIIA is a 47-kDa integral glycoprotein, and FcRIIIB is a heavily glycosylated protein with an apparent molecular mass of 50-80 kDa linked by a glycosyl-phosphatidylinositol anchor to the outer plasma membrane (2). Additionally, neutrophils express two kinds of complement receptors, CR1 and CR3, both of which recognize surface C3b and C3bi, respectively (3, 4).

Cross-linking of the transmembrane FcRIIA initiates a tyrosine kinase cascade-dependent upon the cytoplasmic tail of this receptor, which contains one copy of an immunoreceptor tyrosine-based activation motif, a substrate for phosphorylation by members of the Src tyrosine kinase family (5). The phosphorylated immunoreceptor tyrosine-based activation motif can bind to and activate Syk-tyrosine kinase, which subsequently activates a number of effector pathways (6, 7). FcRIIA aggregation has been shown to be associated with enhanced activity of PI-3-kinase and of Ca²⁺/calmodulin-dependent protein kinase II in human neutrophils (8, 9). Cross-linking of human FcRII on monocytes results in stimulation of several signal transduction events, including inositol phosphate metabolism (10), an increase in cytoplasmic calcium concentration (11, 12), and tyrosine phosphorylation of several cellular proteins including phospholipase C 1/2 (10, 13) and FcRII itself (14). The role of FcRIIIB in triggering neutrophil function is controversial because it has no intracytoplasmic domain for direct association with the cytosolic signal transduction cascade. Some studies have suggested the inability of FcRIIIB to independently transduce signals (15-19). On the other hand, others have suggested that cross-linking of FcRIIIB by itself is able to mediate various functions, such as calcium mobilization (20, 21), translocation of Src-related tyrosine kinase Hck (22), actin polymerization (23), activation of PKB by the PI-3-kinase dependent pathway (9, 24), release of -hexosaminidase (25), and superoxide production in human neutrophils (26, 27).

Despite all this accumulated data, the precise transduction pathway initiated by FcRs leading to activation of p85 cytosolic phospholipase A₂ (cPLA₂) remains obscure. We have previously shown the involvement of the MAP kinase, ERK1/2, in activating cPLA₂ and superoxide production in human neutrophils stimulated with opsonized zymosan (OZ) as a model for the immune complex (28). The MAP kinase family includes the 42-44-kDa ERK, the stress-activated protein kinases: 38-kDa MAP kinase and c-Jun N-terminal kinase, and big MAP kinase (BMK, ERK5) (29). In various systems, both ERK and p38 MAP kinase have been shown to phosphorylate p85 cPLA₂ on serine residue (505) and thus render it active (30, 31). We also provided evidence for the essential requirement of arachidonic acid signals generated by p85 cPLA₂ in activation of the phagocyte NADPH oxidase (32). ERK, cPLA₂, and NADPH oxidase activities stimulated by OZ were all shown to be tyrosine kinase-dependent (28). To present, the specific tyrosine kinase-dependent pathways leading to activation of MAP kinases by FcRs have not been identified. The regulation of some of the elements upstream to ERK1/2 have been elucidated and are best understood for tyrosine kinase growth factor receptor signaling (33). Receptor activation initiates a cascade of events leading to ERK1/2 activation and involves protein-protein interactions between the adaptor proteins Grb2 and Shc. The proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase constitute a distinct family of nonreceptor protein-tyrosine kinases (34). In hematopoietic cells, Pyk2 is activated by a variety of extracellular stimuli such as the inflammatory cytokine tumor necrosis factor , T and B lymphocyte antigen receptor, CD-28 ligation, interleukin-2 receptor, FcRI, and chemokine receptors (35-40). Several reports have shown that activation of Pyk2 is necessary for the activation of ERK, p38 MAP kinase, and/or c-Jun N-terminal kinase in different cell lines and in response to diverse stimuli (36, 41-44).

The present study was designed first to evaluate the relative role of p38 MAP kinase and ERK in cPLA₂ activation by OZ. Because this agent binds to FcRIIA, FcRIIIB, and C3bR, the role of each receptor was defined. Second, the involvement of tyrosine kinase-dependent elements in these signaling pathways was studied.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- Zymosan A, p-nitrophenylphosphate, cytochrome c, PMSF, leupeptin, benzamidine, 1,2-dioleoyl-sn-glycerol, ATP, and DTT were obtained from Sigma. [-³²P]ATP (3000 Ci/mmol) was purchased from NEN Life Science Products. Anti-rabbit and anti-mouse IgG horseradish peroxidase and ECL detection kit for immunoblotting were obtained from Amersham Pharmacia Biotech. The myelin basic protein peptide (MBP) was synthesized at the Weizmann Institute (Rehovot, Israel). The MEK inhibitor PD-098509 was purchased from Biomol and the p38 inhibitor SB-203580 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-MAP kinase (sc-154 and sc-153) and anti-Pyk2 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-p38 MAP kinase, anti-active p38 MAP kinase and anti-active ERK were from Sigma. Anti-Sos 1/2 antibodies were obtained from Transduction Laboratories (Lexington, KY). Anti-phosphotyrosine monoclonal antibody, 4G10, was from Upstate Biotechnology, Inc. The F(ab')₂ fragment goat anti-mouse was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti-FcRII mAb IV.3 Fab and anti-FcRIII mAb 3G8 F(ab')₂ were obtained from Medarex Inc. (Annandale, NJ). The anti-Grb2 and anti-Shc polyclonal antibodies were provided by Dr. J. Schlessinger (New York University Medical Center). Rabbit anti-cPLA₂ antibodies raised against a glutathione S-transferase fusion with cPLA₂ as described earlier (28).

