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J. Biol. Chem., Vol. 275, Issue 46, 35692-35698, November 17, 2000

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Human Calcium-independent Phospholipase A₂ Mediates Lymphocyte Proliferation^*

Amy K. Roshak, Elizabeth A. Capper, Christopher Stevenson, Christopher Eichman, and Lisa A. Marshall

From the Department of Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

Received for publication, March 16, 2000, and in revised form, August 25, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activation of lymphocytes induces blastogenesis and cell division which is accompanied by membrane lipid metabolism such as increased fatty acid turnover. To date little is known about the enzymatic mechanism(s) regulating this process. Release of fatty acids such as arachidonic acid requires sn-2-deacylation catalyzed by a class of enzymes known as phospholipases A₂ (PLA₂, EC 3.1.1.4). Herein, we confirm that human peripheral blood B or T lymphocytes (PBL) do not possess measurable levels of 85-kDa PLA₂ as assessed by Western immunoblot. Low levels of 14-kDa PLA₂ protein and activity were detectable in the particulate fraction of PBL and Jurkat cells. Western immunoblot analysis indicates that PBLs possess the calcium-independent PLA₂ (iPLA₂) protein. Calcium-independent sn-2-acylhydrolytic activity was measurable in PBL cytosols and could be inhibited by the selective iPLA₂ inhibitor bromoenol lactone. Mitogen activation of PBLs resulted in maintenance of activity levels which remained constant over 72 h suggesting an important role for iPLA₂ in this proliferative process. Indeed, evaluation of iPLA₂ activity in cell cycle-arrested Jurkat T cell fractions revealed the highest iPLA₂ levels occurring at the G₂/M phase. Addition of the iPLA₂ inhibitors, bromoenol lactone, or arachidonyl trifluoromethyl ketone (AAOCF₃), inhibited both mitogen-induced PBL as well as Jurkat T cell proliferation. Moreover, specific depletion of iPLA₂ protein by antisense treatment also resulted in marked suppression of cell division. Inhibition of Jurkat cell proliferation was not associated with arrest at a particular phase of the cell cycle nor was it associated with apoptosis as assessed by flow cytometry. These findings provide the first evidence that iPLA₂ plays a key role in the lymphocyte proliferative response.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activation of resting lymphocytes occurs by ligation of antigen-specific co-receptors or artificially through mitogen stimulation. This in turn results in the formation of blast cells which undergo cellular proliferation, differentiation, and clonal expansion. Preparation for cell division requires not only synthesis of nucleotide pools and new protein but also increases in phospholipid (PL)¹ content and remodeling of existing PL in support of new membrane formation (1). Several studies have demonstrated that changes in membrane PL (1, 2) and arachidonic acid (AA) metabolism (3, 4) occur during the cell cycle. More specifically, mitogen activation of lymphocytes is accompanied by an increase in fatty acid turnover and an enrichment of PL with AA, the major substrate of proinflammatory eicosanoids (3-6). The AA comes from internal pools since the enrichment occurs in serum-free conditions (3, 4). The fact that this phenomenon, requiring deacylation-reacylation of PL, is AA specific and targeted toward selected PL classes (e.g. phosphatidylethanolamine and phosphatidylinositol) suggests regulation through specific enzyme systems (4). Indeed, the lysophosphatide acyltransferase, a reacylating enzyme, preferentially incorporates polyunsaturated fatty acid (i.e. AA) into membrane PL. The molecular mechanism regulating these events is not fully elucidated. It is conceivable that the family of enzymes which catalyze the acylhydrolysis of fatty acid from the sn-2 position of membrane PL, namely the phospholipase(s) A₂ (PLA₂) would be important.

The two best characterized mammalian PLA₂ enzymes, are the calcium-dependent 14-kDa PLA₂ (types IIa, V and X) and 85-kDa PLA₂ (type IV) (7). The 14-kDa PLA₂(s) exist as extracellular forms in inflammatory fluids (8, 9) and in a cell-associated form (10-13) and requires calcium for catalysis. The cytosolic 85-kDa PLA₂ is structurally distinct and unlike the 14-kDa PLA₂(s) exhibits a preference for AA in the sn-2 position of PL and is regulated by physiological intracellular Ca²⁺ concentrations and phosphorylation (14-16). We and others have shown that both the 14- and 85-kDa enzymes are induced by inflammatory cytokines and growth factors (17-20) and that both enzymes influence cellular AA release and subsequent eicosanoid production in a variety of cell types (17, 21-24). More recently, a calcium-independent PLA₂ (iPLA₂) has been cloned from Chinese hamster ovary cells, murine P388D1 macrophages, and a human B cell line (25-27). Although little data exists regarding its function, antisense depletion of the murine iPLA₂ caused inhibition of AA incorporation into membrane PL with or without stimulation but did not affect receptor-coupled arachidonate mobilization suggesting a housekeeping role for this enzyme in basal PL remodeling in P388D1 macrophages (28). Conversely, studies by Ramanadham and co-workers (29) demonstrated the lack of a role for iPLA₂ in islet cell remodeling.

