Cytosolic phospholipase A2 is responsible for ...

Cytosolic phospholipase A2 is responsible for prostaglandin E2 and leukotriene B4 formation in phagocyte-like PLB-985 cells: studies of differentiated cPLA2-deficient PLB-985 cells I. Furstenberg Liberty,*,†,1 L. Raichel,*,1 Z. Hazan-Eitan,* I. Pessach,* N. Hadad,* F. Schlaeffer,† and R. Levy*,2 Infectious Diseases Laboratory, Departments of *Clinical Biochemistry and †Internal Medicine, Faculty of Health Sciences, Ben-Gurion University of the Negev and Soroka Medical Center, Beer Sheva, Israel

Abstract: Our previously established model of cytosolic phospholipase A2 (cPLA2)-deficient, differentiated PLB-985 cells (PLB-D cells) was used to determine the physiological role of cPLA2 in eicosanoid production. Parent PLB-985 (PLB) cells and PLB-D cells were differentiated toward the monocyte or granulocyte lineages using 5 ⴛ 10ⴚ8 M 1,25 dihydroxyvitamin D3 or 1.25% dimethyl sulfoxide, respectively. Parent monocyte- or granulocyte-like PLB cells released prostaglandin E2 (PGE2) when stimulated by ionomycin, A23187, opsonized zymosan, phorbol 12-myristate 13-acetate, or formylMet-Leu-Phe (fMLP), and monocyte- or granulocytelike PLB-D cells did not release PGE2 with any of the agonists. The kinetics of cPLA2 translocation to nuclear fractions in monocyte-like PLB cells stimulated with fMLP or ionomycin was in correlation with the kinetics of PGE2 production. Granulocyte-like PLB cells, but not granulocyte-like PLB-D cells, secreted leukotriene B4 (LTB4) after stimulation with ionomycin or A23187. Preincubation of monocyte-like parent PLB cells with 100 ng/ml lipopolysaccharide (LPS) for 16 h enhanced stimulated PGE2 production, which is in correlation with the increased levels of cPLA2 detected in these cells. LPS preincubation was less potent in increasing PGE2 and LTB4 secretion and did not affect cPLA2 expression in granulocyte-like PLB cells, which may be a result of their lower levels of surface LPS receptor expression. LPS had no effect on monocyte- or granulocyte-like PLB-D cells. The lack of eicosanoid formation in stimulated, differentiated cPLA2-deficient PLB cells indicates that cPLA2 contributes to stimulated eicosanoid formation in monocyte- and granulocyte-like PLB cells. J. Leukoc. Biol. 76: 176 –184; 2004.

cleaved by PLA2, serves as a precursor of proinflammatory lipid mediators. These lipid mediators are synthesized in a broad range of tissue types and serve as autocrine or paracrine mediators to signal changes within the immediate environment. They play a role not only in inflammation, but they also regulate other critical, physiological responses [2]. Several types of PLA2 have been identified in phagocytic cells: cytosolic PLA2 group IV (cPLA2) [3, 4], Ca2⫹-independent PLA2 group VI (iPLA2) [5], and several secreted PLA2 (sPLA2-IIA, -V, and -X) [4, 6 – 8]. cPLA2 is present in the cytosol [9] and translocates to the membranes in a calcium-dependent manner [3]. It is activated by phosphorylation on serine residue (505) mediated by mitogen-activated protein kinase [10, 11]. The preference of cPLA2 for highly unsaturated fatty acids suggests that this enzyme may be a key contributor to cellular AA mobilization and lipid mediator formation. The low molecular weight (13–16 kDa) sPLA2 enzymes possess a broad selectivity to phospholipids and an absolute catalytic requirement for micromolar concentrations of Ca2⫹ [12, 13]. sPLA2-IIA is considered to be one of the more potent mediators of inflammatory processes in that its local and systemic levels are elevated in numerous inflammatory diseases [14] and because of its in vitro bactericidal activity [15–17]. sPLA2-V is expressed in P388D1 macrophage-like cells and mast cells, and its level dramatically changes upon endotoxin challenge [18, 19]. The novel group X has been cloned in humans and was shown to be expressed mainly in tissues and cells of the immune system [20, 21]. sPLA2-X was shown to elicit marked release of AA and to act as a high-affinity ligand of the PLA2 receptor [22]. iPLA2 has been detected in a variety of cells and tissues [5]. It has been proposed that iPLA2-VI in mouse macrophage-like P388D1 participates in phospholipid remodeling rather than in stimulus-induced AA or in prostaglandin formation [23–26]. In contrast, another study has shown that

Key Words: PGE2 䡠 LTB4 䡠 cPLA2 䡠 PLB cells 䡠 LPS 1

These authors contributed equally. Correspondence: Infectious Diseases Laboratory, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel. E-mail: [email protected] Received October 1, 2003; revised March 10, 2004; accepted March 13, 2004; doi: 10.1189/jlb.1003453. 2

