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(The FASEB Journal. 2003;17:1068-1080.)
© 2003 FASEB

Interaction of low molecular weight group IIA phospholipase A2 with apoptotic human T cells: role of heparan sulfate proteoglycans

ERIC BOILARD{ddagger}, SYLVAIN G. BOURGOIN{ddagger}, CHANTALE BERNATCHEZ{ddagger}, PATRICE E. POUBELLE{ddagger} and MARC E. SURETTE{ddagger},§,1

{ddagger} Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Pavillon CHUL, and Faculté de Médecine, Université Laval, Québec, Canada G1V 4G2; and
§ Pilot Therapeutics Inc., Charleston, South Carolina, USA

1Correspondence: Pilot Therapeutics Inc., 2000 Daniel Island Dr., Suite 440, Charleston, SC 29492, USA. E-mail: MarcS{at}pilott.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human group IIA phospholipase A2 (hIIA PLA2) is a 14 kDa secreted enzyme associated with inflammatory diseases. A newly discovered property of hIIA PLA2 is the binding affinity for the heparan sulfate proteoglycan (HSPG) glypican-1. In this study, the binding of hIIA PLA2 to apoptotic human T cells was investigated. Little or no exogenous hIIA PLA2 bound to CD3-activated T cells but significant binding was measured on activated T cells induced to undergo apoptosis by anti-CD95. Binding to early apoptotic T cells was greater than to late apoptotic cells. The addition of heparin and the hydrolysis of HSPG by heparinase III only partially inhibited hIIA PLA2 binding to apoptotic cells, suggesting an interaction with both HSPG and other binding protein(s). Two low molecular weight HSPG were coimmunoprecipitated with hIIA PLA2 from apoptotic T cells, but not from living cells. Treatment of CD95-stimulated T cells with hIIA PLA2 resulted in the release of arachidonic acid but not oleic acid from cells and this release was blocked by heparin and heparinase III. Altogether, these results suggest a role for hIIA PLA2 in the release of arachidonic acid from apoptotic cells through interactions with HSPG and its potential implication in the progression of inflammatory diseases.—Boilard, E., Bourgoin, S. G., Bernatchez, C., Poubelle, P. E., Surette, M. E. Interaction of low molecular weight group IIA phospholipase A2 with apoptotic human T cells: role of heparan sulfate proteoglycans.


Key Words: lymphocytes • apoptosis • IIA phospholipase A2 • heparan sulfate proteoglycan • arachidonic acid • arthritis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PHOSPHOLIPASES A2 (PLA2) are enzymes that catalyze the hydrolysis of fatty acids from the sn-2 position of phospholipids leading to the liberation of free fatty acids and 2-lysophospholipids. These products can then be metabolized into potent bioactive lipid mediators such as platelet-activating factor, lyso-phosphatidic acid, leukotrienes, prostaglandins, thromboxanes, and lipoxins. Several forms of PLA2 have been cloned and are well documented (1 , 2) . Although some PLA2 like the arachidonate-specific 85 kDa group IV cytosolic PLA2 are cell associated, others with a low molecular mass (~14 kDa) are secreted PLA2 (sPLA2). The sPLA2 are further subdivided according to their amino acid sequence, position of disulfide bridges, and homology (3 4 5) .

One of the most studied sPLA2 is the group IIA sPLA2 (6) , which possesses important antibacterial properties (7) and is found in high levels in the circulation of patients with acute pancreatitis and certain cancers and in the synovial fluid from patients with rheumatoid arthritis (1 , 8 9 10 11 12) . Among the secreted mammalian PLA2, group IIA, group V, and group X PLA2 have all been implicated in the release of arachidonic acid (AA) for the generation of inflammatory lipid mediators (2) . Group V PLA2 and most of group II PLA2, including group IIA PLA2, have been shown to bind with high affinity to cell surface heparan sulfate proteoglycans (HSPG), which may enhance their ability to hydrolyze fatty acids from mammalian cell membranes (2 , 13 14 15 16 17) . Whereas the heparin binding site of the human group IIA (hIIA) PLA2 appears to be essential for AA release in stimulated HEK-293 cells (17) , the enzymatic activity of human group V and group IIA PLA2 toward human neutrophils, CHO-K1, or unstimulated HEK-293 cells was not correlated with their affinity for heparanoids (18 19 20 21) . Therefore, the relationship between hIIA PLA2 binding to HSPG and its catalytic activity on cell membranes has not been clearly elucidated.

Exogenous group IIA PLA2 hydrolyzes little measurable AA from mammalian cells likely because of the low accessibility of anionic phospholipids on the cell surface. However, during programmed cell death, membrane asymmetry is lost leading to the externalization of phosphatidylserine (PS) and phosphatidylethanolamine (PEth) (22) . This exposure of anionic phospholipids on the cell surface was suggested to enhance the affinity of mammalian group IIA PLA2 for apoptotic cells (23) . Like mammalian group IIA PLA2, monomeric aspartate-49 group II PLA2 from snake venom has increased activity toward the perturbed membranes of calcium ionophore-treated S49 lymphoma cells independent of newly exposed anionic phospholipids substrate (24) , and the susceptibility of apoptotic S49 cells to venom group II PLA2 activity appears to begin before the PS flip occurs, suggesting that other factors are responsible for this enhanced activity (25) . Therefore, the reasons why apoptotic cells become susceptible to group II PLA2 have not been clearly established.

