FcγRI-triggered Generation of Arachidonic Acid and Eicosanoids Requires iPLA2 but Not cPLA2 in Human Monocytic Cells*

Aggregation of receptors for immunoglobulin G (FcγRs) on myeloid cells activates a series of events that are key in the inflammatory response and that can ultimately lead to targeted cell killing by antibody-directed cellular cytotoxicity. Generation of lipid-derived proinflammatory mediators is an important component of the integrated cellular response mediated by receptors for the constant region of immunoglobulins (Fc). We have demonstrated previously that, in interferon-γ-primed U937 cells, the high affinity receptor for IgG, FcγRI, is coupled to a novel intracellular signaling pathway that involves the sequential activation of phospholipase D, sphingosine kinase, calcium transients, and protein kinase C isoforms, leading to the activation of the NADPH-oxidative burst. Here, we investigate the nature of the phospholipase that regulates arachidonic acid and eicosanoid production. Our data show that FcγRI couples to iPLA2β for the release of arachidonic acid and the generation of leukotriene B4 and prostaglandin E2. Activation of iPLA2β was protein kinase C-dependent; on the other hand, platelet-activating factor triggered cPLA2α by means of the mitogen-activated protein kinase pathway. These studies demonstrate that intracellular PLA2s can be selectively regulated by different stimuli and suggest a critical role for iPLA2β in the intracellular signaling cascades initiated by FcγRI and its functional role in the generation of key inflammatory mediators.

Receptors for the constant region of immunoglobulins (Fc) 1 play a pivotal role linking the humoral and cellular arms of the immune system. On leukocytes, aggregation of receptors for immunoglobulin G (IgG) leads to a number of cellular responses, including the internalization of immune complexes, release of proteases, activation of the respiratory burst, the release of cytokines, and the generation of eicosanoids. Receptor aggregation can ultimately lead to targeted cell killing through antibody-directed cellular cytotoxicity (1,2). These Fc receptors, therefore, play critical roles in host defense mechanisms against invading pathogens in autoimmune diseases (3) and in cancer surveillance (4). We have recently reported that, in the human monocyte model (cytokine primed U937 cells), aggregation of the high affinity receptor for IgG (Fc␥RI) activates, through non-receptor tyrosine kinases, a novel signaling pathway that involves the sequential activation of phosphatidylinositol 3-kinase, phospholipase D, and sphingosine kinase (5)(6)(7). This pathway is necessary for efficient intracellular trafficking of Fc␥RI-internalized immune complexes to lysosomes for degradation, the release of calcium from intracellular stores, and the activation of the NADPH oxidative burst (6 -8).
The best studied PLA 2 s are Groups IIA, V, and IVA, which for a long time have been shown to be responsible for AA release and prostaglandin generation in different systems (14 -16). iPLA 2 has been shown to be implicated in many cellular functions ranging from basal fatty acid reacylation reactions (17), to playing major roles in intracellular signaling cascades, including its involvement in agonist-induced eicosanoid production (18,19), stimulation of smooth muscle (20) and endothelial cells (21), in lymphocyte proliferation (22), and in endothelium-dependent vascular relaxation (21). Very recently, it has been reported that myocardial ischemia activates iPLA 2 ␤ in intact myocardium, and that this iPLA 2 ␤ activation is sufficient to induce malignant ventricular arrhythmias (23), and also that functional iPLA 2 is required for activation of store-operated channels and capacitative Ca 2ϩ influx in several cell types (24).