Neutrophils Isolation-- Neutrophils were separated by Ficoll/Hypaque centrifugation, dextran sedimentation, and hypotonic lysis of erythrocytes (45).

Superoxide Anion Measurements-- The production of superoxide anion (O₂) by intact cells was measured as the superoxide dismutase inhabitable reduction of ferricytochrome c (46). Cells were suspended (5 × 10⁵ cells/well) in 100 µl of Hanks' balanced salt solution (HBSS) containing 150 µM ferricytochrome c and stimulated by the addition of the appropriate stimulus. The reduction of ferricytochrome c was followed by a change of absorbance at 550 nm every 2 min over a 20-min time course on a Thermomax microplate reader (Molecular Devices, Melno Park, CA). The maximal rates of superoxide generation were determined using extinction coefficient E₅₅₀ = 21 mM¹ cm¹.

Cell Stimulation-- Neutrophils were incubated with OZ or inactivated OZ (iOZ) for the indicated time at 37 °C. OZ was prepared as follows: 20 mg of zymosan was incubated with 1 ml of pooled human serum (LPS-free) for 1 h at 37 °C and washed three times with HBSS buffer. iOZ was prepared in the same way except that the serum was preheated to 56 °C for 30 min to inactivate the complement proteins. Alternatively, neutrophils were placed (5 × 10⁶ cells/ml) on ice for 30 min with 1 µg/ml F(ab')₂ of anti-FcRIIA or anti-FcRIIIB. Excess antibodies were removed by centrifugation, and the cells were resuspended in HBSS buffer, prewarmed to 37 °C. Cross-linking of antibodies was performed with goat anti-mouse F(ab')₂ fragments (10 µg/ml). Adding ice-cold HBSS buffer followed by centrifugation terminated the reaction. Negative control samples were incubated without primary antibody.

Fractionation of Cells into Soluble and Particulate Fractions-- The cells were resuspended at 10⁸ cells/ml in iced buffered sucrose solution (10 mM HEPES, pH 7.5, 100 mM sucrose, 0.5 mM ETDA, 1 mM EGTA, 5 mM p-nitrophenyl phosphate, 50 µg/ml leupeptin, 1 mM PMSF, 10 mg/ml aprotinin, 1 mM benzamidine, 2 mM Na₃VO₄, 25 mM NaF) (47). Cells were disrupted by sonication and then centrifuged at 100,000 × g for 30 min at 4 °C. The cytosolic fraction protein was measured, adjusted, and used for immunoprecipitation of Pyk2 or immunoblotting of Pyk2 and cPLA₂.

MAP Kinase in Vitro Kinase Assay-- Neutrophils (5 × 10⁶ cells/ml) in HBSS buffer were stimulated at 37 °C with 1 mg/ml OZ; the reaction was stopped by 10-fold dilution with cold HBSS and immediate centrifugation at 4 °C. In every experiment 100-µl aliquots were taken from each reaction mixture to measure superoxide production on a microtiter plate. Neutrophils were lysed immediately with lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM ETDA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 10 mM MgCl₂, 20 mM p-nitrophenyl phosphate, 10 mg/ml leupeptin, 1 mM PMSF, 10 mg/ml aprotinin, 1 mM benzamidine, 1 mM Na₃VO₄, 25 mM NaF) and centrifuged (1 min at 15,600 × g) to remove granules, nuclei and unbroken cells. Immunoprecipitation was conducted at 4 °C for 2 h using 300 µg of neutrophil protein lysate plus 30 µl of 50% protein A-Sepharose bound to 10 µl of anti-ERK antibody or 1.5 µl of polyclonal anti-p38 MAP kinase at a final volume of 300 µl. The beads were then washed once with RIPA buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 2 mM ETDA, 20 mM leupeptin, 1 mM PMSF) and once with 500 mM LiCl₂ in 100 mM Tris-HCl, pH 8, and twice with buffer A (50 mM -glycerol phosphate, pH 7.3, 1 mM ETDA, 1.5 mM EGTA, 1 mM DTT, 10 mg/ml leupeptin, 1 mM PMSF, 10 mg/ml aprotinin, 1 mM benzamidine, 1 mM Na₃VO₄). The beads were resuspended in 30 µl of kinase assay buffer containing 25 mM -glycerol phosphate, pH 7.3, 1.25 mM EGTA, 1.5 mM DTT, 0.5 mM Na₃VO₄, 1 mg/ml bovine serum albumin, 1 mM ATP, and 2.5 µCi of [-³²P]ATP using 2 mg/ml MBP peptide as a substrate. Reaction duration time was 20 min at 30 °C and was terminated by addition of 20 µl of Laemmli buffer. After boiling for 5 min, proteins were separated by 15% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose by electroblotting. Phosphorylated proteins were visualized utilizing an autoradiography technique and quantified by a FUJIX BAS 1000 phosphoimager.