In the present study, we sought to understand the binding and activity of hIIA sPLA2 toward primary human T cells that had been stimulated via the T cell receptor complex and induced to undergo apoptosis after CD95 stimulation. We found that CD95 stimulation resulted in an increase in the binding of hIIA PLA2 to apoptotic T cells. This binding was accompanied by the specific release of AA, which was dependent on interactions with HSPG. These results suggest that hIIA PLA2 can generate inflammatory signals after specific interactions with early apoptotic cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
The [5,6,8,9,11,12,14,15-3H]-arachidonic acid (204 Ci/mmol) and 1-stearoyl-2-[1-14C]-arachidonoylphosphatidylcholine (50 mCi/mmol) were purchased from Amersham (Baie d'Urfé, Québec, Canada). [9,10-3H]-oleic acid (14 Ci/mmol) was from Mandel-NEN Life Science Products (Guelph, Ontario, Canada). The fluorogenic PLA2 substrate N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phospho-ethanolamine (PED6) was from Molecular Probes (Eugene, OR, USA). Phosphatidylcholine (PC), PS, PEth, and phosphatidylinositol (PIn) were obtained from Matreya Inc. (Pleasant Gap, PA, USA). Heparin 1-A (H3393) and Flavobacterium heparinum heparinase III (EC 4.2.2.8) were purchased from Sigma Chemical Co. (Oakville, Ontario, Canada). RPMI 1640, fetal bovine serum (FBS), Ficoll type 400 (density of 1.077 g/mL), phosphate-buffered saline (PBS), and Hank's balanced salt solution (HBSS) were obtained from Wisent (Saint-Bruno, Québec, Canada). Essentially fatty acid free bovine serum albumin (BSA) was purchased from ICN Biomedical Co. (Costa Mesa, CA, USA). TLC plates (20x20) were from Fisher (Nepean, Ontario, Canada). The FITC-conjugated annexin V-propidium Iodide (PI) apoptosis detection kit was obtained from R&D Systems (Minneapolis, MN, USA). The phycoerythrin (PE)-conjugated annexin V was from PharMingen (Mississauga, Ontario, Canada). Methyl arachidonyl fluorophosphonate (MAFP) was obtained from Cayman Chemical Co (Ann Arbor, MI, USA) and pyrrophenone was a generous gift from Dr. Kaoru Seno (Shionogi Research Laboratories, Osaka, Japan). The human recombinant group IIA PLA2 (hIIA PLA2) was a generous gift from Dr. Alfred Fonteh (Huntington Medical Research Institutes, Pasadena, CA, USA). The human sPLA2 inhibitor LY311727 (3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid) was a generous gift from Dr. E. Michelich (Ely Lilly, Indianapolis, IN, USA).

Antibodies
The anti-CD3 was produced and purified from the OKT3 hybridoma clone (a gift from Dr. Walid Mourad, CHUL, Québec, Canada). The anti-CD95 (clone CH-11) was from Upstate Biotechnology (Lake Placid, NY, USA). Anti-hIIA PLA2 (polyclonal antibody) was obtained from Cayman Chemical. The anti-poly(ADP-ribose) polymerase (PARP) (clone C2–10) was from Dr. Guy Poirier (CHUL, Québec, Canada). The anti-heparan sulfate (clone 10E4) was from Biolynx Inc. (Brockville, Ontario, Canada). Horseradish peroxidase (HRP) -conjugated goat anti-rabbit immunoglobulin G (IgG) and HRP-conjugated goat anti-mouse IgG were from BioCan Scientific (Mississauga, Ontario, Canada). The anti-actin, the HRP-conjugated mouse anti-goat IgG, FITC-conjugated mouse anti-rabbit IgG, rhodamine-, FITC-, and HRP-conjugated goat anti-mouse IgM were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit IgG and mouse IgM were from Cedarlane (Hornby, Ontario, Canada).

Lymphocyte preparation, incubation, and induction of apoptosis
Human lymphocytes were isolated from heparinized blood of healthy donors. Blood was diluted 1:0.5 with HBSS containing 200 µM EDTA and centrifuged at 400 x g for 20 min on a Ficoll cushion. Interface cells were collected and washed once with HBSS containing 200 µM EDTA and centrifuged on Ficoll/HBSS+200 µM EDTA (1:1, v/v) for 7 min at 170 x g to remove platelets. The pellet was washed twice in RPMI containing 10% FBS and cell suspensions (106 cells/mL) were incubated for 3 days at 37°C in a 5% CO2 atmosphere with anti-CD3 (1 µg/mL). Interleukin-2 (IL-2; 20 U/mL) was added after 1 day of cell culture. Apoptosis was induced by adding 0.5 µg/mL of the anti-CD95 antibody to 200 µL of cultured lymphocytes in a U-bottom 96-well plate for the final 18 h of incubation.

Detection of hIIA PLA2 on surface of lymphocytes
To detect cell-associated hIIA PLA2 by flow cytometry, hIIA PLA2 (0.5 µg) was added to 2 x 105 lymphocytes in 100 µL of PBS and incubated for 15 min on ice. Cells were washed twice, resuspended in 100 µL cold PBS, and incubated with anti-hIIA PLA2 polyclonal antibody (0.2 µL) for 30 min on ice. Cells were then washed twice, resuspended in 100 µL cold PBS, and incubated with the FITC-conjugated goat anti-rabbit IgG (1.5 µg) for 30 min on ice before analysis by flow cytometry (Beckman Coulter, Miami, FL, USA). Controls were obtained by adding the same combination of antibodies but no hIIA PLA2. A minimum of 15,000 cells was analyzed in each gated area. When double coloration in combination with annexin V was performed, cells were incubated with PE-conjugated annexin V in binding buffer according to manufacturer’s instructions.

To detect cell-associated hIIA PLA2 by immunoblot analysis, hIIA PLA2 (0.5 µg) was added to 2 x 105 lymphocytes in 100 µL of PBS for 15 min on ice. Where indicated, heparinase III (50 mU/mL) was added 5 h before hIIA PLA2 addition. Cells were washed twice, proteins were separated by SDS-PAGE on a 15% polyacrylamide gel (26) , and samples were transferred to a polyvinylidene fluoride (PVDF) blotting membrane. The membrane was blocked for 30 min at room temperature (RT) with 5% milk proteins in TBS-Tween (190 mM NaCl, 0.15% Tween 20, 25 mM Tris-HCl, pH 7.6), washed in TBS-Tween, and incubated with the polyclonal anti-hIIA PLA2 antibody (1:1000 dilution) in TBS-Tween for 1 h at RT. The membrane was washed six times in TBS-Tween and incubated 1 h at RT with an HRP-linked goat anti-rabbit IgG (1:15,000) diluted in TBS-Tween. The membrane was then washed in TBS-Tween and signals were visualized using enhanced chemiluminescence (ECL) Plus (Mandel-NEN Life Science Products, Guelph, Ontario, Canada).