Here, we demonstrate that coupling of Fc␥RI to AA generation and production of LTB 4 and PGE 2 absolutely requires iPLA 2 ␤ activation. Although both intracellular forms of PLA 2 (cPLA 2 ␣ and iPLA 2 ␤) are present in U937 cells, only iPLA 2 ␤ functionally couples Fc␥RI to trigger physiological responses, such as the generation of AA and the production of LTB 4 and PGE 2 . Moreover, only iPLA 2 ␤ translocates to the plasma membrane and triggers the generation of AA and eicosanoids after Fc␥RI activation. Furthermore, by using specific antisense oligonucleotides against iPLA 2 ␤ and cPLA 2 ␣, we found that both isoforms can be activated independently by different receptors, because the addition of platelet-activating factor (PAF) triggers cPLA 2 ␣-dependent generation of AA without activating iPLA 2 ␤. Thus, these studies demonstrate that both intracellular PLA 2 s can be selectively regulated by different stimuli and suggest a critical role for iPLA 2 ␤ in the intracellular signaling cascades initiated by immune-receptors and its functional role in the generation of key inflammatory mediators.

MATERIALS AND METHODS
Cell Culture-U937 cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum, 2 mM glutamine, 10 units/ml penicillin, and 10 mg/ml streptomycin at 37°C and 6.8% carbon dioxide in a water-saturated atmosphere. The cells were treated with 200 ng/ml interferon (IFN)-␥ (Bender Wien Ltd, Vienna, Austria) for 16 h. Antisense oligonucleotides were purchased from Oswell DNA Services; 24-mers were synthesized, capped at either end by the phosphorothioate linkages (first two and last two linkages), and corresponded to the reverse complement of the first eight amino acids for either iPLA 2 ␤ or cPLA 2 ␣. The sequences of the oligonucleotides were 5Ј-CAGGCGGCCAAAGAACTGCATCTT-3Ј for iPLA 2 ␤ and 5Ј-GGTA-AGGATCTATAAATGACAT-3Ј for cPLA 2 ␣.
Cells were incubated in 1 M oligonucleotide mixed with 20 l of Superfect (Qiagen) for a total of 36 h (20 h prior to the addition of INF␥, and then incubated for the duration of culture with IFN-␥).
Receptor Stimulation-Fc␥RI aggregation was carried out as described previously (6 -8). Briefly, cells were harvested by centrifugation and then incubated at 4°C for 45 min with 1 M human monomeric IgG (Serotec UK) to occupy surface Fc␥RI in the presence or absence of inhibitors or alcohols. Excess unbound ligand was removed by dilution and centrifugation of the cells. Cells were resuspended in ice-cold RHB medium (RPMI 1640 medium, 10 mM HEPES, 0.1% BSA) and surface immune complexes formed by incubating with cross-linking antibody (sheep anti-human IgG; 1:50), without or in the continued presence of inhibitors. Cells were then warmed to 37°C for the times specified in each assay.
PAF Stimulation-Cells were harvested by centrifugation and resuspended in ice-cold RHB medium, and surface platelet-activating factor receptor was stimulated by the addition of 10 M platelet-activating factor (PAF) (Calbiochem), without or in the continued presence of inhibitors. Cells were then warmed to 37°C for the times specified in each assay.
Immunoprecipitations-iPLA 2 ␤ and cPLA 2 ␣ were immunoprecipitated from cell lysates stimulated by Fc␥RI for the indicated times. Specific goat-polyclonal anti-iPLA 2 ␤ or rabbit-polyclonal anti-cPLA 2 ␣ (Santa Cruz Biotechnology, Inc) were incubated with protein A-agarose (50% slurry from Amersham Biosciences) at 4°C, with rocking for 2 h to form precipitating complexes. Cell lysates were precleared with protein A-agarose (incubated for 30 min under rocking conditions); after the removal of the protein A-agarose, the precleared cell extracts were incubated with either anti-iPLA 2 ␤ or anti-cPLA 2 ␣ precipitating complexes and placed in a tumbler at 4°C for 4 h, after which the precipitates were washed 3ϫ in ice-cold phosphate-buffered saline to discard unbound material. The precipitated proteins were resolved by SDS-PAGE.