The potential contribution of these distinct enzymes in regulating cellular proliferation has only begun to be examined. The prevalence of these three enzymes in B or T lymphocyte populations is still under investigation and their potential involvement in lymphocyte proliferative processes is not known. Herein, we assess the presence of the PLA₂ isoforms in purified human monocytes, B cells and T cells and/or in Jurkat T cells through enzymatic assays, ELISA, and Western analysis. We demonstrate the presence of iPLA₂ in peripheral blood B and T cells and the Jurkat T leukemia cell line. We then evaluate both the effects of isoform selective PLA₂ inhibitors as well as antisense technology on Jurkat and mitogen-induced (phytohemagglutinin; PHA) PBL proliferation and cell cycle progression. Both iPLA₂ inhibition and depletion of iPLA₂ protein using antisense demonstrates that this enzyme plays a key role in lymphocyte proliferation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Isolation and Culture

Mixed Lymphocyte Isolation-- Human monocyte-depleted peripheral blood lymphocytes were isolated from whole blood as described previously (30). The resultant population was greater than 85% pure lymphocytes as assessed by differential staining. Cells were resuspended to 2 × 10⁶/ml and cultured in RPMI 1640 with 10% fetal bovine serum unless otherwise noted.

Purification of Human Monocytes, B or T Lymphocytes-- Human peripheral blood leukocytes (buffy coat) were isolated from whole blood as described previously and a greater than 95% pure population of human monocytes was obtained by separation over a Ficoll gradient followed by adherence (2 h) as described previously (12, 23). Purified B cells from human spleen were isolated through positive immunomagnetic selection using magnetic polystyrene beads coated with monoclonal antibody to the B cell-restricted membrane antigen, CD19 (Dynabeads M-450 Pan-B (CD19), Dynal Lake Success, NY) as described previously (30). Purified human peripheral blood T lymphocytes were obtained by separation over T cell enrichment columns, HTCC (R & D Systems, Minneapolis, MN), as per the manufacturer's instructions. The Jurkat T cell leukemia was acquired from American Type Culture Collection (Rockville, MD). The cells were maintained at a density of 1 to 5 × 10⁵ cells/ml and grown as suspension cultures in RPMI 1640 with 2 mM L-glutamine, 1.5 g/liter NaHCO₃, and 10% fetal bovine serum.

Chemical Cell Cycle Arrest and FACS Analysis of Jurkat T Lymphocytes

Cells were chemically arrested at G₁/S, S, or G₂/M using established protocols (31). Jurkat cells at 5 × 10⁵/ml were treated with either 3 mM hydroxyurea for 16 h to give a G₁/S phase arrest or 0.1 µg/ml nocodazole for 12 h to achieve a G₂/M phase arrest. In addition, cells were also treated with 2 mM thymidine for 17 h, released for 6 h in culture media (no thymidine), then retreated with 2 mM thymidine for 15 h to allow for a broad S-phase arrest. The specific cell cycle phase arrest of the individual populations was verified by measuring the fluorescence intensities using a Becton-Dickinson FACSTAR Plus. Briefly, following the treatments, the cells were centrifuged, the media was removed, and the cells were resuspended in 750 µl of ice-cold phosphate-buffered saline. To fix the cells, 2 ml of 95% ethanol (20 °C) was added and cells were then washed twice with 2 ml of phosphate-buffered saline and resuspended in 1 ml of a solution containing 250 µg/ml propidium iodide and 250 µg/ml RNase A. The stained cells were vortexed, then incubated at room temperature for 1 h in darkness prior to FACS evaluation of cell cycle phase. Forward scatter versus side light scatter and fluorescent area versus width were used to gate for intact, single cells. Results were based on 20,000 gated cell events.

Subcellular Fractionation

Cell pellets were resuspended to 1 × 10⁸ cells/0.5 ml in homogenization buffer containing 0.34 M sucrose, 10 mM HEPES, pH 7.4, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 200 µM leupeptin, 20 µg/ml soybean trypsin inhibitor, and 20 µg/ml aprotinin at 4 °C. The cell suspension was disrupted on ice by sonication (5 s) with a Bransonic probe tip and the homogenate was centrifuged at 400 × g for 10 min at 4 °C to remove unbroken cells and debris. The resulting supernatant fraction was centrifuged at 100,000 × g for 60 min at 4 °C to obtain a supernatant (cytosol) and particulate fraction. The particulate fraction was resuspended in homogenization buffer and both fractions were flash frozen with liquid N₂ and stored at 80 °C until analysis.