Cytosolic Phospholipase A₂ Assay-- Neutrophils (5 × 10⁶ cells/ml) in HBSS buffer were stimulated for the indicated time at 37 °C. The reaction was stopped by 10-fold dilution with cold HBSS and immediate centrifugation at 4 °C. PLA₂ activity was performed immediately after lysate separation using sonicated dispersions of 1-stearoyl-2-[¹⁴C]arachidonyl phosphatidyl choline (30 µM, 50,000 dpm/assay) cosonicated with sn-1,2-dioleoylglycerol at a molar ratio of 2:1 in a assay mixture containing 5 mM DTT with some modifications, as described earlier (28). Briefly, the assay mixture contained the phospholipid substrate in 80 mM KCl, 5 mM CaCl_2, 5 mM DTT, 1 mg/ml bovine serum albumin, 1 mM EDTA, and 10 mM HEPES, pH 7.4 (48). The reaction was started by the addition of 100 µg of neutrophil lysate (within the linear protein range of the reaction) and incubated at 37 °C in a shaking water bath for 30 min. The radiolabeled fatty acids were counted (Packard spectrometry 1900CA), and the specific activity of cPLA₂ was calculated.

Immunoprecipitation-- Equal amounts of neutrophil lysates were subjected to immunoprecipitation with 20 µl of rabbit polyclonal antibodies against Grb2 or Shc cross-linked to protein A-Sepharose beads (Zymed Laboratories Inc.) with dimethylpimelimidade (Pierce) (49). For Pyk2 immunoprecipitation, 40 µl of anti-Pyk2 antibodies (Santa Cruz) were added to 1 mg of soluble fraction. Immunoprecipitation was conducted at a final volume of 1 ml for 4 h at 4 °C with shaking. The beads were then washed six times with lysis buffer (for Grb2 or Shc immunoprecipitation) or three times with sucrose buffer for Pyk2 immunoprecipitation. 20 µl of 2× sample buffer was added to the beads, and the mixture was boiled for 5 min.

Immunoblot Analysis-- Lysates were solubilized in 5× sample buffer and analyzed by electrophoresis on 7.5 or 10% SDS-polyacrylamide gels. The amount of protein in each sample was quantified with the Pierce BCA proteins assay using bovine serum albumin standards. The resolved proteins were electrophoretically transferred to nitrocellulose, which was stained with Ponsue red to detect protein banding, and then blocked in 3% bovine serum albumin in TBS (10 mM Tris, 135 mM NaCl, pH 7.4). The blots were incubated overnight at 4 °C with primary antibodies. After four washings with TBS-T (TBS containing 0.05% Tween 20) the membranes were incubated with the second antibody, peroxidase-conjugated goat anti-rabbit, or anti-mouse (Amersham Pharmacia Biotech) for 1 h at room temperature and developed using the ECL detection system (Amersham Pharmacia Biotech). Changes in ERK or p38 MAP kinase phosphorylation were quantified by densitometry. The quantitative measurements are adequate to determine the changes in the same immunoblot. To reprobe the blots they were incubate in stripping buffer (62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol) at 58 °C for 25 min, washed extensively with TBS, reblocked as described above, and reblotted with appropriate antibodies.

Statistical Analysis-- The differences in means were analyzed by Student's t test. The plots were drawn as least squares regression lines and tested by analysis of variance.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The Role of p38 MAP Kinase and ERK in cPLA₂ Activation by OZ-- In a previous paper (28), we demonstrated that OZ stimulates ERK1 and ERK2 activity in human neutrophils and that ERK is involved in activation of cPLA₂. A number of recent studies have shown stimulation of more than one member of the MAP kinase family by the same agent in phagocyte cells (50-52). To evaluate whether OZ also activates p38 MAP kinase, human neutrophils were stimulated with 1 mg/ml OZ, and both p38 MAP kinase and ERK activities were determined. As presented in Fig. 1A, stimulation of the cells with 1 mg/ml OZ for 2 min caused a significant increase in p38 MAP kinase activity, as detected by an in vitro kinase assay. This activity was totally inhibited by the p38 MAP kinase inhibitor SB-203580 in its optimal concentration (5 µM) to the basal levels that were detected in unstimulated cells. In contrast, p38 MAP kinase activity was not affected by 100 µM of the MEK inhibitor PD-098059. This PD-098059 concentration was found to be optimal for inhibition of ERK activity in human neutrophils stimulated by OZ (data not shown; see Ref. 28). Activation of ERK was determined using phospho-specific antibodies against the phosphorylated form. Stimulation of neutrophils with 1 mg/ml OZ for 2 min caused a significant increase in ERK phosphorylation (Fig. 1B). ERK phosphorylation was inhibited by 100 µM PD-098059 but was not affected by 5 µM SB-203580.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Activation of MAP kinases by OZ. A, p38 MAP kinase activity induced by OZ determined by an in vitro kinase assay. p38 MAP kinase was immunoprecipitated from cell lysates with anti-p38 MAP kinase antibody and assayed for kinase activity using MBP as a substrate by an in vitro kinase assay. The samples were subjected to SDS electrophoresis, and the radioactive bands were quantified by a phosphoimager. The relative phosphorylation units are: Con, 280; OZ, 529; SB, 262; PD, 585. B, immunoblot of ERK phosphorylation using a phospho-specific ERK antibody. Shown are representative blots of three experiments with identical results. Neutrophils were incubated with 5 µM SB-203580 or 100 µM PD-098059 for 40 min at 4 °C before stimulation with 1 mg/ml OZ for 2 min at 37 °C.