INTRODUCTION Phospholipases A2 (PLA2) form a large family of phospholipidhydrolyzing enzymes [1]. Arachidonic acid (AA), which is 176

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iPLA2, in the same cells, is involved in release of AA and prostaglandin [27]. Recent studies, using inhibitors, have revealed that leukotriene B4 (LTB4) formation is mediated by cPLA2 in human neutrophils [28, 29]. Conversely, studies performed using initiation site-directed antisense against cPLA2 expression in human monocytes, which caused partial inhibition-prostaglandin formation, decreased, and leukotriene production did not alter [30, 31]. Moreover, it was suggested that immediate production of prostaglandin E2 (PGE2) is mediated by cPLA2, and delayed production is mediated by sPLA2-IIA [32]. We previously used the RNA antisense technique to create a p85 cPLA2-deficient cell model (PLB-D) in the human phagocyte myeloid cell line PLB-985 and provided evidence indicating an essential requirement for AA signals generated by p85 cPLA2 in activation of the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [33]. These PLB-D cells undergo normal differentiation toward the granulocyte or monocyte lineage, as detected by the expression of membrane activated complex-1 (MAC-1) and the NADPH oxidase components [33, 34]. Furthermore, normal phagocytic functions and receptor expression were detected in differentiated PLB-D cells (not shown). Thus, our model of differentiated PLB-D cells totally lacking the expression of cPLA2 offers a unique tool to determine the physiological role of cPLA2 in proinflammatory lipid mediator formation in monocyte- or granulocyte-like cells.

MATERIALS AND METHODS Cell culture and induction of differentiation PLB-985 cells and selected PLB-D clones were grown in a stationary suspension culture in RPMI-1640 medium, as described previously [33]. Optimal concentrations of 1.25% dimethyl sulfoxide (DMSO) or 5 ⫻ 10–8 M 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] were added to PLB cells or PLB-D cells (2⫻105 cells/ml) at their logarithmic growth phase to induce differentiation toward granulocyte- or monocyte-like cells, respectively [33, 34]. Differentiation was induced for 4 days and was determined by MAC-1 antigen expression detected by indirect immunofluorescence, as described previously [33].

Determination of AA metabolites Prostaglandin and leukotriene formation was measured in stimulated, differentiated PLB and PLB-D cells. Cells were harvested, washed with phosphatebuffer saline (PBS), and suspended in Hanks’ balanced saline solution (HBSS) at a concentration of 2 ⫻ 107 cells/ml. Cell suspensions were stimulated with 1 mg/ml opsonized zymosan (OZ), 10⫺7 M formyl-Met-Leu-Phe (fMLP), 50 ng/ml phorbol 12-myristate 13-acetate (PMA), or 2.5 ␮M A23187 for 20 min at 37°C. The cells were then centrifuged at 4°C, and the supernatants were immediately stored at –70°C. OZ was prepared as follows: Zymosan (20 mg) was incubated with 1 ml pooled human serum [lipopolysaccharide (LPS)-free)] for 1 h at 37°C and washed three times with HBSS buffer. PGE2 and LTB4 levels were determined by radiation immune assay using commercial kits (NEN Life Science Products, Boston, MA). The samples were analyzed within 48 h from the experiments.

Reverse transcription and polymerase chain reaction (RT-PCR) RT-PCR of the various PLA2 was performed, as described previously [35]. Total cellular RNA was extracted from 107 cells by the RNAzol B method of RNA isolation. The RNA pellet was precipitated with isopropanol, washed twice with 70% ethanol, and reprecipitated with 10% sodium acetate (3 M) and

70% ethanol. Total RNA was reverse-transcribed into cDNA at 37°C for 1 h using the Moloney murine leukemia virus RT (Gibco-BRL Life Technologies, Grand Island, NY) and primer p(dT)15 potassium salt (Boehringer Mannheim GmbH, Germany). The RT was then heat-inactivated at 65°C for 10 min, and cDNA was cooled to 4°C. cDNA was amplified via PCR using Thermus aquaticus DNA polymerase in conditions found to amplify cDNA molecules in a linear manner. cPLA2 was detected by amplification of a 628-bp using an upstream primer, 5⬘-CTCTTGAAGTTTGCTCATGCCCAGAC-3⬘, and a downstream primer, 5⬘-GCAAACATCAGCTCTGAAACGTCAGG-3⬘. sPLA2-IIA was detected by amplification of 240 bp using an upstream primer, 5⬘GAGCTAGGCCAGTCCATCT-3⬘, and a downstream primer, 5⬘-GCTCCCTCTGCAG TGTTTAT-3⬘. sPLA2-V was detected by amplification of 270 bp using an upstream primer, 5⬘-CCAAAGAGAACCCAGAGATGAAA-3⬘, and a downstream primer, 5⬘-TGGGGAGGCCTAGGAGCAGAG-3⬘. sPLA2-X was detected by amplification of 410 bp using an upstream primer, 5⬘-CGCGCCCGGCCAAATAAAATAA-3⬘, and a downstream primer, 5⬘-CAGCGACGGCAGTAGCAGGAGCAG-3⬘. The ␤-actin primer pairs amplified 445 bp using an upstream primer, 5⬘-GGGTCAGAAGGATTCCTATG-3⬘, and a downstream primer, 5⬘-GGTCTCAAACATGATCTGGG-3⬘. PCR amplification was performed in a microprocessor-controlled incubation system (Crocodile II, Appligene Inc., Plessanton, CA). The reaction was performed with 1 ␮M 5⬘ and 3⬘ primers in 50 ␮l reaction mixture using a step program (94°C, 1 min; 55°C, 30 s; 72°C, 2 min) followed by a 10-min final extension at 72°C for 25, 35, and 45 cycles. An 8-␮l sample of the completed reaction mixture was run on a 2% agarose gel stained with ethidium bromide.

