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RESEARCH COMMUNICATION |
a Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec and Université Laval.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: priming • polymorphonuclear leukocytes • free arachidonic acid • translocation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Much of the effort aimed at understanding the regulation of the biosynthesis of LTB4 and other arachidonic acid (AA) metabolites in PMNL has involved the investigation of isolated PMNL stimulated with a potent nonphysiological agonist, the calcium ionophore A23187. These studies have led to the observations that, at least in A23187-stimulated PMNL, 5-LO is translocated from the cytosol to nuclear-associated membranes after cell stimulation, i.e., in proximity to the five lipoxygenase-activating protein (FLAP), a protein localized in the nuclear envelope and previously demonstrated to be required for LT biosynthesis (11–14). The exact function(s) of FLAP is not fully understood, but it may facilitate the utilization of arachidonic acid by 5-LO (15). Although agonists like fMLP or PAF can induce the release of AA in unprimed PMNL and activate 5-LO, they are nevertheless poor stimuli for LTB4 biosynthesis. Pretreatment of cells with cytokines enhances agonist-induced 5-LO activation (16, 17), but it is not known by what mechanism(s) such an enhanced activation may occur.
As for AA availability, the enzyme(s) responsible for AA hydrolysis from phospholipids after cell stimulation have not been unequivocally identified. Depending on the cell type and the stimulus, both the 85 kDa AA-specific cytosolic phospholipase A2 (cPLA2) and the 14 kDa type II secreted PLA2 (sPLA2) have been shown to be responsible for stimulated AA release (18–25). In fact, in ionophore A23187-stimulated human monocytes, AA release mediated by cPLA2 appears to be utilized by cyclooxygenases whereas lipoxygenases may preferentially utilize AA released by the action of sPLA2 (26). Human PMNL possess both enzymes, and the question of which PLA2 is responsible for AA release for subsequent LTB4 biosynthesis remains ambiguous (27, 28). Specific inhibitors of sPLA2 block ionophore A23187-induced LTB4 biosynthesis in human PMNL (29, 30) whereas LPS, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor 
(TNF-), as well as other compounds such as hydroxy fatty acids and diacylglycerol (9, 31–36), have been shown to phosphorylate cPLA2, an event associated with an enhancement of the enzyme's activity and necessary for an increase in AA release from stimulated cells (37, 38). Similarly, treatment of PMNL with GM-CSF enhances the subsequent translocation of cPLA2 to cellular membranes after cell stimulation (34). Although these events are associated with the activation of the cPLA2, the respective contributions of cPLA2 and sPLA2 for AA release utilized for subsequent LTB4 biosynthesis by PMNL have still not been clearly delineated.
In the present study, we sought to explain the priming effect of LPS for the enhanced biosynthesis of leukotrienes in PMNL after fMLP stimulation. We have dissected the effect of priming on key events leading to receptor-mediated AA release and its transformation to 5-LO products. These results provide a more complete picture of how agents like LPS can potentiate otherwise refractory human PMNL for lipid mediator biosynthesis at sites of inflammation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A23187 and fMLP were prepared in DMSO that had been filtered on alumin. LPS was dissolved in 0.9% NaCl. Rabbit polyclonal anti-5-LO (5LO 32) was kindly supplied by Drs. Jillian F. Evans and Philip Vickers and anti-cPLA2 (MF-140) by Dr. Philip Weech of Merck Frosst (Dorval, Canada). The enhanced chemiluminescence (ECL) detection kit was purchased from Amersham Canada (Oakville, Ontario, Canada). Immobilon-P PVDF blotting membrane was from Millipore (Mississauga, Ontario, Canada). Monoclonal antibody CC-3 was a generous gift from Dr. Michel Vincent (Université Laval, Québec City, Canada). Ficoll-Paque (Pharmacia, Montréal, Canada) and water used for the preparation of various solutions were tested for endotoxin content using the Limulus amebocyte lysate assay (Whittaker Bioproducts, Walkersville, Md.) and found to contain endotoxin in low pg/ml levels. SB 203347 was a generous gift from Drs. James Winkler and Lisa Marshall of SmithKline Beecham (King of Prussia, Pa.). All solutions and reagents used for cell preparation and priming were sterile.