ELISA Analysis of Type II 14-kDa PLA₂

Cell fractions were assessed for 14-kDa PLA₂ mass using a specific enzyme-linked immunosorbent assay as described previously (22). This ELISA recognizes both the Type IIa and Type V 14-kDa PLA₂ but not the Type X 14-kDa PLA₂.

Immunoblot Analysis

Cell cytosols (50-100 µg of protein) were analyzed by SDS-polyacrylamide gel electrophoresis (10% Tris glycine gels, Bio-Rad). Proteins were transferred to nitrocellulose paper, incubated with either rabbit anti-rh 85-kDa PLA₂ antiserum (1:500) or rabbit anti-iPLA₂ antiserum (1:500; Caymen Chemical) and then incubated with donkey anti-rabbit IgG conjugated to horseradish peroxidase (1:3000; Roche Molecular Biochemicals, Indianapolis, IN). Detection of immunoreactive bands was carried out using the ECL Western blotting system (Amersham Pharmacia Biotech). Rabbit polyclonal antiserum against the rh 85-kDa PLA₂ was prepared as described previously (23).

Phospholipase A₂ Enzyme Assays

Calcium-dependent PLA₂ activity was routinely measured by the acylhydrolysis of [³H]AA Escherichia coli as described previously (12). Briefly, the reaction mixture (50 µl total volume) contained 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl₂, and 100 µM [³H]AA-labeled E. coli (5 nmol of lipid phosphorus (P_i) per assay. The assay was initiated by addition of substrate and assays were incubated at 37 °C for a time predetermined to be on the linear portion of a time versus hydrolysis plot. Calcium-independent PLA₂ activity was measured by the acylhydrolysis of a Triton X-100 (400 µM), 1-palmitoyl-2-[1-¹⁴C]arachidonyl-sn-glycero-3-phosphorylcholine (50 µM; 56 Ci/mol; Amersham Pharmacia Biotech) mixed micelle according to published methods (26). In addition to the substrate, the reaction mixture contained 100 µg of cell fraction, 100 mM HEPES, pH 7.5, 5 mM EDTA, and 1 mM ATP. The reaction was initiated by the addition of substrate and allowed to proceed at 37 °C for 60 min. Dimethyl sulfoxide (Me₂SO) vehicle or drug was added as no greater than 10% of the total assay volume. All drugs or vehicle were added 10 min prior to substrate addition unless otherwise stated. Both reactions were terminated by the addition of 1.0 ml of tetrahydrofuran. Free fatty acid was exclusively separated by elution of the sample over aminopropyl solid phase silica columns with tetrahydrofuran/acetic acid (49:1) and quantitated by liquid scintillation counting as described previously (12).

Cellular Proliferation

Human PBL or Jurkat T cells were resuspended to 2 × 10⁶ cells/ml in RPMI 1640 with 10% fetal bovine serum and 200 µl of the suspension was added to each well of a Nunc 96-well cell culture plate. Drug or Me₂SO vehicle (0.05%) was added to cells for 15 min at 27 °C prior to the addition of PHA (30 µg/ml for PBL cultures) and incubation for 72 h at 37 °C in a 5% CO₂ incubator. [³H]Thymidine (PerkinElmer Life Sciences, Wilmington, DE) was diluted in RPMI 1640 and 0.1 µCi/well was added to the cells for 6 h prior to harvesting. Plates were harvested using a Packard Filtermate 196 cell harvester onto Packard Unifilter GF/C filter plates. Scintillation fluid was added and filters were quantitated using a Packard Topcount Microplate counter. For antisense studies, PBLs were exposed to initiation site-directed antisense phosphorothioate oligonucleotides SB8603 (5'-aaggcgtccgaactgcat-3') or SB 8604 (5'-aaggcggccaaggaactgcat-3'), a scrambled control oligonucleotide, SB8606 (5'-cggggaggacgctagacgatc-3'), an unrelated control oligonucleotide SB9030 (tccgaaggcagaaaggcttca-3') or LipofectAMINE vehicle alone (5 µg/ml) for 24 h at 37 °C prior to stimulation with PHA (30 µg/ml) for an additional 72 h. Cell viability was monitored using either trypan blue exclusion or the Cytotox 96 Assay System (Promega, Madison, WI) for all studies.

Apoptosis Measurement

Apoptosis was measured by TUNEL (32) using the Promega ApopTag kit (Madison, WI) as per the manufacturer's directions. In brief, the enzyme terminal deoxynucleotidyl transferase extends the DNA fragments with digoxigenin-containing nucleotides, which are then detected with a anti-digoxigenin antibody carrying fluorescein to allow detection by fluorescence (494 nm excitation, 523 nm emission). Propidium iodide was used as a counterstain to measure total DNA content and determine distribution of cells in G₀/G₁, S, and G₂/M phases of the cell cycle. Flow cytometric analysis was performed on a Becton-Dickinson FACScan instrument using CellQuest software.

Protein Determination

All protein concentrations were determined by Bradford protein analysis kits (Bio-Rad).