Immunoblot analysis For immunoblot detection of cPLA2, cell lysates were prepared using 1% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 25 mM NaF, 10 ␮M ZnCl2, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 100 ␮M leupeptin [33]. Protein (100 ␮g) from cell lysates was separated by electrophoresis on 7.5% polyacrylamide sodium dodecyl sulfate (SDS) gels and blotted to nitrocellulose. For immunoblot detection of sPLA2, whole cells were lysed, and the samples were analyzed in nonreducing conditions on Tricine gels [36]. cPLA2 antibodies were raised by immunizing rabbits, as described earlier [37]. Recombinant sPLA2 proteins and antibodies against sPLA2-IIA, -V, and -X were kindly given to us by Dr. Michael H. Gelb, University of Washington (Seattle). In addition, immunizing rabbits with the specific C-terminal 20 amino acid sequence for each sPLA2 bound to a keyhole limpet hemocyanin carrier protein raised antibodies against sPLA2-V and sPLA2-IIA (Sigma Chemical Co., St. Louis, MO). The relative changes of the proteins were quantitated by densitometry in a reflectance mode (Hoefer Scientific Instruments, San Francisco, CA).

Preparation of nuclei Nuclei were separated from granulocyte-like PLB cells (2⫻107 cells) before and after stimulation as described for neutrophils [38, 39]. Stimulated cells were pelleted and resuspended in 600 ␮l ice-cold Nonidet P-40 (NP-40) lysis buffer (0.1% NP-40, 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotonin, and 1 mM PMSF). The cells were vortexed for 15 s, kept on ice for 5 min, and centrifuged at 500 g, 10 min, at 4°C. The resulting pellets (the nuclei-containing fractions) were then solubilized immediately in electrophoresis sample buffer and processed for SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot determination of cPLA2 and nuclear lamin B. Nuclear integrity was verified directly by light microscopy, which also revealed that intact cells were rarely observed in nuclei-containing fractions (less than 2%).

Immunofluorescence analysis of the surface expression of LPS receptors The surface expression of LPS receptors (CD14) was determined by mixing 1 ml undifferentiated or differentiated PLB or PLB-D cells at 1 ⫻ 106 cells/ml and 10 ␮g specific mouse anti-human CD-14 R-phycoerythrin (PE)-conjugated antibodies at 4°C for 30 min. For negative controls, PLB or PLB-D cells were stained with 10 ␮g mouse immunoglobulin G (IgG)2 R-PE-conjugated antibodies. The antibodies were bought from Caltag Llaboratories (Burlingame, CA). The cells were then washed three times in ice-cold PBS with 0.05% NaN3 and were analyzed by flow microfluorimetry on fluorescein-activated cell sorter

Liberty et al. Eicosanoid production by cPLA2 in phagocyte-like PLB cells

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Fig. 1. The expression of sPLA2 isotypes in PLB cells. Detection of sPLA2 isotypes sPLA2-IIA, sPLA2-V, and sPLA2-X (A) mRNA by RT-PCR amplification and (B) protein expression by SDS-PAGE immunoblot analysis in undifferentiated PLB cells (–) and in PLB cells differentiated toward the monocyte lineage using 5 ⫻ 10–8 M 1,25(OH)2D3 (Vit D) or the granulocyte lineage using 1.25% DMSO. Amplification of ␤-actin was performed as a control for mRNA levels. rec, Recombinant proteins. Four other experiments showed similar results.

(Becton Dickinson, Mountain View, CA). For each sample, 10,000 light scatter-gated, viable cells were analyzed. The median (median of fluorescence intensity) was calculated by subtracting the nonspecific fluorescence. The percent total is the percent of cells staining positive.

