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J. Biol. Chem., Vol. 281, Issue 16, 10935-10944, April 21, 2006
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11
2
From the
Departments of Chemistry and Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607
Received for publication, November 28, 2005 , and in revised form, February 10, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, -
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) and Ca2+-independent intracellular PLA2s( and ) (2-4) have been identified from mammalian tissues. Among these enzymes, group IVA cytosolic PLA2 (cPLA2) has been shown to play an important role in cellular AA formation (2, 5, 6). However, physiological roles of most of sPLA2 isoforms have not been fully defined. It has been reported that some sPLA2s, most notably group V PLA2 (gVPLA2), work in concert with cPLA2 (7-14) or independently of cPLA2 (15) to induce eicosanoid formation in different mammalian cells. The involvement of gVPLA2 in inflammation was further supported by a recent gene knock-out study (16).
Based on the earlier finding that the level of sPLA2 was elevated in inflammatory exudates (17, 18), it was generally thought that sPLA2s are released to the extracellular medium in response to specific stimuli and act on different target cells. However, Kudo and coworkers (10, 19-24) found that many basic sPLA2s, including group IIA PLA2 (gIIAPLA2) and gVPLA2, remained bound to their originating cells after secretion due to their high affinity for cell surface heparan sulfate proteoglycans (HSPG) and were re-internalized to augment the stimulus-dependent AA release. Furthermore, a recent study suggested that the agonist-induced AA release from gIIAPLA2- and group X PLA2 (gXPLA2)-transfected CHO-K1 and HEK293 cells occurs predominantly during the secretory process (i.e. before secretion) and with the involvement of cPLA2 (25), adding further complexity to the mechanism by which sPLA2s exert their cellular effects. These findings raised a fundamental question as to whether sPLA2s function transcellularly by a paracrine mechanism or act on their originating cells by an autocrine mechanism or simply function intracellularly before secretion under physiological conditions. Previous studies have indicated that sPLA2s can work transcellularly to induce AA production in distal cells (11, 26). However, no direct experimental evidence has been reported for the transcellular action of sPLA2s under physiological conditions.
In this study we systematically measured the intracellular, autocrine, and paracrine actions of various sPLA2s under different conditions by means of a new real-time cellular PLA2 activity assay using two orthogonal fluorogenic phospholipid substrates. Our results provide direct evidence for the transcellular activity of sPLA2s both in model cell systems and under physiologically relevant conditions. The results also indicate that the relative importance of paracrine, autocrine, and intracellular pathways significantly varies depending on the nature of donor and acceptor cells as well as the biochemical properties of sPLA2.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Synthesis of a Red Derivative of PED6—25 mg of N-(6-((2,4-dinitrophenyl)amino)hexanoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (Avanti%20Polar%20Lipids">Avanti Polar Lipids) in CHCl3:CH3OH (9:1) was added to a flask, and the solvent was evaporated under N2. 5 ml of 50 mM Tris-HCl buffer, pH 7.4, containing 10 mM CaCl2, 0.1 M KCl was added to the lipid film and vortexed. To this solution 250 mg of Agkistrodon piscivorus piscivorus venom was added, and the PLA2 reaction was allowed to proceed overnight. The solvent was evaporated in vacuo, and the residue was dissolved in 1 ml CHCl3. Apreparativethinlayer chromatography was performed to purify 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] using CHCl3:CH3OH:H2O (65:25:4; v/v/v) as eluent (Rf = 0.7). 8 mg of this compound was dissolved in 8 ml of dry CH2Cl2 and 5.5 mg of succinimidyl 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a,diaza-s-indacene-3-propionate (Invitrogen), and 1.39 µl of triethylamine (molar ratio 1:1.2:1) was added to the solution. The reaction mixture was stirred for 48 h, and solvent was removed in vacuo. The residue was dissolved in a minimal volume of CH2Cl2, and the product was purified by preparative thin layer chromatography using CHCl3: CH3OH:H2O (65:25:4; v/v/v) as eluent (Rf = 0.85). The product is referred to as Red-PED6 hereafter. The concentrations of PED6 and Red-PED6 were determined spectrophotometrically using the known extinction coefficients of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid (BODIPY®; 
= 96,000 M-1 cm-1) and 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY 576/589®; = 83,000 M-1 cm-1), respectively (Invitrogen).