Calculations and Statistics

Data are expressed as mean ± S.D. of triplicate determinations unless otherwise stated. All experiments were conducted 2-4 times using cells obtained from different donors.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Evaluation of 14-kDa, 85-kDa, and Calcium-independent PLA₂ Isoform Protein Levels in Human Monocytes, B Lymphocytes, and T Lymphocytes-- We have previously demonstrated the presence of 14-kDa PLA₂ protein in isolated human monocytes (22). Human PBL cytosolic and particulate fractions were evaluated for 14-kDa PLA₂ by ELISA. Particulate fractions contained low but detectable levels of 14-kDa PLA₂ (1.4 ng of 14-kDa PLA₂/300 µg of total PBL particulate protein) while no immunoreactive protein was detectable in the cytosolic fraction. Jurkat T cell particulate fractions contained 0.5 µg of 14-kDa PLA₂/300 µg of total particulate protein. RT-PCR analysis of Jurkat RNA for type IIa and type V 14-kDa PLA₂ indicated the existence of only the type V isoform (data not shown).

85-kDa PLA₂ and iPLA₂ levels were evaluated in the subcellular fractions of purified CD19⁺ B lymphocytes, CD4⁺ T lymphocytes, and peripheral blood monocytes as described under "Experimental Procedures." The cytosolic fractions (100 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted using either a rabbit anti-human 85-kDa PLA₂ polyclonal antisera or a rabbit anti-hamster iPLA₂ polyclonal antisera which cross-reacts with human. Fig. 1A shows a Western analysis of 85-kDa PLA₂ protein levels where a 110-kDa immunoreactive band is clearly present in human monocytes as has been previously described (23). However, an appropriately sized immunoreactive protein is not detectable in purified T or B lymphocytes. Calcium-independent PLA₂ immunoreactive protein (~80-85 kDa) was present in the cytosolic fraction of both purified peripheral blood B and T cells but it was not detectable in human monocytes cytosols (Fig. 1B).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Immunoblot analysis of 85-kDa PLA2 or iPLA2 protein levels in purified human monocytes, B lymphocytes, and T lymphocytes. Cytosolic fraction (100 µg) from monocytes (lane 1), human spleen B cells (lane 2), and peripheral blood T cells (lane 3) were prepared as described under "Experimental Procedures," electrophoresed through a 10% SDS-polyacrylamide gel and subjected to Western analysis using rabbit anti-rh 85-kDa PLA2 polyclonal antisera (panel A) or rabbit anti-hamster iPLA2 polyclonal antisera (panel B).

Evaluation of PLA₂ Acylhydrolytic Activity in PBL and Jurkat T Cells-- PBL were next examined for calcium-independent sn-2 acylhydrolytic activity using a Triton X-100 phosphatidylcholine-mixed micelle substrate as has been previously described (25, 26). Cells were incubated for 0-72 h in the absence or presence of the T cell-specific mitogen, PHA (30 µg/ml), prior to preparation of subcellular fractions. Fig. 2A shows that freshly isolated cells possessed 55 ± 0.1 pmol/min/mg calcium-independent sn-2 acylhydrolytic activity. Activity of cultured cells remained the same after 24 h of culture (58 ± 2.0 pmol/min/mg) but then decreased by half after 72 h (26 ± 3.0 pmol/min/mg). Cells exposed to PHA for 24 h (73 ± 15.0 pmol/min/mg) or 72 h (77 ± 19 pmol/min/mg) possessed levels of activity which were not statistically different from those of the freshly isolated cells. Western analysis indicates that the reduction in activity after 72 h in unstimulated cells was due to a significant decrease in iPLA₂ protein, while protein was clearly evident in cells exposed to PHA for 72 h (Fig. 2B). In a second study, calcium-independent activity was measured in the cytosol and was shown to be completely inhibited by addition of the iPLA₂ inhibitor, bromoenol lactone (BEL), (control, 113 ± 10.0 pmol/min/mg; 10 µM BEL, 0.5 ± 0.2 pmol/min/mg).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Evaluation of calcium-independent sn-2 acylhydrolytic activity and protein levels in human PHA-stimulated PBL. Human PBL were incubated in the absence or presence of PHA (30 µg/ml) for various times (0-72 h) prior to preparation of subcellular fractions as described under "Experimental Procedures." Panel A, calcium-independent sn-2 acylhydrolytic activity in cytosolic fractions (100 µg of protein/37 °C for 60 min) was measured via hydrolysis of a Triton X-100 (400 µM)/1-palmitoyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphorylcholine (50 µM; 56 Ci/mol) mixed micelle. Panel B, cytosolic fractions (100 µg) from 72-h samples were electrophoresed through a 10% SDS-polyacrylamide gel and subjected to Western analysis using rabbit anti-hamster iPLA2 polyclonal antisera.