Statistical analysis The mean differences were analyzed by Student’s t-test.

RESULTS Expression of various sPLA2 isotypes in PLB cells Our previously developed model of PLB-D cells lacking the expression and activity of cPLA2 [33] was used to determine the role of cPLA2 in producing the AA metabolites, PGE2 and LTB4. In all experiments, the parent PLB-985 cell line and the G418-resistant clones, transfected with the empty pcDNA3 vector, were used as controls. As no changes were observed between the two controls, for simplicity, we present the results of the clones transfected with the empty pcDNA3 vector only (PLB cells). Undifferentiated PLB cells and PLB cells, differentiated with 5 ⫻ 10⫺8 M 1,25(OH)2D3 toward the monocyte phenotype or with 1.25% DMSO toward the granulocyte phenotype, expressed mRNA and protein of sPLA2-V, sPLA2-IIA, and sPLA2-X, as detected by RT-PCR (Fig. 1A) and immunoblot analysis (Fig. 1B). As shown in the figure, similar to the behavior of cPLA2 in our previous study [33], the levels of the three sPLA2 isotypes did not change during differentiation. Other types of sPLA2 could not be detected by RT-PCR using published, specific primers [28]. PLB cells differ from peripheral blood human neutrophils, which express only sPLA2-V and -X [28], and from human monocytes, which express sPLA2-IIA and -V (data not shown). The presence of the iPLA2-specific inhibitor, bromoenol lactone, did not affect AA release or eicosanoid production in granulocyte- or monocytelike PLB cells, suggesting that iPLA2 is probably not involved in these processes in differentiated PLB cells (not shown).

secreted by differentiated HL-60 cells [40]. PLB-D cells, differentiated to the monocyte lineage, did not release any PGE2 after stimulation with either of the agonists. Addition of 10 ␮M free exogenous AA to PLB and PLB-D cells, differentiated to the monocyte lineage, caused a high and similar secretion of PGE2 (1990⫾120 and 1950⫾176 pg/ml, respectively), indicating that the machinery responsible for production of PGE2 in monocyte-like PLB-D cells is normal, and the inability of these cells to produce PGE2 is a result of the absence of cPLA2. These results clearly demonstrate that cPLA2 is the PLA2 isotype responsible for the immediate production of PGE2 in monocyte-like cells and are in line with our previous study [33], demonstrating that cPLA2 mediates the release of AA in these cells. Incubation of parent monocyte-like PLB cells with 100 ng/ml LPS for 16 h caused a significant (P⬍0.001) secretion of PGE2 after stimulation for 15 min with OZ, PMA, fMLP, A23187, or ionomycin, reaching levels of 185 ⫾ 20, 196 ⫾ 15, 250 ⫾ 15, 910 ⫾ 58, or 1176 ⫾ 102 pg/ml, respectively (Fig. 3A). Moreover, LPS pretreatment by itself caused a basal release of PGE2 from unstimulated cells (65⫾7.8 pg/ml). Incubation of monocyte-like PLB-D cells with LPS did not cause any secretion of PGE2 after stimulation with the various agonists (Fig. 3B), suggesting that cPLA2 also mediates the production of PGE2 in LPS-pretreated monocytelike cells. To support this suggestion, the effect of LPS was studied on protein levels of the four PLA2 isotypes present in these cells. As shown in Figure 4, incubation of parent monocyte-like PLB cells with LPS caused a significant enhancement of cPLA2 expression but did not affect the level of

The role of cPLA2 in eicosanoid production in monocyte-like PLB cells The immediate release of PGE2 stimulated by various agonists was analyzed in parent PLB cells differentiated to the monocyte lineage by 1,25(OH)2D3. As demonstrated in Figure 2, PGE2 was appreciably released by OZ, PMA, fMLP, A23187, or ionomycin to levels of 98 ⫾ 6, 89 ⫾ 7, 100 ⫾ 8, 125 ⫾ 12, or 149 ⫾ 14 pg/ml, respectively, and no significant levels of PGE2 were released by resting cells. The levels of PGE2 secreted by differentiated PLB cells were in the range of those 178


Fig. 2. PGE2 release by monocyte-like PLB and PLB-D cells. PGE2 release was measured in the supernatants of PLB and PLB-D cells differentiated toward the monocyte lineage using 5 ⫻ 10–8 M 1,25(OH)2D3 in response to 15 min of stimulation with 1 mg/ml OZ, 50 ng/ml PMA, 10⫺7 M fMLP, 1 ␮M A23187, or 1 ␮M ionomycin (ION). Addition of 10 ␮M free exogenous AA restored normal PGE2 biosynthesis to monocyte-like PLB-D cells. Shown are the means ⫾ SEM of five experiments.