Pulse labeling of lymphocytes and [H3]-fatty acid release
Cells were centrifuged and resuspended in 100 µL of PBS containing [3H]-AA or [3H]-oleic acid (3µCi/107 cells) and incubated for 30 min at 37°C in a shaking water bath. Typically, these cells incorporated 25% of the added radioactivity. The cells were then washed three times with 10% FBS in RPMI, resuspended in 10% FBS in RPMI, and cultured for 5 h at 37°C in a 5% CO2 atmosphere. Where indicated, cells were treated with 50 mU/mL heparinase III during this 5 h period. HIIA PLA2 (1 µg) was then added to 200 µL of cell suspension (3x106 cells/mL) in the presence or absence of LY311727 (10 µM), heparin (50 µg/mL), MAFP (10 µM, added 20 min before hIIA PLA2), or pyrrophenone (100 nM, added 20 min before hIIA PLA2) for 30 min at 37°C in a shaking water bath. The reaction was stopped on ice and total (T) radioactivity contained in 20 µL of cell suspension was determined by liquid scintillation counting (Beckman LS 5000 CE, Fullerton, CA, USA). The remaining 180 µL was centrifuged and the radioactivity contained in 50 µL of supernatant (S) was measured. The percent of total radioactivity release was calculated as follows: % of labeled fatty acid release = 100(S x 4)/T x 10.

RT-PCR detection of human M receptor for sPLA2
RT-PCR analysis was performed using 500 ng of total RNA isolated using Trizol Reagent (Life Technologies Inc., Grand Island, NY, USA) from CD3-activated human T lymphocytes and human kidney tissue (generous gift from Dr. Raynald Roy CHUL, Québec, Canada). Amplification of the membrane-anchored sPLA2 M receptor was achieved with the sense 5'-TGAACACCCAGAGTTGTGCTC-3' and the antisense 5'-GAAACCCTGCAAGTCTCCTG-3' primers using a C. Therm. one-step RT-PCR kit (Roche, Indianapolis, IN, USA).

Flow cytometry staining for HSPG
Since the anti-CD95 and anti-HSPG antibodies were both mouse IgM, anti-CD95 stimulated lymphocytes were incubated 45 min on ice with rhodamine-conjugated goat anti-mouse IgM (15 µg/mL). This completely blocked the ability of FITC-conjugated goat anti-mouse IgM from interacting with any cell-bound anti-CD95. Cells were washed twice and incubated with the anti-HSPG antibody (5 µg/mL) for 30 min on ice. Samples were washed twice and incubated with FITC-conjugated goat anti-mouse IgM for 30 min on ice. The cell suspensions were then washed twice before analysis for FITC by flow cytometry. Gates for living or apoptotic cells were performed according to cell size scatter. For controls, cells were incubated as above, the anti-CD95 included, but an isotype-matched antibody (mouse IgM) replaced the anti-HSPG.

PLA2–Phospholipid interactions
Phospholipids (1 nmol in chloroform/methanol 1:1) were spotted on Hybond-C extra membranes (Amersham). The membranes were blocked in 3% (w/v) fatty acid-free BSA in TBS-Tween for 1 h and incubated overnight at 4°C with 0.1 µg/mL hIIA PLA2. The membranes were washed five times and incubated with polyclonal anti-hIIA PLA2 antibody (1/1000 dilution) for 1 h at 4°C. After six washes in TBS-Tween, membranes were incubated 1 h at 4°C with an HRP-linked goat anti-rabbit IgG (1:15,000) in TBS-Tween containing 3% BSA. Membranes were washed and signals were revealed using ECL Plus.

PLA2–HSPG interactions in affinity columns
HIIA PLA2 (5 µg) was incubated at room temperature in 100 µL of binding solution (100 mM HEPES, 137 mM NaCl, 1 mM CaCl2) alone or in the presence of 50 µg heparin or 100 µM LY311727. After 30 min, 900 µL of binding solution was added and the total volume was injected onto 1 mL HiTrapTM affinity columns (Amersham). These affinity columns are filled with beads covalently coupled to sulfated glucosaminoglycans extracted from porcine intestinal mucosa. Columns were washed with 6 mL of binding solution, the flow-through was collected, and 100 µL underwent SDS-PAGE on a 15% polyacrylamide gel. HIIA PLA2 was detected by immunoblotting as described above.

Determination of PLA2 activity
hIIA PLA2 activity was assessed using radiolabeled PC or PED6, a PEth with a fluorescent dye–labeled sn-2 acyl chain, as substrate. The assay mixture using PC as substrate consisted of 5 µM of 1-stearoyl-2-[1-14C]-arachidonoyl-PC substrate, 4 mM CaCl2, 1 mg/mL BSA, 80 mM KCl, and 10 mM HEPES (pH 7.4). To prepare the assay mixture the substrate was dried under a nitrogen stream, resuspended in the assay solution, sonicated, and vortexed. The reaction was started by adding the assay mixture to 1 µg of hIIA PLA2 in a final volume of 100 µL. After 15 min at 37°C, the reaction was stopped by adding 300 µL chloroform/methanol (1:2, v/v). The lipids were extracted (27) and separated by TLC using hexane/ether/acetic acid (60:40:2) as the mobile phase. Spots were analyzed using a PhosphorImager BAS-1800 (Fuji Medical Systems, Stanford, CT, USA). hIIA PLA2 catalytic activity using PED6 as substrate was measured in 96-well microtiter costar plates (Corning Inc., NY, USA). PLA2-mediated hydrolysis of this substrate generates a fluorescent product. The assay mixture contained 10 ng of hIIA PLA2 and 15 µM of PED6 in 100 mM HEPES pH 7.5 with 1 mM CaCl2 and 1 mg/mL BSA. The assay was initiated by addition of substrate and fluorescence emission (excitation, 500 nm; emission 512 nm) was monitored using a Bio-Tek FL600 microplate fluorescence reader (ESBE Scientific, Montréal, Québec).

Immunoblot detection of HSPG
Cells (75 µg protein) were boiled in sample buffer (26) , separated by SDS-PAGE on a 12% polyacrylamide gel, and transferred to a PVDF blotting membrane that was blocked 30 min at RT with 5% milk proteins in TBS-Tween. The membrane was then washed in TBS-Tween and incubated with the anti-HSPG mAb (2 µg/mL final concentration) in TBS-Tween for 18 h at 4°C. The membrane was washed six times in TBS-Tween and incubated for 1 h at RT with an HRP-linked goat anti-mouse IgM (1:15,000) in TBS-Tween. The membrane was then washed six times with TBS-Tween and signals were revealed using ECL Plus.