Because both ERK and p38 MAP kinase are capable of phosphorylating and activating cPLA₂ (30, 31), we utilized the MEK and the p38 MAP kinase inhibitors (whose specificity is demonstrated in Fig. 1) to evaluate the relative role of these two MAP kinases in cPLA₂ activation. As demonstrated in Fig. 2, preincubation of neutrophils with 100 µM PD-098059, which caused total inhibition of ERK activity, caused partial inhibition of cPLA₂ activity following stimulation with OZ for 2 min (0.13 ± 0.041 pmol/µg/30 min compared with 0.31 ± 0.068 pmol/µg/30 min induced by OZ). Similarly, preincubation of neutrophils with 5 µM SB-203580, which caused total inhibition of p38 MAP kinase activity, caused partial inhibition of cPLA₂ activity following stimulation by OZ for 2 min (0.15 ± 0.04 pmol/µg/30 min compared with 0.31 ± 0.068 pmol/µg/30 min induced by OZ). Pretreatment of the cells with both inhibitors resulted in total inhibition of cPLA₂ activity induced by OZ. These results clearly indicate that both ERK and p38 MAP kinase are required to induce complete activation of cPLA₂ by OZ in human neutrophils.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effect of p38 MAP kinase and/or ERK inhibitors on cPLA2 activity stimulated by OZ. Neutrophils were preincubated for 40 min at 4 °C with 100 µM PD-098059 and/or 5 µM SB-203580. Cells were stimulated for 2 min with 1 mg/ml OZ at 37 °C, and cPLA2 activity was determined in neutrophil lysates using labeled phosphatidylcholine vesicles as a substrate. The results expressed as specific activity are the means ± S.E. of five experiments done in duplicate. Each inhibitor significantly (p < 0.001) reduced the activity induced by OZ. Treatment of the cells with both inhibitors caused total inhibition, which is significantly higher (p < 0.001) than the effect of each inhibitor alone. There is no significant difference between the activity of unstimulated cells and cells treated with both inhibitors.

To differentiate between the role of these two types of MAP kinases in activation of cPLA₂, the time course activation of each isotype by OZ was determined and compared with that of cPLA₂. As shown in Fig. 3A, stimulation of neutrophils with 1 mg/ml OZ caused a temporary and transient phosphorylation of ERK1/2, reaching a maximal level at 1-2 min and decreasing thereafter. ERK1/2 phosphorylation could not be detected 10 min after stimulation. This blot also illustrates, by electrophoretic mobility shift, the same pattern of ERK activation that is demonstrated in Fig. 3A. Phosphorylated ERK2 with reduced electrophoretic mobility was detectable as early as 15 s after stimulation and showed a maximum activation at 1-2 min. Phosphorylation of p38 MAP kinase induced by 1 mg/ml OZ was detected 15 s after stimulation, peaked 2-5 min later, stayed elevated for 10 min, and slightly decreased at 20 min (Fig. 3C). Western blot analysis with anti-ERK2 or anti-p38 MAP kinase antibody confirmed the presence of an equal amount the MAP kinase in each sample (Fig. 3, B and D).

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Time course activation of ERK and p38 MAP kinase stimulated by OZ. Neutrophils were stimulated for the indicated times with 1 mg/ml OZ at 37 °C, and ERK or p38 MAP kinase phosphorylation were detected by immunoblot with phospho-specific antibodies. A and C, immunoblots of ERK or p38 MAP kinase phosphorylation induced by 1 mg/ml OZ using phospho-specific ERK or p38 MAP kinase antibodies, respectively. B and D, equal amounts of ERK2 or p38 MAP kinase in each sample were evaluated by immunoblotting the same blots with anti-ERK2 or anti-p38 MAP kinase antibodies, respectively. The results are from one representative experiment of three with identical results.