Fig. 3. The effect of preincubation with LPS on PGE2 secretion by monocytelike PLB and PLB-D cells. PLB cells (A) and PLB-D cells (B), differentiated toward the monocyte lineage, were preincubated with 100 ng/ml LPS for 16 h. PGE2 release was measured in the supernatants of cells stimulated for 15 min with 1 mg/ml OZ, 50 ng/ml PMA, 10⫺7 M fMLP, 1 ␮M A23187, or 1 ␮M ionomycin (ION). Shown are the means ⫾ SEM of five experiments.

either of the sPLA2 isotypes. The surface expression of LPS receptors (CD14) was similar in monocyte-like PLB and in monocyte-like PLB-D cells (Table 1), indicating that cPLA2 does not regulate LPS receptor expression. Taken together, the absence of PGE2 production in PLB-D cells lacking cPLA2 and the correlation between cPLA2 expression and PGE2 formation imply that cPLA2 is responsible for stimulated production of PGE2 in monocyte-like PLB cells before and after pretreatment with LPS. Monocyte-like PLB cells did not produce LTB4 when stimulated by either of the agonists before or after LPS pretreatment.

The role of cPLA2 in eicosanoid production in granulocyte-like PLB cells We next studied the role of cPLA2 in production of LTB4 and PGE2 in PLB cells differentiated with DMSO toward the granulocye lineage. As demonstrated in Figure 5A, stimulation of granulocyte-like cells with OZ, PMA, fMLP, A23187, or ionomycin induced secretion of PGE2 to levels of 72 ⫾ 8, 102 ⫾ 15, 494 ⫾ 50, 3105 ⫾ 210, or 2183 ⫾ 180 pg/ml, respectively. Secretion of PGE2 by granulocyte-like PLB cells was higher than that secreted by monocyte-like cells (Fig. 2), which is in contrast to the higher production of PGE2 by peripheral blood monocytes compared with granulocytes [41]. This finding implies that the model system does not absolutely reflect the normal counterparts, as was the case for superoxide production in differentiated HL-60 cells [42]. Granulocyte-like PLB-D cells did not produce any PGE2 after stimulation, but addition of 10 ␮M AA caused a significant PGE2 secretion, similar to that secreted by granulocyte-like PLB cells. Preincubation of

Fig. 4. The effect of preincubation with LPS on PLA2 isotype expression in monocyte-like PLB and PLB-D cells. (A) SDS-PAGE immunoblot analysis of cPLA2-IV, sPLA2-V, sPLA2-IIA, and sPLA2-X in monocyte-like PLB or PLB-D cells with 5 ⫻ 10– 8 M 1,25(OH)2D3 before and after preincubation with 100 ng/ml LPS for 16 h. Protein (100 ␮g) cell lysates were applied per lane. (B) Densitometry of the different protein levels is expressed in arbitrary units. Four other experiments showed similar results.

granulocyte-like PLB cells with 100 ng/ml LPS for 16 h resulted in a significant increase in PGE2 secretion when stimulated with OZ, PMA, fMLP, A2387, or ionomycin to levels of 120 ⫾ 21, 180 ⫾ 25, 980 ⫾ 72, 4950 ⫾ 310, or 3325 ⫾ 280 pg/ml, respectively (Fig. 5B). In contrast to LPS-treated monocyte-like cells, which secreted basal levels of PGE2 (Fig. 3B), resting granulocyte-like PLB cells pretreated with LPS did not secrete basal levels of PGE2. Preincubation of granulocyte-like PLB-D cells with LPS did not cause any secretion of PGE2 (Fig. 5C), suggesting that similar to monocyte-like PLB cells, cPLA2 is responsible for production of PGE2 in untreated and LPS-treated granulocyte-like cells. Although granulocyte-like cells stimulated with calcium ionophors secreted higher levels of PGE2 than monocyte-like cells, they were less affected by preincubation with LPS (about a 1.5-fold increase compared with a tenfold increase, respectively). This difference in the effect of LPS may be a result of the level of LPS receptor expression in both type of cells, which was much higher in PLB

TABLE 1.

Surface Expression of CD14 in Differentiated PLB and PLB-D Cells PLB % Total

PLB-D Median

% Total

Median

Undifferentiated 42.5 ⫾ 2.1 12.9 ⫾ 4.2 47 ⫾ 3.2 11.7 ⫾ 3.2 Vit D 95.6 ⫾ 2.9 295 ⫾ 12.3 96.2 ⫾ 1.9 304 ⫾ 5.7 Cells were differentiated for 4 days with 1,25(OH)2D3(Vit D) or with DMSO. The results are the mean ⫾ SEM of three independent experiments.