Hypotonic lysis of human T cells and coprecipitation of hIIA PLA2 and HSPG
After incubation of living or apoptotic lymphocytes (2x105) with hIIA PLA2 (0.5 µg/mL), cells were washed three times in PBS, the pellet lysed for 5 min at 4°C in hypotonic lysis buffer A (HLB: 0.1% NP-40, 20 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1 mM EDTA, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 2 mM PMSF) and centrifuged at 600 x g for 10 min at 4°C. In some experiments, the resulting lysate supernatant and pellet were prepared for SDS-PAGE and immunoblot detection of hIIA PLA2 as described above. For immunoprecipitation experiments, 3 x 107 cells were used and were incubated with 15 µg hIIA PLA2/mL. The HLB lysate supernatant was removed, adjusted to an isotonic salt concentration (buffer B, final concentration 137 mM NaCl, 0.75% Triton X-100, 0.75% NP-40, 1 mM EDTA, and 20 mM Tris HCL, pH 7.5), and precleared with protein G-Sepharose beads (Amersham) at 4°C for 45 min. Protein G-Sepharose beads previously incubated for 1 h by rotation at 4°C with 10 µL of anti-hIIA PLA2 in buffer B were mixed with the lysates and incubated together by rotation for 2.5 h at 4°C. After three washes in buffer B, the beads were boiled in sample buffer (26) and samples were analyzed by immunoblot after separation of proteins by SDS-PAGE on a 12% polyacrylamide gel. HSPG detection was performed with the anti-HSPG mAb as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of apoptosis in human T cells and hIIA PLA2 binding
During programmed cell death, membrane asymmetry is altered and anionic phospholipids like PS and PEth are externalized rendering apoptotic cells susceptible to phospholipid hydrolysis by group IIA PLA2 (23 , 25 , 28) . To compare the binding of hIIA PLA2 to resting, activated and apoptotic human T cells, freshly isolated human T cells were stimulated via the T cell receptor complex using anti-CD3 and apoptosis was induced with anti-CD95. Freshly isolated cells were typically 70% CD3-positive (data not shown) and weakly expressed CD95. After incubating cells for 48–72 h with anti-CD3 in the presence of IL-2, a pure CD3-positive culture expressing high CD95 levels was obtained (Fig. 1 A, B). When these activated T cells were incubated with anti-CD95, ~50% of the cells became apoptotic according to annexin V/PI staining (data not shown).



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  		Figure 1. CD3 and CD95 expression in resting and activated human lymphocytes. Human lymphocytes from peripheral blood were isolated and stimulated for up to 72 h with anti-CD3 and IL-2 as described in Materials and Methods. Cells were then analyzed for CD3 (A) and CD95 (B) expression by flow cytometry after incubating washed cells on ice with anti-CD3 or anti-CD95, followed by the appropriate FITC-labeled secondary antibody. A minimum of 10,000 cells from the total cell population were analyzed. A) Cells were stimulated for 72 h before analysis. B) The indicated mean fluorescent channel value represents the difference between the fluorescence of CD95-labeled cells and that of isotype control-labeled cells. The isotype controls were performed by adding an equivalent amount of isotype matched antibodies instead of the anti-CD95 or anti-CD3 antibodies. These experiments are representative of 4 separate experiments.

Neither of these T cell populations expressed detectable levels of hIIA PLA2 as assessed by immunoblot analysis (Fig. 2 A). When the T cells were incubated with exogenous hIIA PLA2, little or no enzyme was bound to resting or CD3-stimulated cells but significant quantities remained bound to anti-CD95-treated cells. To determine whether the hIIA PLA2 was binding to all anti-CD95-treated cells or only to apoptotic cells, the anti-CD95-treated population of T cells was sorted into two populations based on cell size and granularity since cells become smaller and more granular as they progress through apoptosis (29 30 31) . The sorting of apoptotic from nonapoptotic T cells was confirmed by evaluating PARP cleavage in these two cell populations (Fig. 2B ), and immunoblot analysis of hIIA PLA2 showed that the enzyme only remained bound to the smaller more granular apoptotic population of cells. Having established that hIIA PLA2 binds to apoptotic human T cells, we wanted to verify whether this binding was influenced by the apoptotic cell state. The top left panel in Fig. 3 shows the typical cell scatter distribution of CD3-stimulated human T cells that have been treated with anti-CD95. This mixed population of cells can be gated based on cell size (forward scatter) and granularity (side scatter) resulting in three subpopulations that are 1) mostly nonapoptotic cells, 2) early apoptotic annexin V-positive cells, and 3) late apoptotic annexin V-positive cells. A two-color cell staining was performed on these three gated cell populations using hIIA PLA2 and PE-conjugated annexin V to compare hIIA PLA2 binding to non-, early, and late apoptotic cells. Figure 3(i) shows that very few cells in the nonapoptotic population bind hIIA PLA2, confirming the results obtained by immunoblot analysis. Additionally, a much greater proportion of early apoptotic cells (gated area ii) bound significant quantities of hIIA PLA2 compared with the nonapoptotic T cell population. Surprisingly, hIIA PLA2 bound a much smaller number of late apoptotic T cells (gated area iii) than the early apoptotic cells. Both populations were equally annexin V-positive (~85–90% of cells) and the intensity of annexin V staining was similar with an average staining intensity of cells in the left quadrants of 4.4 and 4.0 for early and late apoptotic cells, respectively. These results indicate that the large difference in binding of hIIA PLA2 to apoptotic cells is not solely dependent on PS externalization and suggest that other hIIA PLA2 binding site(s) may be expressed, especially in the early stages of apoptosis.



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  		Figure 2. Human group IIA phospholipase A2 binds apoptotic T cells. A) Human lymphocytes were incubated with or without anti-CD3 (OKT3) and IL-2 for 72 h as described in Materials and Methods. Where indicated, anti-CD95 (0.5 µg/mL) was added to the incubation medium during the final 18 h of incubation. Cells (2 x 105) were then incubated on ice with or without 0.5 µg hIIA PLA2 for 15 min, washed, and lysed. Total cellular proteins in equal sample volumes were separated by SDS-PAGE and cell-associated hIIA PLA2 was detected by immunoblot analysis as described in Materials and Methods. B) Activated lymphocytes that were stimulated for 18 h with the anti-CD95 mAb were sorted based on cell size and granularity by flow cytometry into two cell populations termed living and smaller denser apoptotic cells. The two populations of cells were collected, proteins were separated by SDS-PAGE, and poly(ADP-ribose) polymerase was determined by immunoblot analysis. The two cell populations (2x105 cells) were also incubated on ice with 0.5 µg hIIA PLA2, washed, and the entire cell lysate analyzed for cell-associated hIIA PLA2 by SDS-PAGE and immunoblot analysis as described in Materials and Methods. These experiments are representative of 4 separate experiments.