Preparation of HEK293 Cell Lines Stably Expressing sPLA2s—The cDNAs for human gIIAPLA2, gVPLA2, and gXPLA2 were cloned from the human brain cDNA library (Clontech) and ligated into the pFLAG vector (Sigma) to yield sPLA2s with C-terminal FLAG tags. Two µg of each plasmid was mixed with 4 µl of LipofectamineTM 2000 in 250 µlof Opti-MEM medium for 30 min and then added to cells that had attained 40-60% confluence in 6-well plates (Nunc) containing 0.5 ml of Opti-MEM medium. After incubation for 16 h, the medium was replaced with 1 ml of fresh culture medium. After overnight culture, the medium was replaced with 1 ml of fresh medium, and cells were incubated at 37 °C in an incubator flushed with 5% CO2 in humidified air. The cells were cloned by limiting dilution in 96-well plates in the culture medium supplemented with 1 mg/ml Geneticin (Invitrogen). After culture for 3-4 weeks, wells containing a single colony were chosen, and the expression of each sPLA2 was assessed by Western blotting using the FLAG antibody. The established clones were expanded and used for further experiments.
Western Blotting Analysis—HEK293 (or BEAS-2B) cells were collected from a 100-mm culture dish (Nunc). After washing with phosphate-buffered saline, the pellet was collected by centrifugation, then lysed in 70 µl of lysis buffer (20 mM Tris-HCl, 30 mM Na4P2O7, 50 mM NaF, 40 mM NaCl, 5 mM EDTA, pH 7.4) containing 1% Nonidet P-40, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, and 0.5% deoxycholic acid. After 10 min on ice, the cell lysates were centrifuged at 12,000 x g for 3 min to remove the cell debris. The supernatants were then mixed with 14 µl of gel loading buffer (0.125 M Tris-HCl, pH 6.8, 20% (v/v) glycerol, 4% sodium dodecyl sulfate, 0.005% bromphenol blue), and the mixtures were boiled for 5 min. The samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions using 16% acrylamide gels. The electrotransfer of proteins from the gels to polyvinylidene fluoride membrane was achieved using a semidry system (400 mA, 120 min). The membrane was blocked with 2% BSA for 60 min, then incubated with 1 µg/ml amounts of either the anti-human gVPLA2 monoclonal antibody (30) or FLAG antibody diluted in Tris-buffered saline plus 0.05% Tween 20 (TBS-T) overnight. The membranes were washed 3 times for 20 min with TBS-T. Goat anti-mouse IgG conjugated with horseradish peroxidase was diluted 3000-fold in TBS-T and incubated with polyvinylidene fluoride membrane for 60 min. The membrane was washed 3 times with TBS-T and assayed with a Super-Signal West Femto enhanced chemiluminescence system (Pierce).
AA Release from HEK293 Cells—Radiolabeling of non-transfected HEK293 cells was achieved by incubating the cells (106) with 0.05 µCi/ml [3H]AA (Amersham Biosciences) for 20 h at 37 °C. Unincorporated [3H]AA was removed by washing the cells 3 times with Dulbecco's modified eagles medium (DMEM) containing 0.2% BSA. These radiolabeled HEK293 cells were detached from the culture dishes by gentle agitation with phosphate-buffered saline containing 0.1 mM EDTA and added to HEK293 cells stably expressing gIIAPLA2, gVPLA2, and gXPLA2, respectively. Mixed cells were stimulated by 10 µM ionomycin for 20 min, and the stimulation was quenched by adding 3 ml of ice-cold DMEM. Cells and the medium were separated by centrifugation, and the radioactivity of pellet and supernatant was separately measured by liquid scintillation counting.
Spectrofluorometric sPLA2 Activity Assay Using PED6 and Red-PED6 Substrates—The PLA2-catalyzed hydrolysis of PED6 and Red-PED6 in sonicated mixed vesicles of POPS/cholesterol/POPG/PED6 (or Red-PED6) (107:31:20:1 in mole ratio; 50 µM final concentration) was carried out at 37 °C in 2 ml of 10 mM HEPES, pH 7.4, containing 0.16 M KCl and 1.2 mM Ca2+. The progress of hydrolysis was monitored as an increase in fluorescence emission at 520 nm with the excitation wavelength set at 488 nm for PED6. For Red-PED6, excitation and emission wavelengths were set at 579 and 589 nm, respectively. Spectral bandwidth was set at 10 nm for both excitation and emission. Values of specific activity were determined from the initial rates of hydrolysis.