Isolated cell preparations
Venous blood was obtained from healthy donors and collected into 10 cc glass tubes (100x16 mm Vacutainer, Becton Dickinson, Rutherford, N.J.) containing 143 USP units of heparin. All donors had normal differential leukocyte counts. PMNL were isolated from peripheral blood after dextran sedimentation and centrifugation on Ficoll-Paque cushions as previously described (39). Final preparations contained 95% PMNL and viability was above 95% as assessed by trypan blue exclusion.
Priming and stimulation of cells
The cells were suspended in HBSS supplemented or not with 10% autologous plasma (heparinized) and incubated in the presence or absence of LPS (1 µg/ml) for 30 min at 37°C, unless otherwise indicated. The cells were washed once by centrifugation, resuspended in HBSS (37°C), and stimulated with fMLP (0.1 µM) for the indicated periods of time. In some experiments, cells were washed and resuspended in HBSS containing 0.1% BSA and then stimulated or not with fMLP. For determination of 5-LO products, reactions were stopped by the addition of 1 vol of ice-cold methanol/acetonitrile (1:1, v/v) containing 12.5 ng each of PGB2 and 19-hydroxy-PGB2 as internal standards; the samples were processed and analyzed by reverse phase-high-performance liquid chromatography (RP-HPLC) using an on-line extraction procedure as described previously (40).
Assay of cellular 5-lipoxygenase activity
The activity of cellular 5-LO was determined as previously described (1). Briefly, isolated PMNL that had been primed with LPS or not for the indicated periods of time were washed and resuspended in HBSS at 37°C; 15-HpETE (3.0 µM) was added to the cell suspensions 5 s prior to the addition of fMLP or its diluent. After 3 min, the reactions were stopped with 1 vol of ice-cold methanol/acetonitrile (1:1, v/v) containing 12.5 ng of the internal standards PGB2 and 19-OH-PGB2. In some experiments, the 15-HpETE was added to the reaction mixtures at the same time as fMLP, or at various times after stimulation with fMLP, and the reactions were allowed to proceed for another 2 min before being stopped as described above. The samples were stored at -20°C until analysis by RP-HPLC.
Determination of free arachidonic acid in isolated PMNL
Isolated PMNL were primed with LPS and washed as described above. Cells were then stimulated with 0.1 M fMLP at 37°C for 2 min and the incubations were terminated by the addition of 2 vol of ice-cold methanol containing 20 ng of D8-AA as an internal standard. Samples were processed for HPLC analysis and the HPLC fractions containing AA (determined by using a 14C-AA standard) were collected. The samples were evaporated under reduced pressure (using a Speed Vac model SVC 100D, Savant Instruments Inc., Farmingdale, N.Y.) and redissolved into 100 µl of acetonitrile. AA was assayed by liquid chromatography-mass spectrometry using a nebulizer-assisted electrospray (ion spray) interface coupled to a triple-quadrupole MS (API-III, PE Sciex, Thornhill, Ont., Canada), as previously described (3).
SDS-PAGE analyses and immunoblot procedures
For the preparation of cellular proteins for immunoblotting with anti-cPLA2, cells were incubated under the conditions described at 37°C and reactions were stopped by the addition of 2 vol of acidified (0.01 M HCL) ice-cold methanol (41). Samples were stored at -70°C for at least 3 h and then centrifuged at 2000 x g for 15 min; the resulting pellet was resuspended in sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM dithiothreitol, 10% glycerol, 0.01% bromophenol blue, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF) and boiled for 5 min. Samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (42) on 9% acrylamide gels for cPLA2 and 5-LO determination. Proteins were then transferred at 0.5 A for 4–15 h at 4°C onto an Immobilon-P PVDF blotting membrane. Transfer efficiency was visualized by Ponceau red staining. For the determination of cPLA2 or 5-LO, the membranes were soaked for 30 min at 25°C in TBS (25 mM Tris-HCl, pH 7.6, 0.2 M NaCl, 0.15% Tween 20) containing 5% dried milk (w/v), blotted with anti-cPLA2 or anti-5-LO, and revealed by using a horseradish peroxidase-coupled monoclonal antibody and the ECL detection kit.