Gel Electrophoresis and Western Blotting-Proteins were resolved as described previously (6). Briefly, immunoprecipitates were resolved on 10% polyacrylamide gels (SDS-PAGE) under denaturing conditions and then transferred to 0.45 m nitrocellulose membranes. After blocking overnight at 4°C with 5% nonfat milk in Tris-buffered saline, 0.1% Tween 20 and washing, the membranes were incubated with the relevant antibodies (rabbit-polyclonal anti-phosphoserine, Chemicon International; goat-polyclonal anti-iPLA 2 ␤, Santa Cruz Biotechnology; or rabbit-polyclonal anti-cPLA 2 ␣, Santa Cruz Biotechnology) for 4 h at room temperature. The membranes were washed extensively in the washing buffer and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (anti-goat IgG-peroxidase conjugate, Sigma) or anti-rabbit IgG-peroxidase conjugate (Sigma) for 3 h at room temperature. The membranes were washed extensively in the washing buffer, and bands were visualized using ECL Western blotting detection system (Amersham Biosciences). On separate experiments, the eluted proteins from the immunoprecipitation were resolved as above, and the gels were subjected to silver staining.
Measurement of Arachidonic Acid Release-AA release was measured as described previously (25). Briefly, cells were labeled (2 ϫ 10 6 cells/ml) with [ 3 H]AA (1 Ci/ml, Amersham Biosciences) in the cell culture medium for 16 h. After washing, the cells were incubated at 37°C for 30 min in RPMI 1640 medium, 1% fetal calf serum containing or not the specific inhibitors. Fc␥RI was stimulated and reactions were stopped at the indicated times. After stimulation, the cells were spun down at 4°C and supernatants were removed to measure the released [3H]AA, whereas the cell pellet was resuspended to measure total cellular [3H]AA incorporation. AA release was measured as the percentage of the total [ 3 H]AA incorporated into the cell membranes.
Measurement of LTB 4 Generation-LTB 4 production was measured after receptor stimulation by the Biotrak TM leukotriene B 4 enzyme immunoassay system from Amersham Biosciences. Briefly, the assay is based on the competition between unlabelled LTB 4 and a fixed quantity of peroxidase-labeled LTB 4 for a limited number of binding sites on an LTB 4 -specific antibody. The amount of LTB 4 in the experimental sample will be inversely proportional to the signal generated by the fixed amount of peroxidase-labeled LTB 4 .
Measurement of PGE 2 Synthesis-PGE 2 production was measured after receptor stimulation by the Biotrak TM PGE 2 system from Amersham Biosciences. Briefly, the assay is based on the competition between unlabelled PGE 2 and a fixed quantity of peroxidase-labeled PGE 2 for a limited number of binding sites on a PGE 2 -specific antibody. The amount of PGE 2 in the experimental sample will be inversely proportional to the signal generated by the fixed amount of peroxidase-labeled PGE 2 .
Fluorescence Microscopy-After receptor aggregation, suspended cells were fixed in 4% paraformaldehyde and deposited on microscope slides using a cytospin centrifuge; they were then permeabilized for 5 min in 0.1% Triton X-100 in phosphate-buffered saline. The permeabilized cells were blocked for nonspecific binding with 5% fetal calf serum for 10 min at room temperature. Fluorescent labeling was performed by incubating the cells with goat-polyclonal anti-iPLA 2 ␤ or rabbit-polyclonal anti-cPLA 2 ␣ (Santa Cruz Biotechnology) and primary antibodies for 1 h at room temperature. The cells were washed with phosphatebuffered saline and secondary antibodies (anti-goat IgG-TRITC conjugate or anti-rabbit-FITC conjugate, Sigma) were added. To a set of cells, only the secondary antibodies were added for control. Stainings were analyzed with an inverted fluorescence Leica DM IRB microscope and recorded by a Leica DC 300F digital camera; pictures were analyzed with the Leica IM500 Image Manager software.

RESULTS
iPLA 2 ␤ and cPLA 2 ␣ Are Both Endogenously Expressed in the Monocytes-U937 cells express cPLA 2 ␣ and iPLA 2 ␤ but not sPLA 2 (26 -28). To ascertain whether the expression patterns of either cytosolic form of PLA 2 changed with the cytokine differentiation of U937 to human monocytes, we performed Western blotting analysis. Relative levels of protein expression were compared in untreated cells and in cytokine (IFN-␥) differentiated cells.