Spectrofluorometric Assay of Transcellular sPLA2 Activity—sPLA2-transfected HEK293 cells (donor cells; 106 cells/ml) and non-transfected HEK293 cells (acceptor cells; 106 cells/ml) were seeded into each of six wells on a sterile Nunc Lak-TeKIITM chambered cover glass filled with 2 ml of DMEM containing 10% fetal bovine serum. Both sPLA2-transfected and non-transfected cells were incubated at 37 °C with 5% CO2 for 1 day. After the cells were washed once with Hank's balanced salt solution (HBSS), non-transfected cells were overlaid with 50 µl of 2 mM POPS/cholesterol/POPG/PED6 (107:31:20:1)-sonicated mixed vesicles in HBSS, whereas sPLA2-transfected HEK293 cells were overlaid with the corresponding Red-PED6 vesicle solution. After both types of cells were incubated for 1 h at 37°C with 5% CO2, labeled cells were rinsed 6 times with HBSS containing 1.2 mM Ca2+ and resuspended in DMEM without phenol red containing 10% fetal bovine serum. This medium was selected to minimize background fluorescence signals. sPLA2-transfected (106 cells/ml) and non-transfected (1 to 5 x 106 cells/ml) cells were mixed in a cuvette, and 1.2 mM Ca2+ (final concentration) was added to the medium. 10 µM ionomycin (final concentration) was then added to the mixture to initiate the sPLA2 release. The progress of hydrolysis of Red-PED6 was monitored as an increase in fluorescence emission (F) at 589 nm with the excitation wavelength set at 579 nm using a Hitachi F4500 fluorescence spectrophotometer. For the same cell mixture, the progress of hydrolysis of PED6 was separately measured with excitation and emission wavelengths set at 488 and 520 nm, respectively. The relative fluorescence intensity was then calculated as F/Fmax where Fmax was the fluorescence emission intensity (F) value obtained when cells were treated with an excess amount (0.1 µM) of N. n. atra PLA2.
Transcellular Activity of gVPLA2 in Heparinase-treated HEK293 Cells— Non-transfected HEK293 cells and gVPLA2-transfected HEK293 cells were seeded into 6-well plates (106 cells/ml) as described above. Both transfected and non-transfected cells were incubated at 37 °C with 5% CO2 for 1 day and were separately treated with heparinase I (1 unit/ml) for 90 min in phosphate-buffered saline at 37 °C. Heparinase-treated cells were washed 6 times with HBSS. gVPLA2-transfected and non-transfected HEK293 cells were then labeled with PED6 and Red-PED6, respectively, and mixed for sPLA2 activity measurements, as described above.
Confocal Microscopy Imaging of Transcellular sPLA2 Activity—BEAS-2B, RBL-2H3, and DLD-1 cells (106 cells/ml) were seeded into each of 8 wells on a sterile Nunc Lak-TeKIITM chambered cover glass that was placed with filled with 400 µl of DMEM and 10% fetal bovine serum and incubated at 37 °C with 5% CO2 for 1 day. Separately, human neutrophils or eosinophils were overlaid with 10 µl of POPS/cholesterol/POPG/PED6 (107:31:20:1) vesicle solution and incubated for 1 h at 37 °C with 5% CO2. After rinsing the labeled cells 6 times with HBSS containing 1.2 mM Ca2+, these cells were added to BEAS-2B, RBL-2H3, or DLD-1 cells in each chamber, and the mixed cells were incubated for 10 min. After cell stimulants (1 µM endothelin-1 for BEAS-2B or DLD-1 activation and 1 µg/ml 2,4-dinitrophenyl-specific IgE and 10 ng/ml 2,4-dinitrophenyl-conjugated BSA for RBL-2H3 activation) were added, imaging was performed with a Zeiss LSM 510 laser-scanning confocal microscope with the detector gain adjusted to eliminate the background autofluorescence. The fluorescence from the hydrolyzed PED6 was monitored with a 568 helium/neon laser and with a 580 line-pass filter. A63x (1.2 numerical aperture) water immersion objective was used for all experiments. The images were analyzed using the analysis tools provided in the Zeiss biophysical software package. Taking into account the diffusion of hydrolyzed products of PED6, the green fluorescence intensity value at a given time was determined as average value (Fav) over the total focal area. The relative fluorescence intensity was then calculated as Fav/Fmax where Fmax was the Fav value obtained when cells were treated with an excess amount (0.1 µM) of N. n. atra PLA2.