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  		Figure 3. Human group IIA phospholipase A2 binds early apoptotic T cells. Human lymphocytes were incubated for 72 h with anti-CD3 and IL-2 as described in Materials and Methods. Anti-CD95 (0.5 µg/mL) was added to the incubations for the final 18 h of incubation. Cells were then incubated on ice with hIIA PLA2 and annexin V as described in Materials and Methods and sorted by flow cytometry into 3 cell populations based on cell size and granularity as shown in the top left panel. Gates containing living cells (i), smaller and more granular early (ii), and late (iii) apoptotic cells were analyzed for bound hIIA PLA2 and annexin V by flow cytometry, with a minimum of 15,000 cells analyzed in each gated area. This experiment is representative of 5 separate experiments. FS = forward scatter; SS = side scatter.

M-type receptor and heparan sulfate expression in apoptotic T cells
In an attempt to characterize the receptors or binding sites responsible for the binding of hIIA PLA2 to early apoptotic human T cells, the expression of proteins known to bind group IIA PLA2 was evaluated. One putative receptor, the M-type receptor, is a protein found in several tissues and can bind, depending on the species studied, various sPLA2s (32 33 34) . The presence of RNA for this receptor was evaluated in CD3-activated T cells incubated or not with anti-CD95. Figure 4 A shows that no RNA coding for the M-type receptor was detected after 35 PCR cycles in human T cells. The same result was obtained with the human Jurkat T cell line (data not shown). Since this gene has been cloned from human kidneys, this tissue was used as a positive control.



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  		Figure 4. Expression of known receptors of group IIA phospholipase A2 in human T cells. Human lymphocytes were incubated for 72 h with anti-CD3 and IL-2 as described in Materials and Methods. Where indicated, anti-CD95 (0.5 µg/mL) was added to the media for the final 18 h of incubation. A) Expression of RNA coding for the M receptor was evaluated using RT-PCR on RNA extracted from both unstimulated and CD95-stimulated lymphocytes, and from human kidney tissue as a positive control. B) Cell surface expression of HSPG was determined in CD95-treated cells by flow cytometry using an anti-HSPG mAb. Gates on living or apoptotic cells were performed according to cell size scatter. Isotype controls of living and early apoptotic lymphocytes indicated by the striped and black filled areas, respectively, were performed as described in Materials and Methods. The experiments in panels A and B are representative of 3 and 5 separate experiments, respectively.

Group IIA PLA2 binds to HSPG, including glypican-1, which has been suggested to be a specific receptor for group IIA PLA2 (13 , 14 , 17 , 35) . HSPG expression on the T cell surface was therefore evaluated by flow cytometry using an antibody (clone 10E4) directed against an epitope common to most HSPG. This antibody does not react with hyaluronan, chondroitin sulfate, dermatan sulfate, or DNA. As shown in Fig. 4B , human T cells express HSPG and this expression is increased ~twofold after incubation with anti-CD95.

Role of HSPG in hIIA PLA2 binding
To determine the role that HSPG may play in the association of hIIA PLA2 to apoptotic human T cells, hIIA PLA2 was incubated with anti-CD95-treated T cells in the presence of heparin, which competes with HSPG for heparin binding proteins, or with anti-CD95-treated T cells that had been pretreated with heparinase III, which degrades heparin sulfate chains. As can be seen in Fig. 5 A, both heparin and heparinase III treatment partially inhibited the binding of hIIA PLA2 to T cells by ~40–60%, indicating that the heparin binding sites are involved in the association of hIIA PLA2 to the cell surface. To verify that heparin was not inhibiting the ability of sPLA2 to interact with externalized PS, binding of hIIA PLA2 to immobilized phospholipids was evaluated using a protein-lipid overlay assay (36) . Figure 5B shows that hIIA PLA2 binds primarily to the PS and PEth and this binding was not affected by the presence of 50 µg/mL of heparin, indicating that heparin should not interfere with the binding of hIIA PLA2 to externalized cellular PS.



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  		Figure 5. Role of heparan sulfate proteoglycans in hIIA PLA2 binding to apoptotic T cells. A) Human lymphocytes were incubated for 72 h with anti-CD3 and IL-2 as described in Materials and Methods. Anti-CD95 (0.5 µg/mL) was added to the incubation medium for the final 18 h of incubation. Cells (2x105) were then incubated on ice with hIIA PLA2 (0.5 µg/mL) for 15 min in the presence or absence of heparin (50 µg/mL) or heparinase III (50 mU/mL, 5 h before addition of hIIA PLA2). Cells were washed and total cellular proteins in equivalent sample volumes were separated by SDS-PAGE; hIIA PLA2 was determined by immunoblot analysis as described in Materials and Methods. B) Different phospholipids (1 nmol per spot) were applied onto a Hybond-C extra membrane that was then incubated with hIIA PLA2 (0.1 µg/mL) in the presence or absence of heparin (50 µg/mL). The membrane was washed and the phospholipid-bound hIIA PLA2 was revealed by immunoblot analysis as described in Materials and Methods. The experiments shown are representative of 4 separate experiments.

Since a site(s) other than HSPG appears to be involved in binding hIIA PLA2 to anti-CD95-treated human T cells, an initial attempt was made to separate these binding activities. Anti-CD95-treated cells were incubated with hIIA PLA2 and the cells were then lysed in a hypotonic lysis buffer (HLB) containing NP-40, which differentially solubilizes cellular structures (37 , 38) . In our cell model, all HSPG detected with the 10E4 mAb, ß-actin, and >90% of tritiated AA in labeled cells (as a marker of phospholipids) were solubilized in the HLB buffer (data not shown). Figure 6 A shows that hIIA PLA2 bound to both the HLB-soluble and -insoluble cellular fractions from anti-CD95-treated cells. However, pretreatment of cells with heparin, anti-heparan sulfate (10E4), or heparinase III blocked only the binding of hIIA PLA2 to the HLB-soluble fraction. Treatment of the cells with the sPLA2 inhibitor LY311727 had no effect on the binding of hIIA PLA2 to the HLB-soluble fraction but partially inhibited the interaction with the HLB insoluble fraction. To confirm that LY311727 did not affect the interaction of hIIA PLA2 with HSPG, hIIA PLA2 was loaded in the presence or absence of heparin or LY311727 onto HitrapTM affinity columns containing sulfated glucosamines and the eluate was analyzed for hIIA PLA2 content. Figure 6B shows that whereas heparin prevented the retention of hIIA PLA2 on the column, LY311727 had no effect on the ability of the column to retain the enzyme.