Preparation of nuclei
Neutrophils (2x107 cells) incubated under the described conditions were pelleted and resuspended in 600 µl of ice-cold 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 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF). The cells were vortexed for 15 s, kept on ice for 5 min, and centrifuged at 300 x g (10 min, 4°C). The resulting supernatants (i.e., the nonnuclear fractions) and pellets (the nuclei-containing fractions) were then immediately solubilized in electrophoresis sample buffer and processed for SDS-PAGE and immunoblot determination of the 5-LO protein content. To assess the purity of these preparations, control immunoblots were routinely performed using a monoclonal antibody (clone 13F6) directed against a PMNL plasma membrane-specific antigen and using CC-3, a nuclear-directed monoclonal antibody as previously reported (14). Nuclear integrity was verified directly by light microscopy, which also revealed that intact cells were rarely observed in the nuclei-containing fraction (less than 3%).
Translocation of cPLA2
PMNL were incubated at 5 x 106 cells/ml for the indicated times at 37°C in the presence of 1 µg/ml LPS or its diluent in HBSS containing 10% plasma. The cells were washed once, resuspended in HBSS (37°C) at 20 x 106 cells/ml, and incubated with fMLP (0.1 µM) or its diluent for 2 min. The reactions were stopped with 1 vol of ice-cold HBSS and the cells were immediately pelleted, resuspended in ice-cold sonication buffer (0.25 M sucrose, 10 mM Hepes, pH 7.4, 1 mM EGTA, 1 mM PMSF, 10 µg/ml leupeptin and 10 µg/ml aprotinin), and sonicated for 20 s using a Branson Sonifier (Branson Ultrasonics, Danbury, Conn.) at a setting of 1.5 with a constant duty cycle. The resulting sonicate was centrifuged for 15 min at 10,000 x g (4°C) to remove cell debris and the supernatant was then centrifuged at 100,000 x g (4°C) for 45 min. The resulting pellet (membrane fraction) was resuspended in electrophoresis sample buffer and boiled for 5 min. The samples were then subjected to electrophoresis and immunoblotting for determination of cPLA2 as described above. The integrated optical density of bands revealed by immunoblotting was measured using a BioImage Visage 110s from Millipore (Ann Arbor, Mich.) with whole band analysis software.
Measurement of intracellular calcium concentration [Ca2+]i
Fura-2 fluorescence was monitored as previously described (43). Briefly, cells (1x107/ml) were incubated for 30 min with 1 µM Fura-2/AM at 37°C. The cells were washed, resuspended at 5 x 106/ml, and then incubated with or without 1 µg LPS/ml for 30 min in HBSS containing 10% plasma. The cells were then washed, resuspended at 5 x 106/ml, and transferred into the thermally controlled (37°C) and magnetically stirred cuvette compartment of the spectrofluorometer (Aminco-Bowman series 2, SLM-Aminco, Urbana, Ill.). The excitation and emission wavelengths for Ca2+ measurements were 340 and 510, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-oxidation products of LTB4, 20-hydroxy-LTB4 (20-OH-LTB4) and 20-carboxy-LTB4 (20-COOH-LTB4). The nonenzymatic hydrolysis products of LTA4, 6-trans-LTB4 and 12-epi-6-trans-LTB4, were not detected in these experiments, suggesting that the LPS/fMLP-generated LTA4 may not be available for transcellular biosynthesis to leukotrienes and lipoxins by other cell types (44, 45). The priming effect of LPS was rapid, a maximal capacity for the biosynthesis of 5-LO products being observed after 30 min of preincubation (
Fig. 1);
the synthetic capacity for 5-LO products decreased when cells were preincubated with LPS for longer times, and the priming effect of LPS was no longer detectable at 120 min. These priming kinetics are essentially identical to those previously described in the LPS-priming of whole blood (3). In the absence of preincubation with LPS, stimulation of 5 x 106 cells with fMLP resulted the biosynthesis of products near or below the detection limits of the assay (2 pmol for LTB4).