Purification of Human Neutrophils and Eosinophils—Human peripheral blood neutrophils were isolated as described previously (13). Human peripheral blood eosinophils were purified by negative selection using magnetic-activated cell sorting (AutoMACS, anti-CD16 magnetic beads, Miltenyi Biotec) from the blood (up to 1 unit) of normal, nonallergic, healthy donors under their informed consent and according to the guidelines established by the Institutional Review Board of the University of Illinois at Chicago as described previously (31). Total cell counts and eosinophil counts were performed using Randolph's stain, and the purity and the viability of purified eosinophils were assessed by Wright's/Giemsa differential staining and trypan blue dye exclusion, respectively. Both the purity and viability of isolated eosinophils was routinely >98%.
Measurement of AA and LTC4 Release from Eosinophils—Human eosinophils (106 cells/group) were labeled with 0.5 µCi of [3H]AA for 4 h at 37 °C. The unincorporated AA was washed 3 times with HBSS containing 1.2 mM CaCl2 and 0.2% BSA. Radiolabeled cells (106) were resuspended in 100 µl of the same buffer and placed onto 1 well of 12-well plates (Nunc) containing BEAS-2B cells (1-2 x 106 cells) and then stimulated with 1 µM endothelin-1, 3 µM ionomycin or 1 µM endothelin-1 plus 40 ng of anti-gVPLA2 antibody (or anti-gIIAPLA2 or anti-gXPLA2 antiserum) for 30 min. As a control, eosinophils were also activated with 3 µM ionomycin without BEAS-2B cells. The reaction was quenched by centrifugation, and the radioactivity in the cell pellet and the supernatant was separately measured by a two-channel liquid scintillation counter. LTC4 levels (pg/106 cells) in the supernatant were determined using an enzyme-linked immunoassay kit (Cayman).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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It has been reported that HEK293 cells contain undetectable amounts of these sPLA2s and that HEK293 cells stably expressing various types of sPLA2s release the enzymes when stimulated with agonists, such as calcium ionophore and interleukin-1 (10, 19-24). We first established three stable cell lines expressing gIIAPLA2, gVPLA2, and gXPLA2, all with a FLAG tag in their C termini. In vitro activity measurements using purified recombinant enzymes showed that the C-terminal FLAG tag did not affect the enzyme activity of the three enzymes (data not shown). Western blot analysis of each cell extract using a FLAG antibody showed that each cell line expressed 14-kDa sPLA2 that was not detectable in control HEK293 cells transfected with the mock FLAG vector (see Fig. 1).
To see if sPLA2s secreted from these cell lines can act on neighboring cells, we labeled non-transfected HEK293 cells with [3H]AA and added these labeled cells to the cultures of unlabeled HEK293 cell lines stably expressing gIIAPLA2, gVPLA2, and gXPLA2 in separate chambers. As the control, [3H]AA-labeled cells were added to unlabeled HEK293 cells transfected with the mock FLAG vector. When these sets of mixed cells were stimulated with 10 µM ionomycin for 30 min, the cell mixture containing gIIAPLA2 exhibited statistically insignificant transcellular AA-releasing activity, whereas the cell mixtures containing gVPLA2 and gXPLA2 displayed the significantly higher transcellular activity (see Fig. 2). A longer incubation (>1 h) led to larger increases in the transcellular activity of gVPLA2 and gXPLA2 over the control while showing a minimal effect on the transcellular activity of gIIAPLA2 (data not shown). Also, changing the order of mixing (i.e. adding sPLA2-transfected HEK293 cells to AA-labeled non-transfected HEK293 cells) did not have any detectable effect on the outcome of transcellular AA-releasing activities of these sPLA2s. These results not only establish that gVPLA2 and gXPLA2 are effectively secreted under our experimental conditions but also indicate that these sPLA2s have a definite ability to act transcellularly on neighboring or distal cells.