Total cell-extracts from untreated or IFN-␥-U937 cells revealed that both iPLA 2 ␤ and cPLA 2 ␣ proteins were readily detectable. Western blot analysis revealed immunoreactive bands corresponding to the predicted molecular weights for iPLA 2 ␤ and cPLA 2 ␣. The intracellular PLA 2 expression profiles did not alter after IFN-␥ differentiation (Fig. 1a).
Fc␥RI Aggregation Triggers AA Release-Fc␥RI aggregation triggers a quick and sustained increase of AA generation over time (Fig. 1b). This Fc␥RI-triggered AA generation was almost completely inhibited in cells pretreated with 30 M of methyl arachidonyl fluorophosphate (MAF), an inhibitor of cPLA 2 and iPLA 2 (16), suggesting the participation of cPLA 2 and/or iPLA 2 in the AA generation (Fig. 1b). To discern which of the two isoforms was activated by Fc␥RI, we examined the effect of E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2-Hpyran-2-one (BEL) (29), a relatively selective inhibitor for iPLA 2 . The Fc␥RI-triggered AA release was inhibited in cells pretreated with 10 M of BEL (Fig. 1c).
Even though the quantity of BEL used has not been shown to inhibit cPLA 2 activity (30), we investigated the role of BEL in the AA release triggered by PAF, a stimulant that we knew activates cPLA 2 (31). Although 30 M of MAF inhibited PAFtriggered AA generation from the IFN␥-primed cells (Fig. 2a), 10 M of BEL did not have an effect at all (Fig. 2b). Taken together, these data suggest that PAF indeed activates cPLA 2 , whereas Fc␥RI couples to iPLA 2 .
Fc␥RI Aggregation Specifically Stimulates iPLA 2 ␤-To gain further proof of the nature of PLA 2 ␤, which plays a major role in the signaling pathways triggered by Fc␥RI, we designed specific antisense oligonucleotides against iPLA 2 ␤ and cPLA 2 ␣ to knockdown specifically the expression of each enzyme. We have shown previously that U937 cells are sensitive to antisense manipulation (7,8). IFN-␥-primed cells were treated with antisense oligonucleotide; AA generation was assayed either in unstimulated cells to measure basal levels of activity or after stimulation with Fc␥RI activation either by immune complexes or with PAF (PAF was used as control). The specificity of the antisense oligonucleotides on relative PLA 2 isozyme expression was checked by Western blot analysis (Fig.  3a). Thus, in cells treated with antisense to iPLA 2 ␤, there was a substantial reduction in iPLA 2 ␤ immunoreactivity (80% reduction measured by densitometry), whereas cPLA 2 ␣ immuno-reactivity was unaffected. Conversely, in cells treated with antisense to cPLA 2 ␣, there was a reduction in cPLA 2 ␣ immunoreactivity (85% reduction measured by densitometry), whereas iPLA 2 ␤ immunoreactivity remained unchanged. Each antisense oligonucleotide, therefore, acted as an internal control for the other.
In cells pretreated with the antisense oligonucleotide to iPLA 2 ␤, the increase in AA generation, observed after Fc␥RI aggregation was significantly reduced, compared with the control cells (Fig. 3b). The reduction in the increase after Fc␥RI activation was about 80% in cells treated with antisense iPLA 2 ␤ compared with control cells and was proportional to the observed reduction in protein expression by Western blot analysis (Fig. 3a). In contrast, treatment of cells with the antisense oligonucleotide to cPLA 2 ␣ had no effect on the Fc␥RI-mediated generation of AA (Fig. 3b). Contrary to the AA generation triggered by Fc␥RI, AA generation stimulated by PAF was significantly reduced by about 80% in cells pretreated with the antisense oligonucleotide to cPLA 2 ␣ but not in cells treated with the antisense against iPLA 2 ␤ (Fig. 3c).