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  		Figure 6. Association of hIIA PLA2 to the hypotonic lysis buffer-soluble and -insoluble fractions of human T cells. A) Human lymphocytes were incubated for 72 h with anti-CD3 and IL-2. Anti-CD95 (0.5 µg/mL) was added to the incubation medium for the final 18 h of incubation. Cells (2x105) were incubated on ice with hIIA PLA2 (0.5 µg/mL) for 15 min in the presence or absence of heparin (50 µg/mL), LY311727 (10 µM), 10E4 mAb (15 µg/mL, 30 min before IIA PLA2 addition), or heparinase III (50 mU/mL added during the last 5 h of incubation before the addition of hIIA PLA2). Cells were then washed and the cell pellet was lysed in HLB buffer and centrifuged as described in Materials and Methods. The supernatant obtained was called the HLB-soluble fraction whereas the pellet was the HLB-insoluble fraction. Total protein from each fraction in equivalent volumes was separated by SDS-PAGE and hIIA PLA2 was determined by immunoblot analysis. B) Affinity of hIIA PLA2 to HSPG in presence or absence of heparin or LY311727 was monitored using HiTrapTM columns as described in Materials and Methods. The eluate from the columns was collected, separated by SDS-PAGE, and hIIA PLA2 was revealed by immunoblot analysis (see text). Results in panels A and B are representative of 4 and 3 separate experiments, respectively.

The initial identification of the HSPG expressed in T cells was performed by immunoblot analyses. Figure 7 A shows the presence of 25, 17, and 15 kDa HSPG in total cell lysates. When cells were treated with anti-CD95, the amount of the 17 kDa protein was increased but not that of the 15 and 25 kDa proteins. The specificity of the anti-HSPG 10E4 mAb was verified using an isotype control and by treating apoptotic cells with heparinase III. To confirm the interaction between hIIA PLA2 and these HSPG, cells were incubated with hIIA PLA2, lysed with HLB buffer, and the cell-associated hIIA PLA2 was then immunoprecipitated using anti-hIIA PLA2. Analysis of the resulting immunoprecipitate with the anti-HSPG 10E4 mAb showed that the 15 and 17 kDa forms of HSPG both coprecipitated with hIIA PLA2 in the lysates prepared from CD95-stimulated cells but not from unstimulated cells (Fig. 7B ).



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  		Figure 7. Immunoblot analysis of HSPG in T lymphocytes. Human lymphocytes were incubated for 72 h with anti-CD3 and IL-2 as described in Materials and Methods. Anti-CD95 (0.5 µg/mL) was added to the incubations for the final 18 h of incubation as indicated. A) Protein (75 µg) from T cells, anti-CD95-stimulated apoptotic T cells, or anti-CD95-stimulated apoptotic T cells treated with heparinase III (50 mU/mL for 5 h) were separated by SDS-PAGE and HSPG were revealed by immunoblot analysis as described in Materials and Methods. B) T cells (3x107 cells) were incubated with hIIA PLA2 (15 µg/mL), washed, and lysed for immunoprecipitation experiments. Protein G beads incubated with anti-hIIA PLA2 were added to the cell lysates and the hIIA PLA2 was precipitated. Beads were boiled and the total proteins in equivalent sample volume were separated by SDS-PAGE; HSPG were detected by immunoblot analysis as described in Materials and Methods. Experiments in panels A and B are representative of 5 and 3 separate experiments, respectively.

HIIA PLA2 specifically releases AA from apoptotic human T cells
Although hIIA PLA2 associated with apoptotic T cells is not fully displaced by strategies that inhibit or prevent its association with HSPG, the interaction of hIIA PLA2 with HSPG may nevertheless perform a functional role. We therefore determined whether the interaction of hIIA PLA2 with apoptotic T cells is associated with the hydrolysis of fatty acids from membrane phospholipids. As shown in Fig. 8 A, treatment of CD3-activated [3H]AA-labeled human T cells with exogenous hIIA PLA2 resulted in no significant release of [3H]AA compared with controls incubated without addition of hIIA PLA2. However, when T cells were also treated with anti-CD95, the incubation with hIIA PLA2 resulted in a significant increase in the amount of [3H]AA released from the cells. Surprisingly, exogenously added hIIA PLA2 to cells did not induce the release of [3H]oleic acid.



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  		Figure 8. [3H]-fatty acid release from cells by hIIA PLA2. Human lymphocytes were incubated for 72 h with anti-CD3 (OKT3) and IL-2 as described in Materials and Methods. A) Where indicated, anti-CD95 (0.5 µg/mL) was added to the media for the final 18 h of incubation. Cells were then labeled with [3H]-AA (black columns) or [3H]-OA (gray columns) as described in text. Human IIA PLA2 (5 µg/mL) or its diluent was added to the cells for 30 min, the reaction was stopped by centrifugation at 4°C, and the radioactivity recovered in supernatant and pellet was quantified by liquid scintillation counting as described in Materials and Methods. B) Where indicated, LY311727 (10 µM), heparin (50 µg/mL), heparinase III (50 mU/mL, 5 h before addition of hIIA PLA2), pyrrophenone (100 nM, 20 min before hIIA PLA2 addition), and MAFP (10 µM, 20 min before addition of hIIA PLA2) were added to the incubation medium of anti-CD95-treated cells immediately before addition of the hIIA PLA2. The control represents cells that were not treated with hIIA PLA2. C) The activity of hIIA PLA2 on phospholipid vesicles containing 1-stearoyl-2-[1-14C]-arachidonoyl-PtdCho or D) the fluorogenic substrate PED6 was determined in the presence or absence of LY311727 (10 µM) or heparin (50 µg/mL) as described in Materials and Methods. The results presented in panels A, B, and D are the means ± SE of 5 separate experiments each performed in duplicate. The results presented in panel C are from 1 experiment representative of 3 separate experiments.