The different temporal patterns of activation of ERK and p38 MAP kinase by OZ would seem to indicate a distinct role for each of these MAP kinases in cPLA₂ activation. Therefore, the time course of cPLA₂ activity induced by OZ was determined by measuring arachidonic acid release from radiolabeled arachidonyl-phosphatidylcholine vesicles and was compared with the time course activation of each MAP kinase isotype. cPLA₂ activity was detected as early as 15 s after stimulation, maintained a plateau for 5 min, and decreased thereafter (Fig. 4). 10 min after stimulation, cPLA₂ activity was still elevated, similar to that of p38 MAP kinase, whereas ERK activity could not be detected. We also demonstrated cPLA₂ activity after 2 min of activation by the retardation in the electrophoretic mobility shift detected by immunoblotting (Fig. 4, inset). The parallel kinetics of p38 MAP kinase phosphorylation and cPLA₂ activity could imply that this type of MAP kinase is necessary for maintaining cPLA₂ activity. If this were the case, 10 min after activation of cPLA₂ (when both cPLA₂ and p38 MAP kinase activities are still elevated), the presence of SB-203580 would reduce most of the cPLA₂ activity, whereas the presence of PD-098059 would cause a lesser effect. However, the results analyzed 10 min after stimulation (Fig. 5) demonstrated that either 5 µM SB-203580 or 100 µM PD-098059 caused about 50% inhibition of cPLA₂, similar to their effect after 2 min of activation by OZ (Fig. 2). Similar results were obtained during the whole time course of stimulation (data not shown). These results clearly indicate that p38 MAP kinase does not play a major role in maintaining cPLA₂ activity but that both types of MAP kinase, ERK and p38, are required for the onset and the maintenance of complete cPLA₂ activity induced by OZ.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Time course of cPLA2 activation by OZ. Neutrophils were stimulated for the indicated times with 1 mg/ml OZ. cPLA2 specific activity was determined in neutrophil lysates using labeled phosphatidylcholine vesicles as a substrate. The results, expressed as specific activity, are the means ± S.E. from three experiments performed in duplicate. Inset, immunoblot of cPLA2 mobility shift in neutrophils stimulated by OZ for 2 min.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   The involvement of ERK and p38 MAP kinase in the maintenance of cPLA2 activation by OZ. Neutrophils were preincubated for 40 min at 4 °C with 100 µM PD-098059 and/or 5 µM SB-203580. Cells were stimulated for 10 min with 1 mg/ml OZ at 37 °C, and cPLA2 activity was determined in neutrophil lysates using labeled phosphatidylcholine vesicles as a substrate. The results expressed as specific activity are the means ± S.E. of three experiments done in duplicate. Each inhibitor significantly reduced (p < 0.001) the activity induced by OZ. Treatment of the cells with both inhibitors caused total inhibition, which is significantly higher (p < 0.001) than the effect of each inhibitor alone. There is no significant difference between the activity of unstimulated cells and cells treated with both inhibitors.

Activation of ERK, p38 MAP Kinase, and cPLA₂ by FcR Cross-linking-- The requirement of two MAP kinase isotypes for activation of cPLA₂ by OZ was queried because various studies have reported that only one type of MAP kinase is sufficient for cPLA₂ stimulation by different agents in human neutrophils. It has been shown that phosphorylation and activation of cPLA₂ in neutrophils stimulated with TNF- were completely abolished by the p38 MAP kinase inhibitor (53). platelet-activating factor stimulation of neutrophils was shown to increase cPLA₂ phosphorylation through activation of ERK 2 (47). Because OZ is ligated by three different receptors in human neutrophils, (FcRIIA, FcRIIIB, and CR3 (1)), the possibility was raised that each receptor initiates a response leading to activation of one type of MAP kinase. Thus, the role of the individual receptors in activation of cPLA₂ and the MAP kinase isotypes was further studied. To evaluate the relative role of C3bR in cPLA₂ activity mediated by MAP kinase, the effect of zymosan opsonized with heat-inactivated pooled human serum (iOZ), which does not contain the complement protein (C3b), or with zymosan opsonized with IgG only, was studied. The time course activation of ERK and p38 MAP kinase by 1 mg/ml iOZ was determined and compared with that of OZ. iOZ induced a time-dependent phosphorylation of ERK (Fig. 6A) and p38 MAP kinase (Fig. 6C) identical to that induced by OZ (Fig. 3, A and C), indicating that the complement receptor does not participate in activating ERK and p38 MAP kinase in neutrophils. The same results were obtained when neutrophils were stimulated by zymosan opsonized with pure IgG (data not shown). A similar pattern of activation of ERK and p38 MAP kinase as that induced by iOZ in our study was shown by cross-linking of FcR in murine macrophage (54). In support of our results demonstrating that the activation of MAP kinases is restricted to FcR stimulation, others studies have shown that in neutrophils, ligation of the complement receptor did not lead to either a detectable increase in tyrosine kinase activity (22) or elevation in intracellular calcium concentration (55). In addition, a recent study in mice genetically deficient in complement components C3 and C4 provided strong evidence that activation of FcRs, but not complements, are required for antibody-triggered murine inflammatory responses (56).

View larger version (46K): [in this window] [in a new window]   Fig. 6.   The time course activation of ERK and p38 MAP kinase stimulated by inactivated OZ. Neutrophils were stimulated with 1 mg/ml iOZ for the indicated times at 37 °C, and ERK or p38 MAP kinase phosphorylation were detected by immunoblot with phospho-specific antibodies. A and C, immunoblots of ERK or p38 MAP kinase phosphorylation using phospho-specific ERK or p38 MAP kinase antibodies, respectively. B and D, equal amounts of ERK or p38 MAP kinase in each sample were evaluated by immunoblotting with anti-ERK2 or anti-p38 MAP kinase antibodies, respectively. The results are from one representative experiment of three with identical results.