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7-fold higher fluorescence emission intensity than that of Red-PED6, presumably due to higher quantum yield of BODIPY®. As summarized in Table 1, all three sPLA2s consistently showed 4-5-fold higher specific activities for Red-PED6 than PED6 in the mixed vesicles. These differences may be due to the structural difference in the sn-2 acyl chain of the two lipids (Fig. 3A). Among sPLA2s, gVPLA2 and gXPLA2 were >10 times more active than gIIAPLA2. Most importantly, all three sPLA2s showed significant activities on these fluorogenic lipids, which resulted in large fluorescence signals. This validated our approach of using these substrates to quantitatively monitor sPLA2 activities on originating and target cells. Furthermore, cPLA2 showed <0.1% of sPLA2 activity toward these substrates, which practically precludes the contribution of cPLA2 in PLA2 activity assays using these substrates.
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To preclude the possibility that the observed transcellular activities were underestimated due to lower activities of sPLA2s toward PED6 than toward Red-PED6 (see Table 1), we performed the same sets of measurements after mixing donor and acceptor (1:3 ratio) cells labeled with PED6 and Red-PED6, respectively. As shown in Fig. 5, essentially the same trend was observed under these conditions; i.e. gIIAPLA2 (Fig. 5A) again showed strong preference for donor cells over acceptor cells, whereas gVPLA2 (Fig. 5B) and gXPLA2 (Fig. 5C) acted on acceptor cells with much higher efficiency. A minor difference was that gVPLA2 (Fig. 5B) and gXPLA2 (Fig. 5C) showed modestly increased transcellular activities than measured with reverse labeling (see Fig. 4, B and C). Collectively, these results demonstrate that gVPLA2 and gXPLA2 can act transcellularly on neighboring cells with high efficiency.
Role of HSPG in Transcellular Activity of sPLA2s—It has been reported that cell surface HSPG plays a critical role in the cellular entry of secreted sPLA2 (10, 19-24). To see how the presence of HSPG on the cell surface affects the transcellular activity of sPLA2s, we measured the effect of removing HSPG from donor and acceptor HEK293 cells by heparinase I treatment. We performed the spectrofluorometric cuvette assay with mixed donor (Red-PED6-labeled) and acceptor (PED6-labeled) cells (donor:acceptor, 1:3) as described above. When acceptor HEK293 cells were pretreated with heparinase I before mixing with gVPLA2 donor cells, PED6 hydrolysis in acceptor cells was dramatically reduced (Fig. 6A), underscoring the importance of HSPG binding in transcellular actions of gVPLA2. Interestingly, however, pretreatment of donor (i.e. gVPLA2-transfected) HEK293 cells with heparinase I before mixing with acceptor cells had only a modest (i.e. 20% reduction) effect on the Red-PED6 hydrolysis in donor cells (Fig. 6B). This suggests that Red-PED6 hydrolysis seen in HEK293 donor cells is mainly from the catalytic action of gVPLA2 before its secretion and that the autocrine re-internalization of gVPLA2 may not significantly contribute to overall Red-PED6 hydrolysis.
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Transcellular Activity of Endogenous gVPLA2 in Mixed Cells—To test if endogenous (i.e. not overexpressed) gVPLA2 can also perform transcellular actions, we measured the transcellular activities of gVPLA2 in two mixed-cell systems; RBL-2H3-neutrophils and DLD-1-eosinophils. In these mixed cell pairs, RBL-2H3 and DLD-1 cells serve as endogenous gVPLA2 donors, whereas neutrophils and eosinophils function as acceptor cells. It has been reported that RBL-2H3 (37) and DLD-1 (38) cells contain significant amounts of endogenous gVPLA2. Human eosinophils serve as an ideal gVPLA2 acceptor because they lack endogenous gVPLA2 (15). Human neutrophils contain endogenous gVPLA2 (39), but the release of endogenous gVPLA2 from neutrophils can be suppressed by selective stimulation of donor cells.