The Jurkat cell line is a transformed, immortalized T cell line and as such does not require activation to induce proliferation. RT-PCR analysis of Jurkat mRNA was first performed and indicated the presence of iPLA₂ message (data not shown). These cells were chemically synchronized at G₁/S, S, or G₂/M phase of the cell cycle, collected, and fractionated as described under "Experimental Procedures." The fractions were analyzed for either calcium-dependent (14-kDa PLA₂) or -independent (iPLA₂) activity. Recombinant human (rh) type II 14-kDa PLA₂ was used as an assay control and gave a robust signal of 29 µmol/mg/min [³H]AA E. coli hydrolyzed. Consistent with ELISA and RT-PCR data, calcium-dependent sn-2 acylhydrolytic activity was detectable in the particulate fraction of control asynchronous cells (100 pmol/mg/h [³H]AA E. coli hydrolyzed). Analysis of cell cycle phase arrested particulate fractions showed 250, 125, and 200 pmol/mg/h [³H]AA E. coli hydrolzyed in the G₁/S, S, and G₂/M phase fractions, respectively (S.E. 25-50 pmol/mg/h; n = 3). Fig. 3 shows the calcium-independent sn-2 acylhydrolysis measured in the cytosolic fraction of cell cycle phase-arrested Jurkats. As expected, the calcium-dependent rh 14-kDa PLA₂ showed no activity in this assay and served as a negative control. Asynchronous cells possessed measurable calcium independent activity (46 pmol/mg/h 1-palmitoyl-2-[1-¹⁴C]arachidonyl-sn-glycero-3-phosphorylcholine mixed micelle hydrolyzed). Enzymatic activity was not significantly different when assessed using the cytosols of cells arrested in G₁/S or S (35 and 65 pmol/mg/h). However, the cytosols of G₂/M-arrested cells exhibited 3-4-fold greater calcium independent PLA₂ activity (168 pmol/mg/h).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Evaluation of calcium-independent sn-2 acylhydrolytic activity in cell cycle phase arrested Jurkat T cells. Jurkat T cells were chemically synchronized at G1/S, S, or G2/M phase and subcellular fractions were prepared, and cytosolic fractions (100 µg/assay) were assayed as described under "Experimental Procedures." Data represent mean ± S.D., n = 3, one representative of three studies.

Effect of Phospholipase A₂ Inhibitors on Jurkat T Cell and PHA-induced Human Lymphocyte Proliferation-- The transition state inhibitor, arachidonyl trifluoromethyl ketone, AAOCF₃ (IC₅₀ versus recombinant human 85-kDa PLA₂, 100 nM; IC₅₀ versus semi-purified iPLA₂, 15 µM (22, 33)), the iPLA₂ inhibitor, BEL (IC₅₀ P388D iPLA₂; 60 nM (33)), and the 14-kDa PLA₂ selective inhibitor SB203347 (IC₅₀ versus rh 14-kDa PLA₂, 500 nM (34)) were evaluated for their effect on PHA-induced human PBL proliferation. Isolated PBL were exposed to increasing concentrations of AAOCF₃, BEL, or SB203347 (10-30 µM) or Me₂SO vehicle alone (0.05%) for 15 min prior to the addition of PHA (30 µg/ml) for 72 h. Proliferation was measured by [³H]thymidine incorporation as described under "Experimental Procedures." Fig. 4 shows one representative of three studies where treatment with either AAOCF₃ or BEL caused a concentration-dependent inhibition of PHA-induced proliferation. Pretreatment with the 14-kDa PLA₂ inhibitor, SB203347, however, had no effect on lymphocyte proliferation induced by PHA.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   iPLA2 inhibitors, AAOCF3, and BEL decrease PHA-induced human PBL proliferation. Human PBL (2 × 106/ml) were treated with increasing amounts of AAOCF3, BEL, or SB203347 (10-30 µM) or with Me2SO vehicle (0.05%) alone for 15 min at room temperature prior to a 72-h stimulation with PHA (30 µg/ml) (unstimulated vehicle control, 263 cpm; PHA vehicle control, 21173 cpm). [3H]Thymidine (0.1 µCi/well) was added 6 h prior to harvesting and liquid scintillation counting as described under "Experimental Procedures." * indicates significant difference from control values at p < 0.05. Data represent mean ± S.D., n = 3, of one representative of three studies.