The research was then focused on MAP kinase and cPLA₂ activation induced by occupancy of FcRIIA or FcRIIIB. FcRIIA cross-linking (as described under "Experimental Procedures") resulted in a transient phosphorylation of ERK2 mainly, which was detectable 30 s after stimulation, reached a maximal level at 1-2 min, and decreased at 5 min. ERK2 phosphorylation could not be detected 10 min after stimulation (Fig. 7A). FcRIIIB cross-linking also resulted in a transient phosphorylation of ERK2 and was detected 30 s after stimulation but was shorter than that induced by FcRIIA and could no longer be detected as early as 5 min after stimulation (Fig. 7C). Cross-linking of either FcRIIA or FcRIIIB resulted in similar and significant levels of p38 MAP kinase phosphorylation that were detected within 15 s of stimulation, reaching maximal phosphorylation at 2-5 min and declining only after 20 min (Fig. 8, A and C). The results demonstrate that cross-linking of each receptor, FcRIIA or FcRIIIB, resulted in activation of both ERK and p38 MAP kinase. The activation of ERK by occupancy of FcRIIA has already been reported in neutrophils (57), but the present study demonstrates that p38 MAP kinase is activated as well. Our results are in accordance with an earlier study in human platelets reporting that ligation of FcRIIA activated these two MAP kinases (58). In addition, our results are the first to demonstrate that FcRIIIB activates both ERK and p38 MAP kinase. The ability of FcRIIIB by itself to activate MAP kinases is in accordance with others who reported that FcRIIIB is able to mediate various functions including calcium mobilization (20, 21), translocation of Src-related tyrosine kinase Hck (22), or superoxide production in human neutrophils (26, 27).

View larger version (46K): [in this window] [in a new window]   Fig. 7.   Time course of ERK stimulation by FcRIIA or FcRIIIB in human neutrophils. Neutrophils were incubated with antibodies (1 µg/ml) against human FcRIIA or FcRIIIB at 4 °C for 30 min followed by cross-linking with F(ab')2 mouse anti-human (10 µg/ml) for the indicated times at 37 °C. ERK phosphorylation was detected by immunoblot with phospho-specific ERK antibodies. A, immunoblot of ERK phosphorylation induced by FcRIIA. C, immunoblot of ERK phosphorylation induced by FcRIIIB. B and D, equal amounts of ERK in each sample were evaluated by immunoblotting with anti-ERK antibodies. Shown are representative blots of three experiments with identical results. Con, resting neutrophils; C.L, neutrophils incubated with cross-linker only.

View larger version (41K): [in this window] [in a new window]   Fig. 8.   Time course of p38 MAP kinase stimulation by FcRIIA or FcRIIIB in human neutrophils. Neutrophils were incubated with antibodies (1 µg/ml) against human FcRIIA or FcRIIIB at 4 °C for 30 min followed by cross-linking with F(ab')2 mouse anti-human (10 µg/ml) for the indicated times at 37 °C. p38 MAP kinase phosphorylation detected by immunoblot with phospho-specific p38 MAP kinase antibodies. A, immunoblot of p38 MAP kinase phosphorylation induced by FcRIIA. C, immunoblot of p38 MAP kinase phosphorylation induced by FcRIIIB. B and D, equal amounts of p38 MAP kinase in each sample were evaluated by immunoblotting with anti-p38 MAP kinase antibodies. Shown are representative blots from three experiments with identical results. Con, resting neutrophils; C.L, neutrophils incubated with cross-linker only.

To compare the effect of the individual receptors on the extent of MAP kinase phosphorylation and cPLA₂ activation, these activities were simultaneously analyzed in neutrophils stimulated for 2 min with either of the individual Fc Rs, OZ or iOZ. As demonstrated in Fig. 9A, cross-linking of FcRIIA caused moderate phosphorylation of ERK, which was significantly lower than that induced by OZ or iOZ. Cross-linking of FcRIIIB was the least efficient in stimulating ERK phosphorylation. ERK phosphorylation was severalfold higher when both receptors were ligated, as demonstrated by iOZ or OZ stimulation. This synergistic enhancement of ERK phosphorylation was clearly demonstrated at the early time points of activation. Although ERK phosphorylation could hardly be detected 15 s after stimulation with each Fc receptor (Fig. 7, A and C), ligation of both receptors resulted in a significant and high phosphorylation at this time point (Figs. 3A and 6A). Synergistic cooperation between FcRIIA and FcRIIIB when ligated together, as would occur when neutrophils bind to immune complexes, has been demonstrated for the phagocytic process (59), respiratory burst (60), and intracellular calcium elevation (61, 62). In contrast with the activation of ERK, a significant p38 MAP kinase phosphorylation was induced by cross-linking of either FcRIIA or FcRIIIB. Occupancy of both receptors did not induce a significant augmentation of p38 MAP kinase phosphorylation as that stimulated by the individual receptors (Fig. 9C). A significant and similar stimulation of cPLA₂ activity was induced by cross-linking of either FcRIIA or FcRIIIB (0.115 and 0.129 pmol/µg/30 min, respectively), although it was about 2-fold higher when induced by OZ or iOZ (0.304 and 0.284 pmol/µg/30 min, respectively) (Fig. 9E). To determine the role of the MAP kinase isotypes in mediating cPLA₂ activation by each of the Fc Rs, the effect of PD-098059 or SB-203580 was studied. As shown in Fig. 10, the presence of 5 µM SB-203580 or 100 µM PD-098059 similarly and significantly (p < 0.001) inhibited cPLA₂ activity induced by the individual receptors. The combined effect of both inhibitors caused total inhibition of cPLA₂ activity induced by either of the receptors. Thus, each FcR induced cPLA₂ activity is mediated by both ERK and p38 MAP kinase. Although the phosphorylation of ERK induced by each FcR is low compared with that of p38 MAP kinase, it has a similar importance to that of p38 MAP kinase in inducing cPLA₂ activity.