We first labeled the membranes of human neutrophils with PED6 and measured the hydrolysis of PED6 when these cells were mixed with RBL-2H3 cells, which were selectively stimulated with IgE and antigen. As shown in Fig. 7A, stimulation of RBL-2H3 cells led to the hydrolysis of PED6 incorporated in neutrophil membranes within 10 min. When labeled neutrophils alone were stimulated with the same agonist in the absence of RBL-2H3 cells, no PED6 hydrolysis was detected (data not shown). Consistent with our previous studies showing that exogenously added gVPLA2 induce phospholipid hydrolysis mainly at the outer plasma membrane of human neutrophils (13, 14), the green fluorescence signal was observed largely on the plasma membrane of neutrophils. In this case we were able to directly quantify the PED6 hydrolysis on the plasma membrane of neutrophils by microscopy (see the right panel of Fig. 7A). When the mixed cells were treated with the gVPLA2 antibody, which was shown not to cross-react with gIIaPLA2 and gXPLA2 (30, 38), the PED6 hydrolysis signal was greatly reduced (Fig. 7A), indicating that gVPLA2 was mainly responsible for the hydrolysis of PED6 in the plasma membrane of neutrophils. This notion was confirmed by the insignificant inhibitory effects by gIIaPLA2 and gXPLA2 antisera (data not shown). Antibodies for human sPLA2s were used for these measurements because those for rat sPLA2s were not available.
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because PED6 is a poor substrate for cPLA2 (15, 34) (see also Table 1). Again, the PED6 hydrolysis on eosinophils was not seen without DLD-1 cells under the same conditions (data not shown). Furthermore, the transcellular PED6 hydrolysis was largely abrogated by the gVPLA2 antibody (Fig. 7B, right panel), but not by gIIaPLA2 and gXPLA2 antisera (data not shown). Thus, although it is unknown whether DLD-1 cells contain endogenous gXPLA2, the data indicate that gVPLA2 mainly causes the transcellular lipolytic activity in this system. Collectively, these results show that endogenous gVPLA2 is capable of performing transcellular lipolytic catalysis.
Activation of Human Eosinophils by gVPLA2 from Bronchial Epithelial Cells—To demonstrate that the transcellular actions of gVPLA2 take place under pathophysiological conditions, we measured the effect of gVPLA2 released from human bronchial epithelial cells (BEAS-2B) on neighboring eosinophils. It has been shown that eosinophil-bronchial epithelial cell interactions play an important role in asthmatic airway inflammation (40-43). A recent reverse transcriptase-polymerase chain reaction measurement showed that BEAS-2B cells express mRNAs of gXPLA2, gVPLA2, and group IID PLA2 (44). To determine the amount of gVPLA2 protein expressed in BEAS-2B cells, we quantified the cellular amount of gVPLA2 by Western blotting using varying concentrations of recombinant gVPLA2 as a calibration standard. As shown in Fig. 7D, BEAS-2B cells express a small but definite amount (7 ng/106 cells) of endogenous gVPLA2 in the resting state. Upon activation for 20 min by endothelin-1, which is a powerful vasoconstrictor and bronchoconstrictor peptide that may be involved in the pathogenesis of bronchial asthma (45), endogenous gVPLA2 in BEAS-2B cells was no longer visible (data now shown), indicating that endothelin-1 potently induced the secretion of gVPLA2. Unfortunately, however, determination of gVPLA2 concentration in the medium by immunoassay or activity assay was unsuccessful, presumably because the secreted gVPLA2 concentration was extremely low and because a majority of gVPLA2 remained attached to cell surfaces.