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Kinetics of priming for arachidonic acid release
Having established that preincubation with LPS transiently enhances the capacity for LTB4 and 5-HETE biosynthesis in PMNL, the next series of experiments were performed to determine whether these changes in the capacity for leukotriene biosynthesis were due to changes in AA availability or in the capacity to transform AA (or both). Therefore, the kinetics of the priming effect of LPS for subsequent AA release by fMLP stimulation were determined. The capacity for AA release in PMNL preincubated with LPS gradually increased over 30 min ( Fig. 2),
but then decreased in cells exposed to LPS for longer preincubation times. In the absence of stimulation, the basal levels of free AA were the same in primed and unprimed cells, whereas fMLP stimulation in the absence of priming resulted in little or no detectable increase in free AA levels. Based on the maximal quantities of AA release and biosynthesis of 5-LO products, one could estimate that approximately 40% of the release AA is converted to 5-LO products. Although the nonconverted AA is eventually reesterified under these experimental conditions, it is possible that under physiological conditions the remaining AA could be available for potential extracellular processes and be utilized by other cells.
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Kinetics of LPS priming of 5-LO activity
After determining that priming for the release of AA followed the same time course as that of LTB4 biosynthesis, the effect of LPS priming on 5-LO activation was investigated. In the absence of stimulation with agonist, the basal measured activity of cellular 5-LO was not different in LPS-primed and unprimed cells (data not shown). However, in cells stimulated with 0.1 µM fMLP, 5-LO activity (measured during the first 3 min poststimulation) was greater in LPS-primed cells, with maximal activity observed in cells primed with LPS for 30 min prior to stimulation ( Fig. 3A).
As with leukotriene biosynthesis and AA release, the kinetics of LPS priming for the activation of 5-LO was transient although the resolution of this priming effect was more gradual than that observed for leukotriene biosynthesis and AA release. Little priming effect for 5-LO activity remained when preincubations with LPS were extended to 4 h (data not shown). These results indicate that the increased biosynthesis of 5-LO products in LPS primed cells is due to a combination of an enhanced availability of free AA coupled to an increased capacity of 5-LO to transform AA.
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We previously demonstrated that the release of AA after fMLP stimulation of LPS-primed cells is immediate, with maximal release after 2 min, but transient (basal levels at 5 min) (3); therefore, it was important to determine whether 5-LO activation occurs in the same time frame as AA release after stimulation with fMLP. The kinetics of 5-LO activation after cell stimulation were determined by addition of the exogenous substrate (15-HpETE) simultaneously with fMLP or at various times after fMLP addition. Stimulation of unprimed cells with 0.1 µM fMLP resulted in maximal activation of 5-LO within 10 s and then in a rapid decline to baseline (unstimulated) levels within 1 min poststimulation (
Fig. 3B). In cells that were preincubated with LPS (1 µg/ml) for 30 min, stimulation with 0.1 µM fMLP resulted in a similarly rapid but greater (two- to fivefold, depending on the donor) increase in cellular 5-LO activity than that of nonprimed cells. The subsequent decrease in activity observed with time after stimulation was attenuated in LPS-treated cells, and 5-LO activity remained significantly above baseline (unstimulated) levels for at least 4 min after stimulation with fMLP. Therefore, in addition to increasing the availability of AA and 5-LO activation, the preincubation of PMNL with LPS results in an increased temporal overlap of AA release and 5-LO activation after fMLP stimulation, thus resulting in a greater capacity to synthesize 5-LO products.
Inhibition of phospholipase(s) A2
Both the 14 kDa sPLA2 and the 85 kDa cPLA2 have been implicated in AA release from stimulated PMNL (30, 46, 47). To determine which PLA2 isotype is responsible for the enhanced release of AA by LPS, LPS-primed PMNL were treated with sPLA2 (SB 203347) (30) or cPLA2 (MAFP) (48) inhibitors prior to their stimulation with fMLP. As shown in Fig. 4,
the treatment of PMNL with the cPLA2 inhibitor MAFP caused a concentration-dependent inhibition of AA release in primed stimulated cells, whereas the sPLA2 inhibitor SB 203347 was without effect on AA release at concentrations up to 5 x 10-6 M. These experiments clearly suggest that AA release after the stimulation of LPS-primed cells with fMLP is mediated by the AA-specific 85 kDa cPLA2.
