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

2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês

10.1074/jbc.m308788200

1083-351X

Hwee Kee Tay, Alirio J. Melendez,

Eicosanoids and Hypertension Pharmacology

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. 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. Retraction: FcγRI-triggered generation of arachidonic acid and eicosanoids requires iPLA2 but not cPLA2 in human monocytic cells.Journal of Biological ChemistryVol. 288Issue 14PreviewVOLUME 279 (2004) PAGES 22505–22513 Full-Text PDF Open Access Receptors for the constant region of immunoglobulins (Fc) 1The abbreviations used are: Fc, immunoglobulin constant region receptors; FcγRI, high affinity receptor for IgG; PGE2, prostaglandin E2; LTB4, leukotriene B4; AA, arachidonic acid; PLA2, phospholipase A2; PAF, platelet-activating factor; IFN, interferon; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; TBS, Tris-buffered saline; BEL, E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2-H-pyran-2-one; MAF, methyl arachidonyl fluorophosphate; Bis, bisindolylmaleimide I; CaM, calmodulin. 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 (1Graziano R.F. Fanger M.W. J. Immunol. 1987; 139: 3536-3541PubMed Google Scholar, 2Fanger M.W. Shen L. Graziano R.F. Guyre P.M. Immunol. Today. 1989; 10: 92-99Abstract Full Text PDF PubMed Scopus (313) Google Scholar). These Fc receptors, therefore, play critical roles in host defense mechanisms against invading pathogens in autoimmune diseases (3Hulett M.D. Hogarth P.M. Adv. Immunol. 1994; 57: 1-127Crossref PubMed Scopus (431) Google Scholar) and in cancer surveillance (4Ely P. Wallace P.K. Givan A.L. Guyre P.M. Fanger M.W. Blood. 1996; 86: 3813-3821Crossref Google Scholar). We have recently reported that, in the human monocyte model (cytokine primed U937 cells), aggregation of the high affinity receptor for IgG (FcγRI) activates, through non-receptor tyrosine kinases, a novel signaling pathway that involves the sequential activation of phosphatidylinositol 3-kinase, phospholipase D, and sphingosine kinase (5Allen J.M. Seed B. Science. 1989; 243: 378-381Crossref PubMed Scopus (205) Google Scholar, 6Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 7Melendez A. Floto R.A. Cameron A.J. Gillooly D.J. Harnett M.M. Allen J.M. Curr. Biol. 1998; 8: 210-221Abstract Full Text Full Text PDF PubMed Google Scholar). This pathway is necessary for efficient intracellular trafficking of FcγRI-internalized immune complexes to lysosomes for degradation, the release of calcium from intracellular stores, and the activation of the NADPH oxidative burst (6Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 7Melendez A. Floto R.A. Cameron A.J. Gillooly D.J. Harnett M.M. Allen J.M. Curr. Biol. 1998; 8: 210-221Abstract Full Text Full Text PDF PubMed Google Scholar, 8Melendez A.J. Bruetschy L. Floto R.A. Harnett M.M. Allen J.M. Blood. 2001; 98: 3421-3427Crossref PubMed Scopus (40) Google Scholar). Eicosanoids (such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4)) are important mediators of inflammation. Eicosanoids generation originates from arachidonic acid (AA), a 20-carbon, unsaturated fatty acid that is hydrolyzed from membrane phospholipids by phospholipase A2 (PLA2) (9Yang V.W. Gastroenterol. Clin. North Am. 1996; 25: 317-332Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). At present, 14 different PLA2 groups have been identified (10Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1223) Google Scholar, 11Balsinde J. Winstead M.V. Dennis E.A. FEBS Lett. 2002; 531: 2-6Crossref PubMed Scopus (407) Google Scholar). These include 10 groups of enzymes utilizing a catalytic histidine, which show millimolar requirements for Ca2+ and are collectively referred to as the secreted PLA2s (Groups I, II, III, V, IX, X, XI, XII, XIII, and XIV) (10Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1223) Google