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We also measured the release of AA and LTC4 from human eosinophils when gVPLA2 secretion from neighboring BEAS-2B cells was induced by endothelin-1. For these measurements, eosinophils were labeled with [3H]AA, and the release of AA and LTC4 was quantified by radioactivity measurement and enzyme-linked immunoassay, respectively. As shown in Figs. 8, A and C, eosinophils themselves did not release either AA or LTC4 in response to endothelin-1. Only when ionomycin was added to eosinophils to activate cPLA2 was the release of AA and LTC4 observed. In contrast, endothelin-1 caused large increases in AA (Fig. 8B) and LTC4 (Fig. 8D) release when eosinophils were mixed with BEAS-2B cells. These increases were greater than or comparable with those caused by the direct activation of eosinophils by ionomycin. Importantly, the AA and LTC4 release was abrogated by the treatment with the gVPLA2 antibody but not by anti-gIIAPLA2 and anti-gXPLA2 antisera. Collectively, these studies indicate that gVPLA2 can act transcellularly to induce inflammatory responses under physiologically relevant conditions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To provide more direct evidence for the transcellular activity of different sPLA2 isoforms under physiological conditions, we employed a systematic and quantitative approach. Our model cell studies using sPLA2-transfected and non-transfected HEK293 cells clearly show that gVPLA2 and gXPLA2, but not gIIAPLA2, are fully capable of acting on neighboring cells to liberate AA and other fatty acids. Ionomycin-induced secretion of gIIAPLA2 causes only a slight increase in AA release from neighboring cells over controls (Fig. 2) and also resulted in the preferential hydrolysis of fluorogenic substrates incorporated in donor cell membranes (Figs. 4 and 5). Together, these results indicate that gIIAPLA2 is not optimized for transcellular actions on mammalian cells. It should be noted that, however, gIIAPLA2 has been shown to have much higher transcellular activity on microbial cells that have high concentrations of anionic lipids and phosphatidylethanolamine on the cell surface membranes (46, 47).
Both gVPLA2 and gXPLA2 demonstrate high transcellular activities on mammalian cells under various conditions. The fact that high lipolytic activities of these sPLA2s were consistently seen with mixed suspended cells (see Figs. 4, 5, 6) indicates that these activities reflect true transcellular activities and do not require direct cell-cell contact. Because gXPLA2 has extremely low affinity for cell surface HSPG, the high transcellular activity of this sPLA2 should derive from its capability of binding and hydrolyzing phosphatidylcholine-rich membranes. Consistent with this notion, the transcellular activity of this sPLA2 is independent of the HSPG content on the surface of acceptor HEK293 cells. gVPLA2 is unique among all sPLA2s in that it can act on both the outer plasma membrane and internal membranes of mammalian cells due to its high activity on phosphatidylcholine membranes and high affinity for HSPG (13-15, 34). Strong dependence of the transcellular activity of gVPLA2 on the HSPG content of acceptor cells shows that HSPG binding is a critical step in the transcellular action of gVPLA2 and also suggests that the main site of transcellular gVPLA2 lipolytic action is internal membranes of acceptor cells. Our studies using heparinase-treated HEK293 cells also suggest that the hydrolysis of fluorogenic substrates in donor HEK293 cells by the three sPLA2s occurs largely before secretion, which is consistent with a recent report (25). It is not clear, however, whether this is a general phenomenon in all sPLA2-expressing mammalian cells or a non-physiological process specific to those cells overexpressing sPLA2s.
High transcellular activity of gVPLA2 in conjunction with its unique ability to act on both the outer plasma membrane and internal membranes of mammalian cells would allow this sPLA2 to play diverse and versatile roles under different physiological conditions. Our previous studies showed that exogenously added recombinant human gVPLA2 could induce AA liberation and leukotriene biosynthesis in unstimulated human neutrophils in a cPLA2-dependent manner (13, 14, 48). In the RBL-2H3-neutrophils mixed cell system, selective activation of RBL-2H3 cells causes the secretion and transcellular lipolytic action of gVPLA2 that is greatly inhibited by the gVPLA2-specific antibody. This suggests that neutrophils can be activated by gVPLA2 released from their neighboring mast cells under physiological conditions.
Eosinophils play a key role in airway inflammation and hyperresponsiveness. However, the mechanism by which eosinophil activation is triggered under pathophysiological conditions is not fully understood. We have recently shown that exogenously added human gVPLA2 can trigger AA and LTC4 release from human eosinophils in a cPLA2-independent manner (15, 48). To our knowledge, eosinophils may be the only cell type in which any sPLA2 has been shown to liberate AA totally independently of cPLA2. Because human eosinophils lack endogenous gVPLA2 (15), the transcellular activity of gVPLA2 on human eosinophils may play a key role in eosinophil activation. DLD-1 cells are colon epithelial cells that have been well characterized in terms of gVPLA2 secretion and regulation (38). Our results show that activation of DLD-1 cells leads to the strong transcellular lipolytic action of gVPLA2 on neighboring eosinophils. As reported previously, gVPLA2 seems to hydrolyze PED6 in both the plasma membrane and internal membranes of eosinophils (15). Because eosinophils may interact with epithelial cells under gastrointestinal inflammatory conditions, the observed transcellular activity of gVPLA2 in the DLD-1-eosinophil system may have some physiological relevance.