The release of [3H]AA was inhibited when heparin was included in the incubation medium or when cells were pretreated with heparinase III, suggesting that the interaction of hIIA PLA2 with HSPG is required for hydrolysis of [3H]AA. Heparin was likely inhibiting the association of hIIA PLA2 with HSPG rather than inhibiting its catalytic activity since heparin had no inhibitory effect on the hIIA PLA2-catalyzed release of AA from phospholipid vesicles (Fig. 8C, D ); in fact, heparin increased the measured activity as has been reported by others using comparable concentrations of heparin (13) . The release of cellular [3H]AA induced by treating cells with hIIA PLA2 was also inhibited by the specific group IIA PLA2 inhibitor LY311727 but not by the group IV PLA2 inhibitor pyrrophenone or the dual group IV/group VI PLA2 inhibitor MAFP (Fig. 8B ).

Hydrolysis of PS after prolonged incubation of T cells with hIIA PLA2
The experiments described above indicated that the initial short-term exposure of early apoptotic cells to hIIA PLA2 results in a specific release of AA. However, a more prolonged exposure of apoptotic cells to hIIA PLA2 could result in a more pronounced hydrolysis of cellular anionic phospholipids. Because annexin V specifically binds to PS, FITC-conjugated annexin V was used to monitor the hydrolysis of PS after a longer exposure of CD95-treated T cells to hIIA PLA2. Figure 9 A illustrates that an 18 h exposure to hIIA PLA2 caused a decrease in the population of annexin V-positive cells compared with cells incubated in the absence of hIIA PLA2, suggesting that hIIA PLA2 hydrolyzed the cell surface PS. Although PI+/annexin V- cells are normally characterized as necrotic because they become permeable without PS externalization, a time course indicated that these cells were originally annexin V+ cells that, upon exposure to hIIA PLA2, gradually lost their ability to bind annexin V and became permeable to PI (data not shown).



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  		Figure 9. Phosphatidylserine hydrolysis and cell membrane permeabilization of apoptotic cells treated with hIIA PLA2. Human lymphocytes were incubated for 72 h with anti-CD3 and IL-2 (see Materials and Methods). Where indicated, anti-CD95 (0.5 µg/mL) was added to the media for the final 18 h of incubation. A) Cells treated with the indicated stimuli were stained with annexin V-PI as described in Materials and Methods and evaluated by flow cytometry. B) Quantification of annexin V-negative/PI-positive cells (found in the top-left quadrant) when cells treated with the indicated stimuli or reagents were stained with annexin V/PI. Where indicated, LY311727 (10 µM), heparin (50 µg/mL) or anti-HSPG antibody (10E4, 15 µg/mL) were added to the incubation medium immediately before the addition of the hIIA PLA2. The experiment in panels A is representative of 5 separate experiments. The results presented in panel B are the means ± SE of 5 separate experiments.

The generation of PI+/annexin– cells induced by a longer exposure to hIIA PLA2 was blocked by heparin and partially blocked by 10E4 (Fig. 9B ). The group IIA PLA2 inhibitor LY311727 completely blocked the disappearance of annexin V-positive cells. Therefore, the interaction of hIIA PLA2 with HSPG and its catalytic activity were essential for the PS hydrolysis and membrane permeabilization induced by a prolonged incubation of T cells with hIIA PLA2.

HIIA PLA2 in human tissues
The concentrations of hIIA PLA2 used in the present studies fall within ranges reported in various diseased tissues (9) . The concentrations of hIIA PLA2 used were nevertheless directly compared with those found in several diseased tissues. Ten nanograms of hIIA PLA2 was therefore compared by immunoblot analysis to the hIIA PLA2 content measured in 4 µL of the synovial fluids from patients with rheumatoid arthritis (RA), osteoarthritis, and gout and to that measured in 4 µL of serum from a normal healthy individual and from a patient with chronic myelogenous leukemia (CML). Figure 10 shows that very little if any hIIA PLA2 was detected in the sera obtained from healthy subjects or from the synovial fluid of osteoarthritic patients. However, the quantities of hIIA PLA2 detected in fluids obtained from patients with gout, RA, and CML were comparable to those used in the present experiments.



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  		Figure 10. Human group IIA PLA2 in serum or inflammatory fluids. Proteins contained in 4 µL of the indicated fluids obtained from human volunteers or recombinant hIIA PLA2 (10 ng) were separated by SDS-PAGE, transferred on PVDF membrane, and hIIA PLA2 was detected by immunoblot analysis. RA, OsA, and gout represent the synovial fluids obtained from the knee joint of patients with rheumatoid arthritis, osteoarthritis, and gout, respectively. Leukemia indicates serum obtained from a patient suffering from acute myelogenous leukemia and normal represents the serum from a healthy volunteer.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
T cells play an important role in the pathogenesis of autoimmune diseases, including rheumatoid arthritis, which is characterized by the accumulation of neutrophils and T lymphocytes within the synovial compartment. Human group IIA PLA2 is highly expressed in hematopoietic cells and large quantities of the protein are found in inflammatory tissues like the synovial fluids from rheumatoid arthritis patients (1 , 9 , 11 , 12) , where it was thought that this enzyme plays a role in the release of AA and the synthesis of lipid inflammatory mediators like platelet-activating factor, leukotrienes, and prostaglandins. However, whereas group IIA PLA2 can release fatty acids from anionic phospholipids like PS or PEth in phospholipid vesicles (18 , 21 , 39 40 41 42) , its ability to hydrolyze phospholipids when presented to intact cells is variable; indeed, after cell stimulation more AA is released from HEK cells transfected with murine IIA PLA2 than control cells, but exogenous hIIA PLA2 does not release fatty acids from intact membranes of adherent HEK 293 and CHO-K1, Swiss-3T3, CFTL-15, or THP-1 cells (20 , 43) and shows only little activity on bone marrow-derived mast cells (43) and human neutrophils (40) . In the present study, exogenous hIIA PLA2 had little or no measurable interaction with either resting or activated human T cells and was incapable of catalyzing the measurable release of AA from their membranes. This observation was consistent with the limited activity of the human group IIA PLA2 with other primary cells of hematopoietic origin like human neutrophils and murine bone marrow-derived mast cells (40 , 43) . In addition to activated T cells and neutrophils, hIIA PLA2 is in contact with significant numbers of apoptotic cells in inflammatory sites and the efficient removal of these cells is important for the resolution of inflammation. Apoptotic cells have an increased susceptibility to the action of hIIA PLA2 since they lose their ability to maintain membrane asymmetry, resulting in the exposure of anionic phospholipids on the outer cell membrane. However, loss of membrane asymmetry may not be sufficient to allow an association between the membranes and hIIA PLA2, which in turn renders the membrane phospholipids susceptible to PLA2-catalyzed hydrolysis. For example, scramblase-transfected cells become more sensitive to the catalytic activity of wild-type group IIA PLA2 but remain insensitive to the IIA sPLA2 mutant, KE4, which has lost the heparanoid binding site (28) . The present finding that PS exposure on the cell surface was not sufficient for binding of hIIA PLA2 to apoptotic human T cells supports a role for binding sites other than anionic phospholipids on membranes of apoptotic cells. The observation that hIIA PLA2 binding to the apoptotic cell surface was partially inhibited by agents that interfere with hIIA PLA2-HSPG interactions indicated that newly expressed or exposed species of HSPG were partially responsible for the ability to bind apoptotic T cells.