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Immunoblot analyses of cPLA2 phosphorylation and translocation
Since inhibitor studies indicated that the cPLA2 was responsible for the release of AA in LPS-primed PMNL, the next series of experiments was performed in order to assess the mechanism(s) by which LPS priming results in an increased capacity to release AA after stimulation with fMLP. Phosphorylation of cPLA2 on serine 505 results in an increase in the activity of both recombinant cPLA2 (37, 49) and cellular cPLA2 in several cell types (22, 34, 50, 51). Although phosphorylation of cPLA2 can occur at several sites (51, 52), only phosphorylation by MAP kinases on serine 505 has so far been associated with activation of the enzyme. Phosphorylation of the enzyme at serine 505 results in a decreased mobility of the protein on SDS polyacrylamide gel electrophoresis (37). As shown in Fig. 5A,
the incubation of PMNL with 1 µg/ml LPS for 5–30 min resulted in a gradual shift in the mobility of cPLA2 on SDS-polyacrylamide gels as revealed by immunoblot analyses. Pretreatment of PMNL with LPS for as little as 5 min was sufficient to demonstrate this phenomenon, and a seemingly complete band shift is apparent after a 15–30 min treatment with LPS. However, this shift associated with phosphorylation on serine 505 is transient, since the faster-migrating band reappears if the incubation with LPS is extended to 60–120 min. The kinetics of this phosphorylation-dephosphorylation coincide remarkably well with capacity for the release AA in LPS-treated cells and thus are consistent with the inhibitor studies indicating that the cPLA2 is responsible for AA release. Similarly, but in an accelerated fashion, the stimulation of unprimed PMNL with fMLP resulted in a shift in the mobility of cPLA2 within 30 s poststimulation ( Fig. 5B) and was maintained for at least 5 min.
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Although both LPS and fMLP on their own induce a complete band shift of cPLA2, the important liberation of AA is observed only after fMLP stimulation of LPS-treated cells. This indicates that phosphorylation on serine 505 is not sufficient for the enhanced release of AA observed in LPS-primed cells. Therefore, the effects of priming and stimulation were evaluated on the translocation of cPLA2 from the cytosol to cellular membranes where its phospholipid substrate is located. As can be seen in Fig. 6,
when PMNL are incubated with LPS for 30 min without subsequent stimulation, or preincubated without added LPS and stimulated with fMLP, there is no significant increase in cPLA2 associated with the membrane fraction of the cell compared to untreated controls. However, when cells are incubated with LPS for 30 min and then stimulated with fMLP, the density of immunoreactive cPLA2 associated with the membrane fraction of the cells is double that measured in untreated controls. When cells are preincubated with LPS for 120 min, this translocation after fMLP stimulation is no longer observed, which is consistent with the loss of the ability to release AA in cells preincubated with LPS for longer times. Therefore, both cPLA2 phosphorylation and translocation of the enzyme to cellular membranes are enhanced in the same time frame as the capacity to release AA. Furthermore, both these phenomena, as well as the capacity to release AA, are reversed during the resolution of the priming effect in cells incubated with LPS for longer periods of time.
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Immunoblot analysis of 5-LO
Experiments were also performed to investigate whether the enhanced activity of 5-LO measured in LPS-primed fMLP-stimulated PMNL may be associated with the translocation of this enzyme to the nucleus as has been shown to occur in A23187-stimulated PMNL (13). Immunoblot analyses of nuclei isolated from PMNL that have undergone various treatments indicate that LPS or fMLP alone do not induce a measurable increase in nucleus-associated 5-LO compared to untreated cells ( Fig. 7).
Therefore, the activation of 5-LO observed with fMLP alone ( Fig. 3B) is not sufficient to induce a measurable translocation of the enzyme either because too little translocation occurs or the agonist simply activates an enzyme that is constitutively associated with the nucleus. However, a measurable and significant increase in the intensity of immunoreactive 5-LO protein is observed in nuclear preparations of cells that were pretreated with LPS and stimulated with fMLP. Concomitant with the increased nuclear 5-LO in primed stimulated cells, a significant decrease in 5-LO protein was observed in the nonnuclear fraction of solubilized cell preparations. These results suggest that one mechanism by which LPS increase 5-LO activity and thus LTB4 biosynthesis is by enhancing the translocation of the enzyme to the nucleus, where it may interact with the five lipoxygenase activating protein.