Moreover, by immunoprecipitation and Western blotting, we found that in Fc␥RI-stimulated cells, iPLA 2 ␤ is phosphorylated on serine residues (Fig. 4a, upper left panel), whereas cPLA 2 ␣ is not phosphorylated by Fc␥RI engagement (Fig. 4b, top panels). Equal protein loading is shown by stripping the blots and reprobing for iPLA 2 ␤ or cPLA 2 ␣ (Fig. 4a, upper right panel; Fig.  4b, upper right and lower right panels). As a control for the iPLA 2 ␤ immunoprecipitation, a Western blot of cell extracts depleted of iPLA 2 ␤ is shown (Fig. 4a, lower left panel), as well as a silver-stained gel showing the elution of a single band after immunoprecipitation with the anti-iPLA2␤ antibody (Fig. 4a,  lower right panel). To further establish the specificity of the system, we show that PAF stimulation causes the serine-phosphorylation of cPLA 2 ␣ (Fig. 4b, lower panels), whereas PAF stimulation does not cause iPLA 2 ␤ phosphorylation (data not shown). Furthermore, microscopy analysis of the subcellular localization of the different intracellular PLA 2 revealed that, after Fc␥RI aggregation, iPLA 2 ␤ translocates to the plasma membrane (Fig. 4c), whereas the cytosolic localization of cPLA 2 ␣ remained unchanged (Fig. 4d). For all fluorescence microscopy experiments, controls were carried out by adding the secondary antibodies to the cells without giving any signals; the antisense treatment did not influence the levels either of Fc␥RIor PAF-receptor expression (data not shown). These data strongly suggest that only iPLA 2 ␤ is coupled to Fc␥RI activation iPLA 2 ␤ Couples Fc␥RI to the Generation of LTB 4 and PGE 2 -As coupling of Fc␥RI to arachidonic acid release requires iPLA 2 ␤ activation, the role of this enzyme in coupling Fc␥RI to other signaling pathways, such as the production of eicosanoids, was investigated. Reduction in the expression of iPLA 2 ␤ by pre-treatment of cells with the antisense oligonucleotide to iPLA 2 ␤ resulted in a substantial inhibition of peak LTB 4 and PGE 2 observed after aggregation of Fc␥RI (Fig. 5, a   and b, respectively). However, the antisense to cPLA 2 ␣ had no effect on the Fc␥RI-triggered eicosanoids production (Fig. 5, a  and b).
To ensure that the loss of eicosanoid production after Fc␥RI activation in cells treated with the antisense oligonucleotide to iPLA 2 ␤ was a feature of the loss of coupling of the receptor and not some direct effect of the iPLA 2 ␤ antisense oligonucleotide on other members of the signaling pathways (such as cyclooxygenases), LTB 4 and PGE 2 were measured after activation of cells with PAF. Addition of PAF to control cells or cells treated with the antisense iPLA 2 resulted in an identical increase in LTB 4 and PGE 2 production (Fig. 5c); on the other hand, in cells pretreated with the antisense to cPLA 2 ␣, eicosanoid production was substantially inhibited (Fig. 5d). These data indicate that, in cells pretreated with antisense oligonucleotides, the reduction in LTB 4 and PGE 2 after Fc␥RI activation reflects role of iPLA 2 in the generation of eicosanoids.
Role of PKC in Triggering iPLA 2 ␤ after Fc␥RI Aggregation-It has been shown that iPLA 2 ␤ activation requires PKC activity (18). Here we show that Bis, a selective PKC inhibitor, inhibits AA generation triggered by Fc␥RI aggregation (Fig.  6a), whereas a MAPK inhibitor (SB203580) did not have any effect on the Fc␥RI-triggered AA generation (Fig. 6b). In contrast, the AA generation triggered by PAF was not affected by the PKC inhibitor (Fig. 6a), but it was substantially reduced by the MAPK inhibitor (Fig. 6b). These findings indicate that PKC activity indeed may be involved in the Fc␥RI-triggered stimulation of iPLA 2 ␤, and confirm that the PAF-triggered activation of cPLA 2 ␣ is MAPK-dependent.