View larger version (60K): [in this window] [in a new window]   Fig. 9.   Comparison between activation of ERK, p38 MAP kinase, and cPLA2 by OZ, iOZ, or the individual Fc  receptor. Neutrophils were incubated with 1 µg/ml antibodies against human FcRIIA or FcRIIIB at 4 °C for 30 min followed by cross-linking with 10 µg/ml F(ab')2 mouse anti-human for 2 min at 37 °C; alternatively neutrophils were stimulated with 1 mg/ml iOZ or OZ for 2 min at 37 °C. Shown are representative blots and activity of three experiments with identical results. A, immunoblot of ERK phosphorylation using phospho-specific ERK antibody. The relative changes in protein phosphorylation were evaluated by a densitometer. The densitometric units are: Con, 813; C.L, 1025; IIA, 1957; IIIB, 1562; OZ, 5038; iOZ, 4331. C, immunoblot of p38 MAP kinase phosphorylation using phospho-specific p38 MAP kinase antibodies. The densitometric units are: Con, 2589; C.L, 2750; IIA, 3786; IIIB, 3985; OZ, 3753; iOZ, 4192. B and D, the blots were stripped and reprobed with anti-ERK or anti-p38 MAP kinase antibodies, respectively. E, cPLA2 activity was determined in neutrophil lysate using labeled phosphatidylcholine vesicles as a substrate.

View larger version (46K): [in this window] [in a new window]   Fig. 10.   Effect of p38 MAP kinase and/or ERK inhibitors on cPLA2 activity stimulated by FcRIIA or FcRIIIB cross-linking. Neutrophils were preincubated for 40 min at 4 °C with 100 µM PD-098059 and/or 5 µM SB-203580. The cells were incubated with 1 µg/ml antibodies against human FcRIIA (A) or FcRIIIB (B) at 4 °C for 30 min followed by cross-linking with 10 µg/ml F(ab')2 mouse anti-human for 2 min at 37 °C. cPLA2 specific activity was determined in neutrophil lysate using labeled phosphatidylcholine vesicles as a substrate. The results expressed as specific activity are the means ± S.E. of three experiments done in duplicate. Con, resting neutrophils; C.L, neutrophils incubated with cross-linker only. Each inhibitor caused a significant inhibition (p < 0.001) of the activity induced by either FcRIIA or FcRIIIB. Treatment of the cells with both inhibitors resulted in reduced activity similar to that of nonactivated cells (p = not significant) and significantly lower (p < 0.001) than that induced by either of the Fc receptors.

The Upstream Signaling of MAP Kinase Activation by FcRs-- To study the tyrosine kinase elements upstream to MAP kinase and cPLA₂, we investigated whether this agent induces tyrosine phosphorylation of Shc and binding of Shc to the Grb2-Sos complex. Lysates from resting or OZ-stimulated neutrophils were subjected to immunoprecipitation with Grb2 or Shc antiserums and analyzed by Western blotting with Grb2, Shc, Sos, and phosphotyrosine antibodies. As shown in Fig. 11A, human neutrophils contain the 66-kDa isoform of Shc. Immunoblot analysis with anti-phosphotyrosine antibodies revealed detectable levels of tyrosine phosphorylation of 66-kDa Shc in resting neutrophils, with no increase following stimulation of the cells with OZ for 2 min (Fig. 11B). Similar results were obtained during 15 s to 20 min of stimulation with OZ (data not shown). Furthermore, Shc was constitutively associated with Grb2 in resting cells, and stimulation of the cells with OZ for 2 min did not alter the extent of the association between Grb2 and Shc (Fig. 11C). In accordance with the results of others (63), a low level expression of Sos was found in human neutrophils. Sos was undetectable in Grb2 immunoprecipitates in resting or activated neutrophils probably because of its limited expression (Fig. 11D). These results argue against the involvement of the Shc-Grb2-Sos complex in activation of the MEK-ERK pathway in neutrophils stimulated by FcRIIA and FcRIIIB and suggest that an alternate, unknown activating mechanism of ERK exists in human neutrophils. In accordance with our results, the formation of Grb2-Shc complex in human neutrophils was not triggered by oxidants that induce tyrosine-dependent signaling (64). Other members of the FcR family that are not expressed in human neutrophils were shown to promote a complex formation between tyrosine-phosphorylated Shc and Grb2, leading to activation of the Ras signaling pathway, such as ligation of FcRIIIA in NK cells (65), cross-linking of FcRI in interferon -differentiated U937 cells (66) or activation of rat alveolar macrophages by zymosan-activated serum (67).