Scholar, 11Balsinde J. Winstead M.V. Dennis E.A. FEBS Lett. 2002; 531: 2-6Crossref PubMed Scopus (407) Google Scholar), and two groups of intracellular, high molecular mass enzymes, which utilize a catalytic serine (Groups IV and VI). Group IV comprise IVA, IVB and IVC PLA2, also known as cytosolic PLA2 (cPLA2α, cPLA2β, and cPLA2γ, respectively), which are highly regulated, Ca2+-dependent enzymes (10Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1223) Google Scholar, 11Balsinde J. Winstead M.V. Dennis E.A. FEBS Lett. 2002; 531: 2-6Crossref PubMed Scopus (407) Google Scholar). Whereas Group VI PLA2, or iPLA2, are Ca2+-independent enzymes (10Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1223) Google Scholar, 11Balsinde J. Winstead M.V. Dennis E.A. FEBS Lett. 2002; 531: 2-6Crossref PubMed Scopus (407) Google Scholar), also possessing a catalytic serine, yet its structure is far distant from that of the cPLA2 family. iPLA2 occurs in multiple alternative splicing variants, the majority of which are enzymatically functional (12Larsson P.K. Claesson H.E. Kennedy B.P. J. Biol. Chem. 1998; 273: 207-214Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 13Dennis E.A. Trends. Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar). Mammalian iPLA2s are classified as groups VIA and VIB, (iPLA2β and iPLA2γ, respectively) (10Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1223) Google Scholar). The best studied PLA2s 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 (14Balsinde J. Balboa M.A. Insel P.A. Dennis E.A. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 175-189Crossref PubMed Scopus (531) Google Scholar, 15Murakami M. Kudo I. J. Biochem. (Tokyo). 2002; 131: 285-292Crossref PubMed Scopus (442) Google Scholar, 16Fitzpatrick F.A. Soberman R. J. Clin. Invest. 2001; 107: 1347-1351Crossref PubMed Scopus (196) Google Scholar). iPLA2 has been shown to be implicated in many cellular functions ranging from basal fatty acid reacylation reactions (17Winstead M.W. Balsinde J. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 28-39Crossref PubMed Scopus (222) Google Scholar), to playing major roles in intracellular signaling cascades, including its involvement in agonist-induced eicosanoid production (18Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 19Mukrakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), stimulation of smooth muscle (20Jenkins C.M. Han X. Mancuso D.J. Gross R.W. J. Biol. Chem. 2002; 277: 32807-32814Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) and endothelial cells (21Seegers H.C. Gross R.W. Boyle W.A. J. Pharmacol. Exp. Ther. 2002; 302: 918-923Crossref PubMed Scopus (23) Google Scholar), in lymphocyte proliferation (22Roshak A.K. Capper E.A. Stevenson C. Eichman C. Marshall L.A. J. Biol. Chem. 2000; 275: 35692-35698Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and in endothelium-dependent vascular relaxation (21Seegers H.C. Gross R.W. Boyle W.A. J. Pharmacol. Exp. Ther. 2002; 302: 918-923Crossref PubMed Scopus (23) Google Scholar). Very recently, it has been reported that myocardial ischemia activates iPLA2β in intact myocardium, and that this iPLA2β activation is sufficient to induce malignant ventricular arrhythmias (23Mancuso D.J. Abendschein D.R. Jenkins C.M. Han X. Saffitz J.E. Schuessler R.B. Gross R.W. J. Biol. Chem. 2003; 278: 22231-22236Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and also that functional iPLA2 is required for activation of store-operated channels and capacitative Ca2+ influx in several cell types (24Smani T. Zakhalov S.I. Leno E. Csutora P. Trepakova E.S. Bolotina V.M. J. Biol. Chem. 2003; 278: 11909-11915Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Here, we demonstrate that coupling of FcγRI to AA generation and production of LTB4 and PGE2 absolutely requires iPLA2β activation. Although both intracellular