To evaluate further the potential involvement of PKC activity in iPLA 2 ␤ activation, we examined the effect of Bis on the Fc␥RI-triggered iPLA 2 ␤ translocation and phosphorylation patterns. Here we also show that pretreatment of cells with Bis markedly suppressed the Fc␥RI-triggered iPLA 2 ␤ translocation to the cell membranes (Fig. 6c). Furthermore, Bis also completely inhibited the phosphorylation of iPLA 2 ␤ triggered by Fc␥RI (Fig. 6d).
Fc␥RI-triggered iPLA 2 ␤ Activation Is Calcium-independent-It is well established that in immune cells, antigen receptor-induced AA release is, in most cases, calcium-dependent (18,30,31) and even, at least in one case (where iPLA 2 was indeed activated), intracellular calcium depletion prevented the generation of AA (although in this case (18), the authors suggested that this result was due to the inhibition of a calciumdependent PKC). Here we show that chelating intracellular calcium with BAPTA had no significant effect on iPLA 2 ␤ translocation (Fig. 7a), iPLA 2 ␤ phosphorylation (Fig. 7b), or on AA release triggered by Fc␥RI (Fig. 7c), whereas the BAPTA treatment did indeed block the PAF-induced AA release in the same cells.
These data correlate with our previous findings that Fc␥RI triggers calcium-independent PKC activities (32,33) and suggest that calcium-independent PKC(s) may be involved in triggering iPLA 2 ␤ after Fc␥RI aggregation in human monocytes. DISCUSSION The two forms of cytosolic PLA 2 (cPLA 2 and iPLA 2 ) are expressed in U937 cells (26,27), and we found that differentiation with IFN-␥ does not significantly alter the expression levels of either of the two enzymes. Our aim was to find out which PLA 2 was involved in the Fc␥RI intracellular signaling cascades leading to the generation of eicosanoids. In this study, we demonstrated that Fc␥RI is functionally coupled to iPLA 2 ␤, and that this enzyme is required for Fc␥RI-mediated generation of arachidonic acid and the formation of leukotrienes and prostaglandins. iPLA 2 ␤ contains a calmodulin (CaM)-binding domain near the C terminus which binds calcium-activated CaM and regu-lates enzyme activity (34). The binding of CaM to iPLA 2 ␤ results in the inhibition of iPLA 2 ␤ activity, which is reversible through the removal of Ca ϩ2 , and subsequent dissociation of CaM from the C terminus of iPLA 2 ␤ (34). Thus, in some models, it is possible for iPLA 2 ␤ to be regulated through alterations in cellular calcium ion homeostasis and become activated after dissociation from its complex with Ca ϩ2 /CaM when intracellular calcium stores are depleted (e.g. by sarco/endoplasmic reticulum calcium ATPase inhibitors, calcium-ionophores, or agonist stimulation; ref. 35).
Here we report that the Fc␥RI-triggered AA generation was almost completely inhibited in cells pretreated with MAF, an inhibitor of both cPLA 2 and iPLA 2 (36), suggesting the participation of cPLA 2 and/or iPLA 2 in the AA generation. To discern which of the two isoforms was activated by Fc␥RI, we examined the effect of BEL, a relatively selective inhibitor for iPLA 2 (29). The Fc␥RI-triggered AA release was inhibited in cells pretreated with BEL. As a control for the specificity of BEL, we investigated the role of BEL in the AA release triggered by PAF, a stimulant known to activate cPLA 2 (31). We found that, although MAF inhibited PAF-triggered AA generation, treatment of the cells with BEL did not have an effect on the AA release triggered by PAF, showing the selectivity of BEL and suggesting to us the possibility that iPLA 2 was the enzyme involved in the Fc␥RI-triggered AA release.