The observed binding of hIIA PLA2 to HSPG in early apoptotic cells could be of physiological or pathological importance since the ability of hIIA PLA2 to rapidly hydrolyze AA from cell membranes was dependent on the association of the enzyme with HSPG. Although the HSPG glypican-1 was shown to enhance the release of AA from HEK 293 cells transfected with murine group IIA PLA2 (14) , the present results indicate that the interaction of hIIA PLA2 with HSPG on apoptotic human T cells also imparts specificity to the enzyme resulting in the specific hydrolysis of cellular AA, with no effect on the release of cellular oleic acid. In inflammatory sites like the arthritic synovium, elevated concentrations of hIIA PLA2 could possibly release quantities of AA from apoptotic cells sufficient to contribute to the transcellular synthesis of leukotrienes or prostaglandins. Indeed, the exposure of human neutrophils to exogenous AA results in the synthesis of important quantities of the powerful chemoattractant LTB4 even in the absence of other stimuli (44) . It is significant that this release of AA is induced by concentrations of the enzyme that are physiologically relevant.

Although the 60 kDa HSPG glypican-1 has been shown to bind to murine group IIA PLA2 and to contribute to its ability to release AA from HEK 293 cells overexpressing the enzyme (14) , the present study shows that other HSPG bind hIIA PLA2 in early apoptotic human T cells. Immunoblot analyses revealed that at least three HSPG with apparent molecular masses of 25, 17, and 15 kDa are expressed in human T cells and that expression of the 17 kDa protein is enhanced in anti-CD95-treated cells. Immunoprecipitation experiments showed that hIIA PLA2 interacts with at least two of these proteins in anti-CD95-treated cells but not in untreated cells. Whereas HSPG species are expressed in untreated cells, either these HSPG do not bind hIIA PLA2 or they are not accessible to the enzyme on the cell surface. The mechanism by which hIIA PLA2 specifically releases AA from cellular phospholipids after interaction with HSPG is not known. Secreted PLA2 can elicit a specific AA release via a downstream activation of the group IV cytosolic PLA2 when interacting with the M-type receptor (45 46 47) . However, the hIIA PLA2-induced release of AA from apoptotic T cells is more likely due to the direct catalytic action of hIIA PLA2 since the specific group IIA PLA2 inhibitor LY311727 completely inhibited AA hydrolysis whereas both the group IV cytosolic PLA2 inhibitor pyrrophenone and the dual group IV/VI PLA2 inhibitor MAFP were without effect on AA release.

A more prolonged exposure (hours instead of minutes) of apoptotic T cells to relevant concentrations of hIIA PLA2 resulted in a complete reversal of the ability of annexin V to bind to the cells, indicating a loss of cell surface PS. This observation, which was also dependent on hIIA PLA2 interactions with HSPG, has potentially important physiological consequences since cell surface PS triggers apoptotic cell recognition and clearance by phagocytes, which is required for the resolution of inflammation (48) . The impaired clearance of apoptotic cells can lead to the release of cytosolic and nuclear particles with a high antigenic potential, as is the case in several autoimmune diseases (49 50 51 52) ; in fact, the immunization of mice with apoptotic Jurkat T cells results in the production of autoantibodies targeting multiple autoantigens, which is consistent with the hypothesis that the impaired removal of apoptotic T cells can contribute to the development of autoimmunity (53) . It is tempting to speculate that the exposure of apoptotic T cells to elevated concentrations of hIIA PLA2 could contribute to the formation of such autoantibodies.

Whereas the release of AA induced by hIIA PLA2 was dependent on its interaction with HSPG, it was clear that other cell surface binding sites were also expressed in apoptotic T cells. The sPLA2 inhibitor LY311727 partially blocked the interaction of hIIA PLA2 with apoptotic T cells, indicating that these unidentified binding sites may interact with a region adjacent to or at the active site of the enzyme. Indeed, in addition to the binding of hIIA PLA2 to heparin-insensitive sites in the HLB insoluble fraction, other proteins were coimmunoprecipitated with hIIA PLA2 as determined by in-gel protein staining with Sypro RubyTM (data not shown). The functional consequences of hIIA PLA2 interactions with these sites are not known, but mass spectrometry experiments are now under way to identify these other hIIA PLA2 binding proteins, which may yield information on the possible functional or physiological consequences of these interactions.

Many reports investigating interactions between group IIA PLA2 and cell membranes have used group IIA PLA2 from species other than humans. Although these studies have yielded important information on substrate specificities and interactions with putative binding sites expressed on immortalized cell lines, it is important to distinguish between the human form of this enzyme and that from other species since their affinities for these binding sites are often very different (54) . In the present report the study of the interactions between primary human T cells and the human group IIA PLA2 has yielded information that could potentially have direct implications in human inflammatory and autoimmune diseases.


   ACKNOWLEDGMENTS
 
These studies were supported by grants from the Canadian Institutes of Health Research (to M.E.S. and S.G.B). E.B. is the recipient of a Scholarship from Le Fonds de la Recherche en Santé du Québec. The authors wish to thank Dr. Alfred N. Fonteh for his helpful comments.

Received for publication October 9, 2002. Accepted for publication February 12, 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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