forms of PLA2 (cPLA2α and iPLA2β) are present in U937 cells, only iPLA2β functionally couples FcγRI to trigger physiological responses, such as the generation of AA and the production of LTB4 and PGE2. Moreover, only iPLA2β translocates to the plasma membrane and triggers the generation of AA and eicosanoids after FcγRI activation. Furthermore, by using specific antisense oligonucleotides against iPLA2β and cPLA2α, we found that both isoforms can be activated independently by different receptors, because the addition of platelet-activating factor (PAF) triggers cPLA2α-dependent generation of AA without activating iPLA2β. Thus, these studies demonstrate that both intracellular PLA2s can be selectively regulated by different stimuli and suggest a critical role for iPLA2β in the intracellular signaling cascades initiated by immune-receptors and its functional role in the generation of key inflammatory mediators. 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)-γ (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 iPLA2β or cPLA2α. The sequences of the oligonucleotides were 5′-CAGGCGGCCAAAGAACTGCATCTT-3′ for iPLA2β and 5′-GGTAAGGATCTATAAATGACAT-3′ for cPLA2α. Cells were incubated in 1 μm oligonucleotide mixed with 20 μl of Superfect (Qiagen) for a total of 36 h (20 h prior to the addition of INFγ, and then incubated for the duration of culture with IFN-γ). Receptor Stimulation—FcγRI aggregation was carried out as described previously (6Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 7Melendez A. Floto R.A. Cameron A.J. Gillooly D.J. Harnett M.M. Allen J.M. Curr. Biol. 1998; 8: 210-221Abstract Full Text Full Text PDF PubMed Google Scholar, 8Melendez A.J. Bruetschy L. Floto R.A. Harnett M.M. Allen J.M. Blood. 2001; 98: 3421-3427Crossref PubMed Scopus (40) Google Scholar). Briefly, cells were harvested by centrifugation and then incubated at 4 °C for 45 min with 1 μm human monomeric IgG (Serotec UK) to occupy surface FcγRI 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 μm 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. For protein kinase C (PKC) inhibition, cells were pretreated for 20 min prior to receptor stimulation with 50 nm of bisindolylmaleimide I (Bis) (Sigma). For mitogen-activated protein kinase (MAPK) inhibition, cells were pretreated for 20 min prior to receptor stimulation with 10 μm of SB2038580 (Sigma). For chelating intracellular calcium, cells were preincubated with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM; Calbiochem) for 30 min at 37 °C prior to receptor stimulation. Immunoprecipitations—iPLA2β and cPLA2α were immunoprecipitated from cell lysates stimulated by FcγRI for the indicated times. Specific goat-polyclonal anti-iPLA2β or rabbit-polyclonal anti-cPLA2α (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-iPLA2β or anti-cPLA2α precipitating complexes and placed in a tumbler at 4 °C for 4 h, after which the precipitates were washed 3× 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 (6Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Briefly, immunoprecipitates were resolved on 10% polyacrylamide gels (SDS-PAGE) under denaturing conditions and then transferred to 0.45 μm 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-iPLA2β, Santa Cruz Biotechnology; or rabbit-polyclonal anti-cPLA2α, 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 (25Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Briefly, cells were labeled (2 × 106 cells/ml) with [3H]AA (1 μCi/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. FcγRI 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 [3H]AA incorporated into the cell membranes. Measurement of LTB4 Generation—LTB4 production was measured after receptor stimulation by the Biotrak™ leukotriene B4 enzyme immunoassay system from Amersham Biosciences. Briefly, the assay is based on the competition between unlabelled LTB4 and a fixed quantity