The transcellular activity of gVPLA2 is most dramatically demonstrated in the BEAS-2B-eosinophil mixed cell system. This system was selected because eosinophil-bronchial epithelial cell interactions have been shown to play an important role in asthmatic airway inflammation (40-43). Our previous studies showed that at least 10-100 nM exogenously added gVPLA2 was required to cause detectable AA and leukotriene releases from human neutrophils and eosinophils (13-15, 48). Because this relatively high concentration of gVPLA2 has not been detected in extracellular fluids, the physiological relevance of the above findings has been disputed (39). The cellular content of gVPLA2 in BEAS-2B cells is estimated to be 7 ng/106 cells, and thus, the maximal concentration of secreted gVPLA2 in the medium containing BEAS-2B cells (106 cells/ml) should be 0.5 nM under our experimental conditions (note that 30 min incubation is not long enough to induce gVPLA2 synthesis). The fact that the selective activation of BEAS-2B cells resulted in large increases in AA and LTC4 releases from eosinophils, which are abrogated by the gVPLA2-specific antibody, in our mixed cell system demonstrates that the transcellular action of gVPLA2 on neighboring cells is a highly efficient and potent process that does not require a high concentration of protein. The finding that a much lower concentration of gVPLA2 was required for eosinophil activation in this mixed-cell system than in exogenous addition of recombinant gVPLA2 raises an interesting possibility that donor cells may release a yet unidentified factor that assists in the transcellular action of gVPLA2. Taken together, these results imply that gVPLA2 released from human bronchial epithelial cells may readily trigger the activation of human eosinophils under physiological conditions, including asthmatic conditions.
In summary, the present study shows that the transcellular activity of sPLA2 varies among isoforms. Among three sPLA2s tested, gVPLA2 and gXPLA2 are capable of effectively performing transcellular lipolytic actions. In particular, gVPLA2 can be secreted and act on neighboring inflammatory cells under physiologically relevant conditions, thereby playing a key role in triggering the activation of inflammatory cells.
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1 These authors contributed equally to this work.
2 To whom correspondence should be addressed: Dept. of Chemistry (M/C 111), University of Illinois, 845 West Taylor St., Chicago, IL 60607-7061. Tel.: 312-996-4883; Fax: 312-996-2183; E-mail: wcho{at}uic.edu.
3 The abbreviations used are: PLA2, phospholipase A2; sPLA2, secretory PLA2; cPLA2, group IVA cytosolic PLA2; gIIaPLA2, group IIa PLA2; gVPLA2, group V PLA2; gXPLA2, group X PLA2; AA, arachidonic acid; BODIPY, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid; BODIPY 576/589, 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; BSA, bovine serum albumin; DMEM, Dulbecco's modified eagles medium; HBSS, Hanks' balanced salt solution; HSPG, heparan sulfate proteoglycans; LTC4, leukotriene C4; PED6, N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine triethylammonium salt; Red-PED6, a red derivative of PED6; CHO, Chinese hamster ovary; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; HEK cells, human embryonic kidney cells.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, acceptor) hydrolysis are shown as a function of time for different mixed cell systems with non-transfected HEK293 cells as acceptor cells. Donor cells were HEK293 cells stably expressing gIIAPLA2-FLAG (A), gVPLA2-FLAG (B), and gXPLA2-FLAG (C). The ratio of donor and acceptor cells was 1:3 in Fig. 4, A-C. The relative fluorescence intensity was then calculated as F/Fmax, where Fmax was the fluorescence emission intensity (F) value obtained when cells were treated with an excess amount (0.1 µM)of N. n. atra PLA2. Fig. 4, A-C show the data from a representative single set of measurements, but the same patterns were observed for at least three independent measurements. D, relative activity of gIIAPLA2 (•), gVPLA2 (
) on acceptor versus donor cells (expressed in terms of the ratio of maximal F/Fmax values after 30 min) as a function of the ratio of acceptor to donor cell numbers. Averages and S.D. were determined from three independent measurements.