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or fMLP did not induce secretion of LTB4 in granulocyte-like PLB cells pretreated with LPS (not shown). Pretreatment of granulocyte-like PLB cells with LPS did not cause any release of LTB4 in unstimulated cells (Fig. 7), which is in accordance with their inability to produce PGE2 (Fig. 5). Granulocyte-like PLB-D cells pretreated with LPS did not secrete LTB4 before and after stimulation (Fig. 7B), indicating a role for cPLA2 in LTB4 production in LPS-treated cells. Immunoblot analysis of the various PLA2 isotypes in granulocyte-like PLB cells (Fig. 8) demonstrated that the level of cPLA2 did not change after LPS preincubation. There was a slight elevation in the levels of sPLA2-V but not sPLA2-II or sPLA2-X by LPS pretreatment in granulocyte-like PLB and PLB-D cells.

Eicosanoid formation and cPLA2 translocation to the nuclear fractions

Fig. 5. PGE2 release by granulocyte-like PLB and PLB-D cells. (A) PGE2 release measured in the supernatants of PLB and PLB-D cells differentiated toward the granulocyte lineage with 1.25% DMSO. Cells were stimulated for 15 min with 1 mg/ml OZ, 50 ng/ml PMA, 10⫺7 M fMLP, 1 ␮M A23187, or 1 ␮M ionomycin (ION). Addition of 10 ␮M free exogenous AA restored normal PGE2 biosynthesis to granulocyte-like PLB-D cells. (B and C) The effect of preincubation with 100 ng/ml LPS for 16 h on PGE2 release by granulocyte-like PLB and PLB-D cells, respectively, stimulated as in A. Shown are the means ⫾ SE of five experiments.

cells differentiated toward the monocyte lineage with 1,25(OH)2D3 than in PLB cells differentiated toward the granulocyte lineage with DMSO (Fig. 6). The production of LTB4 could be stimulated in granulocytelike PLB cells only with calcium ionophors and not with PMA, OZ, or fMLP (Fig. 7A) as has also been reported for granulocyte-like HL-60 cells [43]. These results are in accordance with findings for unprimed neutrophils, in which agonists such as fMLP have been shown to be poor stimulants for LTB4 biosynthesis [39]. Addition of 1 ␮M A23187 or 1 ␮M ionomycin for 15 min induced LTB4 secretion to levels of 3998 ⫾ 195 pg/ml and 4102 ⫾ 270 pg/ml, respectively, and granulocytelike PLB-D cells did not produce any LTB4 after stimulation. As for PGE2 production, addition of 10 ␮M free exogenous AA to PLB and PLB-D cells, differentiated to the granulocyte lineage, caused a high and similar secretion of LTB4 (5010⫾320 and 5320⫾275 pg/ml, respectively), indicating that the machinery responsible for production of LTB4 in granulocyte-like PLB-D cells is normal, and the inability of these cells to produce LTB4 is a result of the absence of cPLA2. Preincubation of granulocyte-like PLB cells with 100 ng/ml LPS for 16 h caused a low but significant (P⬍0.001) increase in LTB4 secretion to levels of 5545 ⫾ 265 and 5470 ⫾ 310 pg/ml after stimulation with A23187 and ionomycin, respectively (Fig. 7B), which was similar to its effect on PGE2 secretion stimulated by calcium ionophors (Fig. 5B). OZ, PMA, 180


The kinetics of eicosanoid formation was studied in differentiated PLB cells stimulated with ionomycin or fMLP. As shown in Figure 9A, in monocyte-like PLB cells, the production of PGE2 by ionomycin could be detected after 1 min of stimulation and reached a plateau at 3 min, and the production of PGE2 stimulated with fMLP could be detected only after 15 min, as shown previously for granulocyte-like PLB cells [44]. Eicosanoid generation has been shown to be regulated in part by perinuclear envelope localization or translocation of individual enzymes of leukotriene and prostaglandin biosynthesis [45]. In addition, perinuclear translocation of cPLA2, which has been demonstrated in a variety of cells including neutrophils and monocytes [38, 46 – 48], is in agreement with its role in leukotriene and prostaglandin formation. Thus, the kinetics of cPLA2 translocation to the nuclear fractions stimulated by these agonists was studied. As shown in Figure 9A, there is a correlation between the kinetics of PGE2 production and of cPLA2 translocation to the nuclear fractions. An increase in cPLA2 levels could be detected in the nuclear fractions already at 1 min when stimulated with ionomycin but only at 10 –15

Fig. 6. LPS receptor expression in differentiated PLB cells. Expression of LPS surface receptors (CD14) were examined in undifferentiated PLB cells and in differentiated PLB cells with 1,25(OH)2D3 (VitD) or DMSO by immunofluorescence analysis. For negative control, PLB cells were stained only with fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies. The ordinate and the abscissa represent the cell number and the fluorescence intensity in a logarithmic scale, respectively. The median for undifferentiated PLB cells and differentiated PLB cells with 1,25(OH)2D3 or DMSO is: 11.76, 289.03, and 19.46, rescpecively. Percent of cells staining positive for undifferentiated PLB cells and differentiated PLB with 1,25(OH)2D3 or DMSO is: 41%, 96.5%, and 63%, respectively. Data show one representative of three independent experiments.