of peroxidase-labeled LTB4 for a limited number of binding sites on an LTB4-specific antibody. The amount of LTB4 in the experimental sample will be inversely proportional to the signal generated by the fixed amount of peroxidase-labeled LTB4. Measurement of PGE2 Synthesis—PGE2 production was measured after receptor stimulation by the Biotrak™ PGE2 system from Amersham Biosciences. Briefly, the assay is based on the competition between unlabelled PGE2 and a fixed quantity of peroxidase-labeled PGE2 for a limited number of binding sites on a PGE2-specific antibody. The amount of PGE2 in the experimental sample will be inversely proportional to the signal generated by the fixed amount of peroxidase-labeled PGE2. 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-iPLA2β or rabbit-polyclonal anti-cPLA2α (Santa Cruz Biotechnology) and primary antibodies for 1 h at room temperature. The cells were washed with phosphate-buffered 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. iPLA2β and cPLA2α Are Both Endogenously Expressed in the Monocytes—U937 cells express cPLA2α and iPLA2β but not sPLA2 (26Clark J.D. Milona N. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7708-7712Crossref PubMed Scopus (422) Google Scholar, 27Hsu F.F. Ma Z. Wohltmann M. Bohrer A. Nowatzke W. Ramanadham S. Turk J. J. Biol. Chem. 2000; 272: 16579-16589Abstract Full Text Full Text PDF Scopus (48) Google Scholar, 28Burke J.R. Davern L.B. Gregor K.R. Todderud G. Alford J.G. Tramposch K.M. Biochim. Biophys. Acta. 1997; 1341: 223-237Crossref PubMed Scopus (35) Google Scholar). To ascertain whether the expression patterns of either cytosolic form of PLA2 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-γ) differentiated cells. Total cell-extracts from untreated or IFN-γ-U937 cells revealed that both iPLA2β and cPLA2α proteins were readily detectable. Western blot analysis revealed immunoreactive bands corresponding to the predicted molecular weights for iPLA2β and cPLA2α. The intracellular PLA2 expression profiles did not alter after IFN-γ differentiation (Fig. 1a). FcγRI Aggregation Triggers AA Release—FcγRI aggregation triggers a quick and sustained increase of AA generation over time (Fig. 1b). This FcγRI-triggered AA generation was almost completely inhibited in cells pretreated with 30 μm of methyl arachidonyl fluorophosphate (MAF), an inhibitor of cPLA2 and iPLA2 (16Fitzpatrick F.A. Soberman R. J. Clin. Invest. 2001; 107: 1347-1351Crossref PubMed Scopus (196) Google Scholar), suggesting the participation of cPLA2 and/or iPLA2 in the AA generation (Fig. 1b). To discern which of the two isoforms was activated by FcγRI, we examined the effect of E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2-H-pyran-2-one (BEL) (29Lehman J.J. Brown K.A. Ramanandham S. Turk J. Gross R.W. J. Biol. Chem. 1993; 268: 20713-20716Abstract Full Text PDF PubMed Google Scholar), a relatively selective inhibitor for iPLA2. The FcγRI-triggered AA release was inhibited in cells pretreated with 10 μm of BEL (Fig. 1c). Even though the quantity of BEL used has not been shown to inhibit cPLA2 activity (30Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar), we investigated the role of BEL in the AA release triggered by PAF, a stimulant that we knew activates cPLA2 (31McColl S.R. Krump E. Naccache P.H. Poubelle P.E. Braquet P. Braquet M. Borgeat P. J. Immunol. 1991; 146: 1204-1211PubMed Google Scholar). Although 30 μm of MAF inhibited PAF-triggered AA generation from the IFNγ-primed cells (Fig. 2a), 10 μm of BEL did not have an effect at all (Fig. 2b). Taken together, these data suggest that PAF indeed activates cPLA2, whereas FcγRI couples to iPLA2. FcγRI Aggregation Specifically Stimulates iPLA2β—To gain further proof of the nature of PLA2β, which plays a major role in the signaling pathways triggered by FcγRI, we designed specific antisense oligonucleotides against iPLA2β and cPLA2α to knockdown specifically the expression of each enzyme. We have shown previously that U937 cells are sensitive to antisense manipulation (7Melendez A. Floto R.A. Cameron A.J. Gillooly D.J. Harnett M.M. Allen J.M. Curr. Biol. 1998; 8: 210-221Abstract Full Text Full Text PDF PubMed Google Scholar, 8Melendez A.J. Bruetschy L. Floto R.A. Harnett M.M. Allen J.M. Blood. 