To further delineate the mechanism of PLA₂ action in PHA-induced PBL proliferation, cells were cultured in the presence of AAOCF₃ (15 µM), BEL (15 µM), or indomethacin (1 µM) in the presence or absence of exogenous AA (20 µM). Fig. 5 confirms the inhibition of proliferation by AAOCF₃ and BEL. However, treatment with the cycloxygenase inhibitor, indomethacin, did not significantly effect tritiated thymidine uptake in the presence or absence of exogenous AA (20 µM), suggesting the antiproliferative effect of the PLA₂ inhibitors is likely not due to alterations in prostanoid production in the cell culture. Similarly, addition of exogenous AA did not reverse the inhibition induced by AAOCF₃ or BEL.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Neither indomethacin nor fatty acids effect PHA-induced human PBL proliferation. Human PBL (2 × 106/ml) were exposed to Me2SO vehicle alone (0.05%) or AAOCF3 (15 µM), BEL (15 µM), or indomethacin (1 µM) in the presence or absence of AA (20 µM) for 15 min at room temperature. PHA (30 µg/ml) was added to the cultures and incubation proceeded for 72 h. [3H]Thymidine (0.1 µCi/well) was added 6 h prior to harvesting and liquid scintillation counting as described under "Experimental Procedures." * indicates significant difference from control values at p < 0.05. Data represent mean ± S.D., n = 3, of one representative of two studies.

Effect of PLA₂ Inhibitors on Jurkat T Cell Proliferation-- Jurkat T cells were cultured in the presence of varying concentrations of SB203347 (3, 15 µM), BEL (3, 15 µM), or AAOCF₃ (3, 15 µM) for 66 h prior to addition of [³H]thymidine and further incubation for 6 h. Plates were harvested at 72 h and proliferation was assessed as described under "Experimental Procedures." Fig. 6A shows that exposure to AAOCF₃ and BEL caused a concentration-dependent decrease in Jurkat T cell [³H]thymidine incorporation while treatment with the 14-kDa PLA₂ inhibitor, SB203347, had no significant effect compared with untreated control cells. Flow cytometric analysis using propidium iodide and TUNEL staining indicated that inhibition of proliferation by either BEL or AAOCF₃ was not associated with an arrest at a specific cell cycle phase nor was it accompanied by apoptosis over 72 h (data not shown). Cytosolic fractions of Jurkat T cells arrested in G₂/M were prepared as described under "Experimental Procedures" as they have previously been shown to possess the highest amount of iPLA₂ activity (Fig. 3). This fraction was incubated with or without 10 µM BEL for 15 min, prior to evaluation of iPLA₂ enzymatic activity. BEL inhibited activity in this fraction by 50% (Fig. 6B)

View larger version (22K): [in this window] [in a new window]   Fig. 6.   iPLA2 inhibitors block Jurkat T cell proliferation. Panel A, Jurkat T cells (2 × 106/ml) were treated with varying amounts of AAOCF3, BEL, or SB 203347 (3, 15 µM) or with Me2SO vehicle (0.05%) alone for 15 min at room temperature prior to a 72-h incubation. [3H]Thymidine (0.1 µCi/well) was added 6 h prior to harvesting and liquid scintillation counting as described under "Experimental Procedures." * indicates significant difference from control values at p < 0.05. Data represent mean ± S.D., n = 3, of one representative of three studies. Panel B, Jurkat cells were arrested in G2/M, fractionated, and analyzed for iPLA2 activity in the presence or absence of BEL (10 µM) as described under "Experimental Procedures."

Effect of iPLA₂ Antisense on PHA-induced PBL Proliferation-- An antisense phosphorothioate oligonucleotide directed against the initiation site of the human iPLA₂ was synthesized in order to directly evaluate the contribution of the enzyme in PHA-induced PBL proliferation. Fig. 7A shows a Western blot analysis of cytosols (50 µg/lane) from PBL treated with either Lipofectin vehicle alone, SB8603 (3 µM), SB8604 (3 µM), or SB9030 (3 µM) for 24 h prior to addition of PHA and incubation for an additional 72 h. Treatment with PHA maintained iPLA₂ protein levels which is consistent with increased activity observed in Fig. 2 using cells from a different donor. Addition of either antisense SB8603 or SB8604 but not the control oligonucleotide, SB9030, caused a marked reduction in the level of iPLA₂ immunoreactive protein when compared with the PHA-stimulated control cytosols. PBL were pretreated with either antisense oligonucleotides SB8603 or SB8604 (0.3 or 3 µM), the control oligonucleotide, SB9030 (0.3 or 3 µM), or Lipofectin vehicle alone (5 µg/ml) for 24 h prior to exposure to PHA (30 µg/ml). Cells were pulsed with [³H]thymidine (0.1 µCi) at 66 h and thymidine incorporation was quantitated at 72 h. Fig. 7B shows that pretreatment with either SB8603 or SB8604 caused a significant concentration-dependent inhibition of the PHA induced PBL proliferative response. In contrast, exposure to the control oligonucleotide, SB9030, did not effect thymidine incorporation over 72 h when compared with the vehicle control. Similar results were obtained in a second study (unstimulated Lipofectin control: 2,137 ± 517 cpm; PHA stimulated Lipofectin control: 35,725 ± 2,199 cpm; PHA + SB 8604 (3 µM): 23,453 ± 225 cpm (34%); PHA + SB8603 (3 µM): 24,083 ± 249 cpm (- 33%); PHA + SB9030 (3 µM): 34,950 ± 274 cpm; n = 3).