View larger version (40K): [in this window] [in a new window]   Fig. 11.   OZ failed to increase tyrosine phosphorylation of Shc and the association of Grb2-Sos with Shc. Grb 2 (A and B) or Shc (C-E) immunoprecipitation (I.P) were performed from lysates of resting (Con) or stimulated neutrophils with 1 mg/ml OZ for 2 min at 37 °C (OZ). The immunoprecipitates were subjected to SDS-electrophoresis, and immunoblotting was performed with anti-Shc (A and C), anti-phospho tyrosine, 4G10 (B), anti-Sos (D), or anti-Grb2 (E) antibodies. In each blot the first right lane contains neutrophil lysate (Neu lysate) to detect the position of the indicated proteins.

An alternate, tyrosine-dependent, activating mechanism of MAP kinase might be mediated by cytosolic tyrosine kinases. Several reports have shown that activation of the tyrosine kinase Pyk2 is upstream to ERK or p38 MAP kinase in different cell lines and in response to diverse stimuli (36, 41-44). Pyk2 was shown to be activated by addition of calcium ionophore (68). We have reported that stimulation of human neutrophils by OZ induced an elevation in intracellular calcium ion concentration (28). We therefore analyzed the possibility that Pyk2 is involved in the signaling pathway leading to ERK and p38 MAP kinase activation in FcR-stimulated neutrophils. As shown in Fig. 12B, stimulation of neutrophils by cross-linking of either FcRIIA or FcRIIIB or by OZ for 2 min induced a significant tyrosine phosphorylation of Pyk2. The tyrosine kinase inhibitor, genistein (300 µM), totally inhibited Pyk2 phosphorylation induced by either OZ (Fig. 12A) or by cross-linking of each FcR (data not shown). Because we have already shown that NADPH oxidase, cPLA₂, and ERK activities are all inhibited by genistein in human neutrophils stimulated by OZ (28), the inhibition of Pyk2 may imply that this enzyme is involved in the signal transduction pathway leading to MAP kinase activation. In neutrophils, it is more difficult to define the role of a specific enzyme than it is in other types of cells. Neutrophils cannot be transfected and cannot be loaded with dominant negative peptides by osmotic shock or electroporation. In addition, there is no available specific inhibitor of Pyk2. The involvement of Pyk2 in the signaling pathways leading to MAP kinases and cPLA₂ activation by OZ in a leukemic myeloid cell line that can be transfected with dominant negative peptides is currently under investigation in our laboratory.

View larger version (59K): [in this window] [in a new window]   Fig. 12.   Tyrosine phosphorylation of Pyk2 induced by FcRs. Neutrophils were incubated with 1 µg/ml antibodies against human FcRIIA or FcRIIIB at 4 °C for 30 min followed by cross-linking with 10 µg/ml F(ab')2 mouse anti-human for 2 min at 37 °C; alternatively neutrophils were stimulated with 1 mg/ml OZ for 2 min at 37 °C. Cell lysates were immunoprecipitated with anti-Pyk2 and analyzed by immunoblotting with anti-phosphotyrosine 4G10 antibody (A and C) or anti-Pyk2 antibodies (B and D). When indicated, neutrophils were preincubated with 300 µM genistein for 30 min at 4 °C before stimulation (C). Shown are representative blots of three experiments with identical results. Con, resting neutrophils; C.L, neutrophils incubated with cross-linker only.

The present study demonstrates (as summarized in Fig. 13) that activation of cPLA₂ induced by OZ is mediated mainly by FcRs. Cross-linking of either FcRIIA or FcRIIIB induced a high p38 MAP kinase phosphorylation and a low ERK phosphorylation, both of which are required for cPLA₂ activation. Cross-linking of the two FcRs induced a synergized ERK activity and a insignificant elevation of p38 MAP kinase activity that mediated an additive cPLA₂ activity. Therefore, both ERKs and p38 MAP kinase are necessary for the onset and the maintenance of cPLA₂ activation induced by ligation of FcRIIA and FcRIIIB. The signaling pathways initiated by these receptors do not involve the formation of the protein complex Shc-Grb2-Sos but induce the tyrosine phosphorylation of the nonreceptor tyrosine kinase Pyk2.

View larger version (26K): [in this window] [in a new window]   Fig. 13.   Proposed is a schematic for FcRs signaling to cPLA2 in human neutrophils. The scheme is based on present and previous studies (28, 32).

	FOOTNOTES

^* This work was supported by a grant from the Ministry of Health, Israel.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel. Tel.: 972-7-6403186; Fax: 972-7-6467477; E-mail: ral@bgumail.bgu.ac.il.

	ABBREVIATIONS

The abbreviations used are: FcRs, Fc  receptors; cPLA₂, cytosolic phospholipase A₂; ERK, extracellular regulated kinase; MAP, mitogen-activated protein; OZ, opsonized zymosan; DTT, dithiothreitol; HBSS, Hanks' balanced salt solution; iOZ, inactivated OZ; PMSF, phenylmethylsulfonyl fluoride.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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