2001; 98: 3421-3427Crossref PubMed Scopus (40) Google Scholar). IFN-γ-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 FcγRI activation either by immune complexes or with PAF (PAF was used as control). The specificity of the antisense oligonucleotides on relative PLA2 isozyme expression was checked by Western blot analysis (Fig. 3a). Thus, in cells treated with antisense to iPLA2β, there was a substantial reduction in iPLA2β immunoreactivity (80% reduction measured by densitometry), whereas cPLA2α immunoreactivity was unaffected. Conversely, in cells treated with antisense to cPLA2α, there was a reduction in cPLA2α immunoreactivity (85% reduction measured by densitometry), whereas iPLA2β immunoreactivity remained unchanged. Each antisense oligonucleotide, therefore, acted as an internal control for the other. In cells pretreated with the antisense oligonucleotide to iPLA2β, the increase in AA generation, observed after FcγRI aggregation was significantly reduced, compared with the control cells (Fig. 3b). The reduction in the increase after FcγRI activation was about 80% in cells treated with antisense iPLA2β 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 cPLA2α had no effect on the FcγRI-mediated generation of AA (Fig. 3b). Contrary to the AA generation triggered by FcγRI, AA generation stimulated by PAF was significantly reduced by about 80% in cells pretreated with the antisense oligonucleotide to cPLA2α but not in cells treated with the antisense against iPLA2β (Fig. 3c). Moreover, by immunoprecipitation and Western blotting, we found that in FcγRI-stimulated cells, iPLA2β is phosphorylated on serine residues (Fig. 4a, upper left panel), whereas cPLA2α is not phosphorylated by FcγRI engagement (Fig. 4b, top panels). Equal protein loading is shown by stripping the blots and reprobing for iPLA2β or cPLA2α (Fig. 4a, upper right panel; Fig. 4b, upper right and lower right panels). As a control for the iPLA2β immunoprecipitation, a Western blot of cell extracts depleted of iPLA2β 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-iPLA2β antibody (Fig. 4a, lower right panel). To further establish the specificity of the system, we show that PAF stimulation causes the serine-phosphorylation of cPLA2α (Fig. 4b, lower panels), whereas PAF stimulation does not cause iPLA2β phosphorylation (data not shown). Furthermore, microscopy analysis of the subcellular localization of the different intracellular PLA2 revealed that, after FcγRI aggregation, iPLA2β translocates to the plasma membrane (Fig. 4c), whereas the cytosolic localization of cPLA2α 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 FcγRI- or PAF-receptor expression (data not shown). These data strongly suggest that only iPLA2β is coupled to FcγRI activation iPLA2β Couples FcγRI to the Generation of LTB4 and PGE2—As coupling of FcγRI to arachidonic acid release requires iPLA2β activation, the role of this enzyme in coupling FcγRI to other signaling pathways, such as the production of eicosanoids, was investigated. Reduction in the expression of iPLA2β by pre-treatment of cells with the antisense oligonucleotide to iPLA2β resulted in a substantial inhibition of peak LTB4 and PGE2 observed after aggregation of FcγRI (Fig. 5, a and b, respectively). However, the antisense to cPLA2α had no effect on the FcγRI-triggered eicosanoids production (Fig. 5, a and b). To ensure that the loss of eicosanoid production after FcγRI activation in cells treated with the antisense oligonucleotide to iPLA2β was a feature of