View larger version (35K): [in this window] [in a new window]   Fig. 7.   Treatment with iPLA2 antisense blocks PHA-induced human lymphocyte proliferation. Panel A, shows a Western blot analysis of PBL cytosols (50 µg) from cells treated with either Lipofectin vehicle alone (5 µg/ml; lanes 1 and 2), SB8603 (3 µM: lane 3), SB8604 (3 µM: lane 4), or SB9030 (3 µM: lane 5) for 24 h prior to addition of PHA and incubation for an additional 72 h (lanes 2-5). Panel B, human PBL (2 × 106/ml) were exposed to antisense SB8603, SB8604, or control oligonucleotide SB9030 (0.3 or 3.0 µM) or Lipofectin alone (5 µg/ml) for 24 h. Cells were then exposed to PHA (30 µg/ml) for 72 h. [3H]Thymidine (0.1 µCi/well) was added 6 h prior to harvesting and liquid scintillation counting as described under "Experimental Procedures." * indicates significant difference from control values at p < 0.05. Data represent mean ± S.D., n = 3, of one representative of three studies.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lipid metabolism is an integral process of immune cell blast formation and proliferation. In the present study, we show that both freshly isolated T and B lymphocytes and cell lines (e.g. Jurkat T leukemia cells) possess low levels of Ca²⁺-dependent PLA₂ activity but are devoid of 85-kDa PLA₂ protein. Moreover, we show for the first time, that these three cell types possess Ca²⁺-independent PLA₂ protein and activity and demonstrate that it is this enzyme which appears to be critical for lymphocyte proliferation.

Consistent with recent reports, Western analysis of peripheral blood T and B lymphocyte cytosols confirmed the lack of the 85-kDa PLA₂ protein in these cells. The existence of 85-kDa PLA₂ mRNA and protein has been reported in activated thymocytes and immature B cells, but it is not present and its expression cannot be induced in mature B or T cells or several lines suggesting a maturation-specific phenomenon (35). The type IIa 14-kDa PLA₂ mRNA is found in lymphoid tissue, such as human tonsil (8) and spleen (17), and protein has been demonstrated in a number of cell lines including platelets and neutrophils. However, its existence in purified B and T lymphocytes has not been described. More recently, a second 14-kDa PLA₂, type V, has been identified in mast cells and the P388D1 murine macrophage cell line but a detailed analysis of its expression in human lymphocytes has not been conducted (36, 37). In the present study, ELISA analysis of PBL and Jurkat particulate fractions showed low but detectable levels of a 14-kDa PLA₂ protein. Subsequent RT-PCR analysis demonstrated this to be the type V PLA₂ isoform, a finding which has not been previously reported. Expression of iPLA₂ mRNA was recently reported in human granulocytes and CD34+ stem cells as well as the promyelocytic cell line, HL60, and the monoclonal B cell line, Raji (38). To date, however, no reports exist regarding the occurrence of iPLA₂ mRNA or protein in primary human B or T lymphocytes or human monocytes. We clearly demonstrate the presence of iPLA₂ protein in the cytosol of purified human T and B cells but not in human monocytes.

Enzymatic evaluation of iPLA₂ activity in human PBL and Jurkat T cells revealed proliferation and cell cycle dependent expression. In PBL, exposure to the T cell mitogen, PHA, over 72 h was associated with the maintenance of iPLA₂ protein levels, resulting in activity equal to that observed in freshly isolated cells. Alternatively, the absence of such a stimulus coincided with a decrease in iPLA₂ activity and protein levels. This data indicated a potential requirement for the iPLA₂ enzyme when cells are stimulated to transition from quiesence (G₀) to G₁ where proliferative responses are initiated. Interestingly, assessment of iPLA₂ activity in cell cycle arrested Jurkat fractions demonstrated a predominance of activity in the G₂/M phase arrested fractions which may be due to an increase in protein or an enhanced enzymatic rate. While very little is known about PL metabolism at G₂/M, this coincides with the reported destruction and reassembly of the nuclear membrane which occurs in cells preparing for mitotic events (1). As entry and progression through the cell cycle is associated with lipid turnover (2), these data implicate iPLA₂ as having a possible regulatory role in cell division.