Fig. 7. LTB4 release by granulocyte-like PLB and PLB-D cells. (A) LTB4 release was measured in the supernatants of PLB and PLB-D cells differentiated toward the granulocyte lineage with 1.25% DMSO. Cells were stimulated for 15 min with 1 mg/ml OZ, 50 ng/ml PMA, 10⫺7 M fMLP, 1 ␮M A23187, or 1 ␮M ionomycin (ION). Addition of 10 ␮M free exogenous AA restored normal LTB4 biosynthesis to granulocyte-like PLB-D cells. (B) The effect of preincubation with 100 ng/ml LPS for 16 h on LTB4 release by granulocyte-like PLB and PLB-D cells before stimulation (–) and after stimulation with 1 ␮M A23187 or 1 ␮M ionomycin (ION). Shown are the means ⫾ SEM of five experiments.

min when stimulated with fMLP. Similar results were obtained in our recent study [44] in granulocyte-like PLB cells stimulated with fMLP and ionomycin. As LPS caused a significant elevation of stimulated PGE2 production and cPLA2 expression in monocyte-like PLB cells (Figs. 3A and 4), its effect was analyzed during 15 min of stimulation. Preincubation of monocyte-like PLB cells with LPS for 16 h caused a basal secretion of PGE2, which was enhanced after stimulation with ionomycin (Fig. 9B). Pretreatment of the cells with LPS induced translocation of cPLA2 to the nuclear fractions, as shown in the immunoblot analysis (Fig. 9, B and C). This translocation is probably responsible for the basal secretion of PGE2 in resting

Fig. 8. The effect of preincubation with LPS on PLA2 isotype expression in granulocyte-like PLB and PLB-D cells. SDS-PAGE immunoblot analysis of cPLA2-IV, sPLA2-V, sPLA2-IIA, and sPLA2-X in differentiated PLB cells or PLB-D cells with 1.25% DMSO before and after preincubation with 100 ng/ml LPS for 16 h. Protein (100 ␮g) cell lysates were applied per lane. The densitometry of the different proteins is expressed in arbitrary units. Four other experiments showed similar results.

Fig. 9. The kinetics of PGE2 production and perinuclear translocation of cPLA2 in differentiated PLB cells. (A) PGE2 production and SDS-PAGE immunoblot analysis of cPLA2 in nuclear fractions of monocyte-like PLB cells stimulated with 10⫺7 M fMLP or 1 ␮M ionomycin (ION) during 15 min of stimulation. (B and C) The effect of preincubation with 100 ng/ml LPS for 16 h on PGE2 production and cPLA2 translocation to the nuclear fractions as a function of time, stimulated with ionomycin (ION) or fMLP, respectively. Immunoblot analysis of the nuclear marker, lamin B, confirmed equal amounts of nuclear fraction in each sample.


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cells before stimulation (Figs. 3A and 9, B and C). Stimulation of LPS-treated monocyte-like cells with ionomycin caused a significantly increased release of PGE2, although there were no significant changes in the levels of cPLA2 protein detected in the nuclear fractions (Fig. 9B). Stimulation of LPS-treated monocyte-like cells with fMLP not only increased PGE2 levels but also changed the kinetics of its production (Fig. 9C). Under these conditions, an increase in cPLA2 levels could be detected in the nuclear fractions as early as 1 min after stimulation (Fig. 9C). The levels of PGE2 secreted by LPS-treated monocyte-like cells stimulated with ionomycin or fMLP were significantly higher than the levels of cPLA2 detected in the nuclear fractions (Fig. 9, B and C) and may be a result of the effect of LPS on other enzymes participating in PGE2 production.