the loss of coupling of the receptor and not some direct effect of the iPLA2β antisense oligonucleotide on other members of the signaling pathways (such as cyclooxygenases), LTB4 and PGE2 were measured after activation of cells with PAF. Addition of PAF to control cells or cells treated with the antisense iPLA2 resulted in an identical increase in LTB4 and PGE2 production (Fig. 5c); on the other hand, in cells pretreated with the antisense to cPLA2α, eicosanoid production was substantially inhibited (Fig. 5d). These data indicate that, in cells pretreated with antisense oligonucleotides, the reduction in LTB4 and PGE2 after FcγRI activation reflects role of iPLA2 in the generation of eicosanoids. Role of PKC in Triggering iPLA2β after FcγRI Aggregation—It has been shown that iPLA2β activation requires PKC activity (18Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Here we show that Bis, a selective PKC inhibitor, inhibits AA generation triggered by FcγRI aggregation (Fig. 6a), whereas a MAPK inhibitor (SB203580) did not have any effect on the FcγRI-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 FcγRI-triggered stimulation of iPLA2β, and confirm that the PAF-triggered activation of cPLA2α is MAPK-dependent. To evaluate further the potential involvement of PKC activity in iPLA2β activation, we examined the effect of Bis on the FcγRI-triggered iPLA2β translocation and phosphorylation patterns. Here we also show that pretreatment of cells with Bis markedly suppressed the FcγRI-triggered iPLA2β translocation to the cell membranes (Fig. 6c). Furthermore, Bis also completely inhibited the phosphorylation of iPLA2β triggered by FcγRI (Fig. 6d). FcγRI-triggered iPLA2β Activation Is Calcium-independent—It is well established that in immune cells, antigen receptor-induced AA release is, in most cases, calcium-dependent (18Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 30Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 31McColl S.R. Krump E. Naccache P.H. Poubelle P.E. Braquet P. Braquet M. Borgeat P. J. Immunol. 1991; 146: 1204-1211PubMed Google Scholar) and even, at least in one case (where iPLA2 was indeed activated), intracellular calcium depletion prevented the generation of AA (although in this case (18Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), the authors suggested that this result was due to the inhibition of a calcium-dependent PKC). Here we show that chelating intracellular calcium with BAPTA had no significant effect on iPLA2β translocation (Fig. 7a), iPLA2β phosphorylation (Fig. 7b), or on AA release triggered by FcγRI (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 FcγRI triggers calcium-independent PKC activities (32Melendez A.J. Harnett M.M. Allen J.M. Curr. Biol. 2001; 11: 869-874Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 33Melendez A.J. Harnett M.M. Allen J.M. Immunology. 1999; 96: 457-464Crossref PubMed Scopus (30) Google Scholar) and suggest that calcium-independent PKC(s) may be involved in triggering iPLA2β after FcγRI aggregation in human monocytes. The two forms of cytosolic PLA2 (cPLA2 and iPLA2) are expressed in U937 cells (26Clark J.D. Milona N. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7708-7712Crossref PubMed Scopus (422) Google Scholar, 27Hsu F.F. Ma Z. Wohltmann M. Bohrer A. Nowatzke W. Ramanadham S. Turk J. J. Biol. Chem. 2000; 272: 16579-16589Abstract Full Text Full Text PDF Scopus (48) Google Scholar), and we found that differentiation with IFN-γ does not significantly alter the expression levels of either of the two enzymes. Our aim was to find out which PLA2 was involved in the FcγRI intracellular signaling cascades leading to the generation of eicosanoids. In this study, we demonstrated that FcγRI is functionally coupled to iPLA2β, and that this enzyme is required for FcγRI-mediated generation of arachidonic acid and the formation of leukotrienes and