To more directly address this hypothesis, two strategies were employed: (a) use of available inhibitors of the various PLA₂ isoforms and (b) utilization of initiation site-directed antisense oligonucleotides. The use of PLA₂ inhibitors has been employed in numerous cell types to investigate the contributions of distinct PLA₂ family members in cellular AA metabolism. The participation of the type IIa 14-kDa PLA₂ in eicosanoid and platelet activating factor production has been investigated in a host of inflammatory cells including monocytes, neutrophils, and mast cells through the use of the structurally distinct 14-kDa PLA₂ inhibitors, e.g. scalardial (21), BMS 181162 (39), and the active site-directed inhibitor SB 203347 (34). The transition state inhibitor AAOCF₃ was previously thought to be selective for the 85-kDa PLA₂ (40) but recent studies indicate it also has significant inhibitory activity against the iPLA₂ (33). BEL, a mechanism based inhibitor, is specific for iPLA₂ over both the 14-kDa PLA₂ and 85-kDa PLA₂ (33, 41) and has been successfully used to demonstrate the regulatory role of iPLA₂ in phospholipid metabolism (42). It should be noted that BEL is also reported to inhibit PA phosphohydrolase (43). Since no 85-kDa PLA₂ exists in PBL, AAOCF₃ was used as a structurally distinct iPLA₂ inhibitor. Exposure of both PHA-activated PBL or cultured Jurkats to the iPLA₂ inhibitors AAOCF₃ or BEL, but not the 14-kDa PLA₂ inhibitor, SB203347, induced a concentration-dependent suppression of PBL or Jurkat proliferation. Thus, even though 14-kDa PLA₂ Ca²⁺ dependent activity exists in these cells, these data would indicate that it does not play a significant role in proliferative processes. Alternatively, we demonstrate that inhibition of iPLA₂ activity corresponded with a profound interference of cellular proliferation.

Initiation site-directed antisense has been used to demonstrate the critical role of the 85-kDa PLA₂ in prostanoid formation in systems such as LPS-induced monocyte (22, 23) and IL-1-induced fibroblast PGE₂ production (20) and more recently to demonstrate the requirement for this enzyme in vascular smooth muscle cell proliferation (32). In addition, iPLA₂ specific antisense has been successfully used to demonstrate the role of this PLA₂ family member in phospholipid fatty acid remodeling in murine P388D1 macrophages (28). In our studies, exposure of PBL to initiation site-directed iPLA₂ antisense successfully decreased iPLA₂ protein. This correlated with a reduction in proliferation and is consistent with the reduced iPLA₂ protein and activity levels observed in quiescent lymphocytes. These data together with the results from the inhibitor studies provide corroborating evidence implicating the iPLA₂ as a key participant in the molecular processes regulating cell division.

The exact mechanism of iPLA₂ regulation of proliferation is not clear. Reports have implicated iPLA₂ in the release of AA for subsequent metabolism to eicosanoids (i.e. leukotrienes and prostaglandins) in other cell systems where proliferation was not assessed (38, 44, 45). PGE₂ is known to be synthesized upon mitogen stimulation of human PBL. In our hands, pretreatment of mitogen-stimulated PBL with the cyclooxygenase inhibitor, indomethacin which blocks PGE₂ production, had no effect on cell proliferation. Since iPLA₂ has been implicated in free fatty acid generation in other systems, we evaluated the effects of exogenous free fatty acid on PBL proliferation. Addition of free fatty acid alone had no effect on tritiated thymidine incorporation. Interestingly, addition of either AA or oleic acid (data not shown) could not reverse the inhibition obtained by iPLA₂ inhibitors. Taken together the role of iPLA₂ in lymphocyte proliferation appears to be more complex than merely liberation of substrate fatty acids. One possibility is that this PLA₂ family member may orchestrate lymphocyte phospholipid fatty acid turnover in coordination with lysotransferase enzymes. Additional studies are necessary to fully understand the role of iPLA₂ in lymphocyte proliferative processes.

Finally, the involvement of specific PLA₂ isoforms in the regulation of cell division may be a cell-type specific phenomena. We have shown the 85-kDa PLA₂ to be very important and required for vascular smooth muscle cell proliferation (32). Exposure of vascular smooth muscle cell to BEL, the iPLA₂ selective inhibitor, had no effect on proliferation (32). However, in lymphocytes the predominant PLA₂ isoform is the calcium-independent and it is this enzyme which is required for mediating the PL metabolism required to sustain proliferation. Taken together these findings may have interesting implications in terms of defining possible immune cell selective therapeutics directed against iPLA₂ useful as novel immunosuppressive or anti-leukemic agents.

	FOOTNOTES

^* 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 Immunology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., King of Prussia, PA 19406. Tel.: 610-270-4969; Fax: 610-270-5381.

Published, JBC Papers in Press, August 29, 2000, DOI 10.1074/jbc.M002273200

	ABBREVIATIONS

The abbreviations used are: PL, phospholipid; AA, arachidonic acid; PLA₂, phospholipase A2; BEL, bromoenol lactone; iPLA₂, calcium-independent phospholipase A₂; ELISA, enzyme-linked immunosorbent assay; PHA, phytohemagglutinin; PBL, peripheral blood B or T lymphocytes; RT-PCR, reverse transcriptase-polymerase chain reaction; rh, recombinant human; AAOCF₃, arachidonyl trifluoromethyl ketone.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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