DISCUSSION The PLA2 isotype responsible for the release of AA and the production of proinflammtory mediators from stimulated cells has been the subject of some controversy in recent years. The results of the present study demonstrate that cPLA2-deficient, differentiated PLB cells did not release PGE2 or LTB4 before or after LPS treatment. Furthermore, there was a correlation between the kinetics of stimulated eicosanoid production and cPLA2 translocation to the nucleus (Fig. 9A). These results indicate that cPLA2 and not sPLA2 is the enzyme responsible for production of PGE2 and LTB4 in stimulated, phagocyte-like PLB cells before and after LPS treatment, which is in contrast to the suggestion that endogenous sPLA2 is responsible for the immediate [25, 49] or delayed formation of eicosanoid [25, 50, 51]. Similar to our results, cPLA2 has been shown to mediate immediate prostaglandin biosynthesis in human monocytes [30], in rat peritoneal macrophages [52], and in a number of cell systems, such as human platelets [53], rabbit aortic smooth muscle cells [54], and rabbit coronary endothelial cells [55]. As in human neutrophils [28, 29] and bone marrow-derived mast cells, we found that cPLA2 is responsible for the production of LTB4 in granulocyte-like PLB cells (Fig. 7). In addition, similar to the effect of LPS in increasing the expression of cPLA2 and the production of PGE2 in monocyte-like PLB cells (Figs. 3 and 4), preincubation of macrophages [52], monocytes [31], or liver macrophages [56] with LPS caused an elevation of cPLA2 levels and activity and increased PGE2 production, and the expression of sPLA2-IIA and -V gradually declined or was not affected. Correlative evidences for the participation of cPLA2 in growth factor- or cytokine-mediated prostaglandin formation have also been reported. For example, macrophagecolony stimulating factor stimulated cPLA2 activity and increased prostaglandin synthesis in human monocytes [57]. Interleukin-1␤ induced the synthesis and activity of cPLA2 and the release of PGE2 in human lung fibroblasts [58] and in amnion-derived WISH cells [59]. In contrast to monocyte-like PLB cells, the increased production of LTB4 and PGE2 in granulocyte-like PLB cells after LPS treatment did not result from an increase in cPLA2 levels, 182


as this treatment did not affect the expression of cPLA2 in these cells (Fig. 8). The difference in the effect of LPS on monocyte-like and granulocyte-like PLB cells may be a result of the levels of LPS receptors in both types of cells. The low levels of surface expression of LPS receptors in granulocytelike PLB cells may explain why LPS did not affect cPLA2 expression and caused only a moderate increase in eicosanoid formation in comparison with monocyte-like PLB cells. This increase in eicosanoid formation might possibly be a result of a low effect of LPS on other enzymes participating in their production, as has been demonstrated for cyclooxygenase-2 expression in human neutrophils [60]. LPS induced a slight elevation in the level of sPLA2-V in granulocyte-like PLB cells (Fig. 8) in accordance with its effect in alveolar macrophagelike cells and rat mastocytoma RBL-2H3 cells [18, 19]. However, sPLA2-V is probably not responsible for eicosanoid formation in granulocyte-like PLB cells, as it is expressed in granulocyte-like PLB-D cells, which did not produce LTB4 or PGE2. Although sPLA2-V has been shown to induce leukotriene biosynthesis in human neutrophils, in this study, it was added exogenously [61]. Hence, we may conclude that cPLA2 contributes to eicosanoid formation in granulocyte-like PLB cells before and after LPS treatment. The importance of cPLA2 in providing AA for prostaglandin and leukotriene biosynthesis has been demonstrated in two separate reports involving cPLA2 knockout mice [62, 63], where it was shown that peritoneal macrophages from these mice were unable to synthesize prostaglandin or leukotriene after stimulation with A23187 or LPS. Several studies using inhibitors have suggested that prior activation of cPLA2 is necessary to regulate gene expression of sPLA2-IIA in fibroblastic 3Y1 cells [64] and in gastric epithelial cells [65] and of sPLA2-V in mouse macrophage cell line P388D1 cells [66]. Our results, using cPLA2-deficient PLB cells, demonstrated that there are no differences in the expression of the various sPLA2 isotypes in granulocyte-like and monocyte-like PLB cells expressing cPLA2 or in granulocytelike and monocyte-like PLB-D cells lacking the expression of cPLA2 (Figs. 4 and 8). These findings clearly indicate that cPLA2 is not responsible for regulation of sPLA2-IIA, -V, or -X expression in PLB cells differentiated to both phenotypes. In conclusion, the ability of PLB cells to undergo differentiation toward monocyte and granulocyte phenotypes provides a tool to study the production of eicosanoid in both lineages. Using PLB-D cells totally lacking cPLA2, we were able to show that cPLA2 is responsible for production of PGE2 and LTB4 in phagocyte-like cells. Monocyte-like PLB cells produced lower levels of stimulated PGE2 in comparison with granulocyte-like PLB cells, but they were much more affected by LPS, probably as a result of the much higher levels of surface LPS receptor expression. Pretreatment of monocyte-like cells with LPS caused an increase in cPLA2 expression and an enhancement in PGE2 production, and pretreatment of granulocyte-like cells with LPS did not affect cPLA2 levels and caused only a moderate increase in eicosanoid formation. The different regulation of cPLA2 expression and of eicosanoid formation in monocyte- and granulocyte-like PLB cells suggests the specificity of these processes to cell type. http://www.jleukbio.org

ACKNOWLEDGMENTS This research was supported by a grant from the Israel Sciences Foundation founded by the Israel Academy of Sciences and Humanities 438/03 and by a grant from the Ministry of Health, Israel 5374. We thank Dr. M Gelb of the University of Washington (Seattle) for supplying recombinant sPLA2 proteins and sPLA2 antibodies. I. F. L. and L. R. contributed equally to the study.

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