prostaglandins. iPLA2β contains a calmodulin (CaM)-binding domain near the C terminus which binds calcium-activated CaM and regulates enzyme activity (34Jenkins C.M. Wolf M.J. Mancuso D.J. Gross R.W. J. Biol. Chem. 2001; 276: 7129-7135Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The binding of CaM to iPLA2β results in the inhibition of iPLA2β activity, which is reversible through the removal of Ca+2, and subsequent dissociation of CaM from the C terminus of iPLA2β (34Jenkins C.M. Wolf M.J. Mancuso D.J. Gross R.W. J. Biol. Chem. 2001; 276: 7129-7135Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Thus, in some models, it is possible for iPLA2β to be regulated through alterations in cellular calcium ion homeostasis and become activated after dissociation from its complex with Ca+2/CaM when intracellular calcium stores are depleted (e.g. by sarco/endoplasmic reticulum calcium ATPase inhibitors, calcium-ionophores, or agonist stimulation; ref. 35Wolf M.J. Wang J. Turk J. Gross R.W. J. Biol. Chem. 1997; 272: 1522-1526Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Here we report that the FcγRI-triggered AA generation was almost completely inhibited in cells pretreated with MAF, an inhibitor of both cPLA2 and iPLA2 (36Lio Y.C. Reynolds L.J. Balsinde J. Dennis E.A. Biochim. Biophys. Acta. 1996; 1302: 55-60Crossref PubMed Scopus (15) Google Scholar), suggesting the participation of cPLA2 and/or iPLA2 in the AA generation. To discern which of the two isoforms was activated by FcγRI, we examined the effect of BEL, a relatively selective inhibitor for iPLA2 (29Lehman J.J. Brown K.A. Ramanandham S. Turk J. Gross R.W. J. Biol. Chem. 1993; 268: 20713-20716Abstract Full Text PDF PubMed Google Scholar). The FcγRI-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 cPLA2 (31McColl S.R. Krump E. Naccache P.H. Poubelle P.E. Braquet P. Braquet M. Borgeat P. J. Immunol. 1991; 146: 1204-1211PubMed Google Scholar). 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 iPLA2 was the enzyme involved in the FcγRI-triggered AA release. To be more specific, we designed antisense oligonucleotides against iPLA2β and cPLA2α to selectively down-modulate the protein levels of these enzymes. Our data show that the iPLA2β antisense substantially decreased AA release and LTB4 and PGE2 generation induced by FcγRI aggregation, whereas the antisense against cPLA2α had no effect on the FcγRI pathway. Moreover, the antisense to iPLA2β did not affect PAF-induced AA release or LTB4 and PGE2 generation, whereas the antisense against cPLA2α 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 FcγRI triggers the serine phosphorylation and membrane translocation of iPLA2β but not of cPLA2α. We found that the selective PKC inhibitor Bis substantially decreased the FcγRI-triggered 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 FcγRI triggers iPLA2β activation by means of PKC, whereas PAF-triggers cPLA2α via the activation of MAP-kinases. Moreover, the PKC inhibitor also blocked iPLA2β translocation to the cell periphery and completely blocked the phosphorylation of iPLA2β that follows FcγRI aggregation. Taking these data together, we suggest that PKC is involved in triggering the activation of iPLA2β in the FcγRI signaling cascade by phosphorylating and thus promoting the translocation of iPLA2β to the cell's plasma membrane. Different stimuli induce AA release in monocytes and macrophages in a Ca+2-dependent and phosphorylation-dependent manner because of the activation of cPLA2 (37Qui Z.