To be more specific, we designed antisense oligonucleotides against iPLA 2 ␤ and cPLA 2 ␣ to selectively down-modulate the protein levels of these enzymes. Our data show that the iPLA 2 ␤ antisense substantially decreased AA release and LTB 4 and PGE 2 generation induced by Fc␥RI aggregation, whereas the antisense against cPLA 2 ␣ had no effect on the Fc␥RI pathway. Moreover, the antisense to iPLA 2 ␤ did not affect PAF-induced AA release or LTB 4 and PGE 2 generation, whereas the antisense against cPLA 2 ␣ inhibited the PAF-triggered generation of AA, showing that both pathways utilize different phospholipases as well as the selectivity of each antisense oligonucleotide.
Furthermore, our data demonstrate that aggregation of Fc␥RI triggers the serine phosphorylation and membrane translocation of iPLA 2 ␤ but not of cPLA 2 ␣. We found that the selective PKC inhibitor Bis substantially decreased the Fc␥RItriggered AA generation, whereas the MAPK inhibitor (BS203580) did not. In contrast, the PAF-triggered AA generation was inhibited by the MAPK inhibitor but not by the PKC inhibitor. These results suggest that Fc␥RI triggers iPLA 2 ␤ activation by means of PKC, whereas PAF-triggers cPLA 2 ␣ via the activation of MAP-kinases. Moreover, the PKC inhibitor also blocked iPLA 2 ␤ translocation to the cell periphery and completely blocked the phosphorylation of iPLA 2 ␤ that follows Fc␥RI aggregation. Taking these data together, we suggest that PKC is involved in triggering the activation of iPLA 2 ␤ in the Fc␥RI signaling cascade by phosphorylating and thus promoting the translocation of iPLA 2 ␤ to the cell's plasma membrane.
Different stimuli induce AA release in monocytes and macrophages in a Ca ϩ2 -dependent and phosphorylation-dependent manner because of the activation of cPLA 2 (37,38). However, PGE 2 generation by zymosan-stimulated macrophages is significantly attenuated by BEL or iPLA 2 ␤ antisense (30). Paradoxically, in these cells, iPLA 2 ␤ activation seems to be regulated by protein kinase C and is Ca ϩ2 -dependent, although in this case, the authors (18) suggested that this result was due to a calcium-dependent PKC, which, in turn, activated iPLA 2 ␤. In contrast, other studies have shown ligand-stimulated eicosanoid production in cells that have been treated with calcium chelators such as BAPTA and EDTA (35). In agreement with the latter, we show here that chelating intracellular calcium with BAPTA had no significant effect on iPLA 2 ␤ translocation, phosphorylation, or AA release triggered by Fc␥RI, whereas the same BAPTA-AM treatment completely blocked the PAFinduced AA release.
Based upon the effects of BEL, it has been suggested for many years that iPLA 2 mediates AA in different cells stimulated by various agonists (29, 39 -42), including during IgGmediated phagocytosis of human monocytes, where AA release was shown to be triggered in a calcium-independent manner (41,42). For iPLA 2 ␤, several important signaling functions have been suggested, including its role in agonist-induced stimulation of smooth muscle (20) and endothelial cells (21), in lymphocyte proliferation (22), and in endothelium-dependent vascular relaxation (21). Very recently, it was reported that myocardial ischemia activates iPLA 2 ␤ in intact myocardium, and that iPLA 2 ␤ activation is sufficient to induce malignant ventricular arrhythmias (23). Another recent study shows that functional iPLA 2 ␤ is required for activation of store-operated channels and capacitative Ca 2ϩ influx in several cell types (24). We show here that in a human monocytic cell line, iPLA 2 ␤ plays a critical role in the intracellular signaling cascades initiated by the high affinity receptor for IgG (Fc␥RI) and in its functional role to coordinate the response to antigen stimulation for the production of lipid-derived proinflammatory mediators such as leukotrienes and prostaglandins. These observations strongly suggest iPLA 2 ␤ as a potential therapeutic candidate for treating human conditions ranging from ischemia to antigen-mediated inflammatory diseases.