-H. Gijón M.A. de Carvalho M.S. Spencer D.M. Leslie C.C. J. Biol. Chem. 1998; 273: 8203-8211Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 38Lennartz M.R. Int. J. Biochem. Cell Biol. 1999; 31: 415-430Crossref PubMed Scopus (89) Google Scholar). However, PGE2 generation by zymosan-stimulated macrophages is significantly attenuated by BEL or iPLA2β antisense (30Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Paradoxically, in these cells, iPLA2β activation seems to be regulated by protein kinase C and is Ca+2-dependent, although in this case, the authors (18Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) suggested that this result was due to a calcium-dependent PKC, which, in turn, activated iPLA2β. In contrast, other studies have shown ligand-stimulated eicosanoid production in cells that have been treated with calcium chelators such as BAPTA and EDTA (35Wolf M.J. Wang J. Turk J. Gross R.W. J. Biol. Chem. 1997; 272: 1522-1526Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In agreement with the latter, we show here that chelating intracellular calcium with BAPTA had no significant effect on iPLA2β translocation, phosphorylation, or AA release triggered by FcγRI, whereas the same BAPTA-AM treatment completely blocked the PAF-induced AA release. Based upon the effects of BEL, it has been suggested for many years that iPLA2 mediates AA in different cells stimulated by various agonists (29Lehman J.J. Brown K.A. Ramanandham S. Turk J. Gross R.W. J. Biol. Chem. 1993; 268: 20713-20716Abstract Full Text PDF PubMed Google Scholar, 39Ramanadham S. Gross R.W. Han X. Turk J. Biochemistry. 1993; 32: 337-346Crossref PubMed Scopus (123) Google Scholar, 40Gross R.W. Rudolph A.E. Wang J. Sommers C.D. Wolf M.J. J. Biol. Chem. 1995; 270: 14855-14858Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 41Lennartz M.R. Lefkowith J.B. Bromley F.A. Brown E.J. J. Leukocyte Biol. 1993; 54: 389-398Crossref PubMed Scopus (40) Google Scholar, 42Karimi K. Lennartz M.R. J. Immunol. 1995; 155: 5786-5794PubMed Google Scholar), including during IgG-mediated phagocytosis of human monocytes, where AA release was shown to be triggered in a calcium-independent manner (41Lennartz M.R. Lefkowith J.B. Bromley F.A. Brown E.J. J. Leukocyte Biol. 1993; 54: 389-398Crossref PubMed Scopus (40) Google Scholar, 42Karimi K. Lennartz M.R. J. Immunol. 1995; 155: 5786-5794PubMed Google Scholar). For iPLA2β, several important signaling functions have been suggested, including its role in agonist-induced stimulation of smooth muscle (20Jenkins C.M. Han X. Mancuso D.J. Gross R.W. J. Biol. Chem. 2002; 277: 32807-32814Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) and endothelial cells (21Seegers H.C. Gross R.W. Boyle W.A. J. Pharmacol. Exp. Ther. 2002; 302: 918-923Crossref PubMed Scopus (23) Google Scholar), in lymphocyte proliferation (22Roshak A.K. Capper E.A. Stevenson C. Eichman C. Marshall L.A. J. Biol. Chem. 2000; 275: 35692-35698Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and in endothelium-dependent vascular relaxation (21Seegers H.C. Gross R.W. Boyle W.A. J. Pharmacol. Exp. Ther. 2002; 302: 918-923Crossref PubMed Scopus (23) Google Scholar). Very recently, it was reported that myocardial ischemia activates iPLA2β in intact myocardium, and that iPLA2β activation is sufficient to induce malignant ventricular arrhythmias (23Mancuso D.J. Abendschein D.R. Jenkins C.M. Han X. Saffitz J.E. Schuessler R.B. Gross R.W. J. Biol. Chem. 2003; 278: 22231-22236Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Another recent study shows that functional iPLA2β is required for activation of store-operated channels and capacitative Ca2+ influx in several cell types (24Smani T. Zakhalov S.I. Leno E. Csutora P. Trepakova E.S. Bolotina V.M. J. Biol. Chem. 2003; 278: 11909-11915Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). We show here that in a human monocytic cell line, iPLA2β plays a critical role in the intracellular signaling cascades initiated by the high affinity receptor for IgG (FcγRI) 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 iPLA2β as a potential therapeutic candidate for treating human conditions ranging from ischemia to antigen-mediated inflammatory diseases. We thank A.-K. Fraser-Andrews for editing the manuscript.