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Calcium mobilization
Since both cPLA2 and 5-LO require an increase in intracellular calcium for their activation and translocation, the effect of LPS priming on fMLP-induced intracellular calcium flux was investigated. As previously reported, neutrophils exposed to fMLP (0.1 µM) showed a rapid and transient increase in intracellular calcium. However, cells preincubated with LPS for 30 min showed no difference in the amplitude or the duration of this response to fMLP compared to cells preincubated in the absence of added LPS (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The isotype of PLA2 responsible for the release of AA from stimulated cells has been the subject of some controversy in recent years due partly to differences between different cell types and the nature of the stimuli. Human neutrophils possess both the 14 kDa type II sPLA2 and the 85 kDa type IV cPLA2 (28), which have both been implicated in AA release after activation in several cell types (19–25, 53) and implicated in AA release after stimulation of human PMNL (9, 14, 29, 30, 33, 34, 36, 46, 47, 54). The present study shows that the release of AA in primed stimulated cells is mediated by the 85 kDa cPLA2 but not the 14 kDa sPLA2. In accord with inhibitor studies, the phosphorylation and translocation of cPLA2 induced by LPS follow the same kinetics as that for the enhanced ability to release AA. Furthermore, the phosphorylation of cPLA2 is reversed and the potential to translocate the enzyme is lost when incubations are extended to more than 60 min, coinciding with the loss of the priming effect for AA release. The importance of cPLA2 in providing AA for both prostaglandin and leukotriene biosynthesis was recently demonstrated in two separate reports of PLA2 knockout mice (55, 56). Both studies showed that peritoneal macrophages, which express several forms of PLA2 in wild-type mice, were unable to synthesize prostaglandins or leukotrienes after stimulation of cells from cPLA2 -/- mice with A23187 or LPS.
Although fMLP stimulation of unprimed cells resulted in a shift in the electrophoretic mobility of cPLA2, only small amounts of AA were released. A possible contribution to the important AA release observed after the stimulation of primed cells is that the cPLA2 is already phosphorylated when fMLP induces its early and transient increase in intracellular calcium levels, as was suggested by Doerfler et al. (9) in opsonized zymosan-stimulated PMNL. However, the acquired capacity of primed but not unprimed cells to translocate cPLA2 to a membrane fraction of the cell is likely critical for this greatly enhanced ability to release AA. In fact, studies with cPLA2 mutants have shown that both activation by phosphorylation on serine 505 and translocation of the enzyme mediated by the calcium-dependent phospholipid binding domain are essential for AA release (38). Therefore, the combination of cPLA2 phosphorylation by LPS prior to fMLP stimulation and the enhanced capacity to translocate the enzyme to cellular membranes likely both contribute to the greatly enhanced capacity for fMLP-induced AA release in LPS-treated cells.
The translocation of cPLA2 and 5-LO is calcium dependent; however, the mechanism by which this translocation occurs is not known. One can speculate that the priming of neutrophils may reduce the calcium requirement for translocation since no significant translocation of cPLA2 or 5-LO was observed after the fMLP-stimulation of unprimed cells, although the calcium transient was identical to that of primed cells. Alternatively, priming may occur through a distinct mechanism that is not functional in unprimed cells. It has been clearly shown using cPLA2 mutants that phosphorylation on serine 505 is not required for the Ca2+-dependent translocation of the enzyme; however, the potential contribution of other less-studied phosphorylation sites on cPLA2 translocation remain to be explored. Recent studies have clearly indicated the involvement of phosphorylation events in the translocation of 5-LO; indeed, in A23187-stimulated HL-60 cells, a very small proportion of cellular 5-LO associated with the nucleus was phosphorylated on undetermined sites, and the activation and translocation of 5-LO were inhibited by a panel of distinct tyrosine kinase inhibitors (57). These studies suggest that phosphorylation of proteins, perhaps of the translocated protein itself, is associated with the translocation process.
It is clear that the activation of 5-LO is dependent on its translocation to the nucleus, an event likely necessary for association with the nuclear membrane-associated FLAP, which may optimize the delivery of released AA to the 5-LO (15). Treatment of human PMNL with MK-886, a compound that binds to FLAP (12), inhibits agonist-induced formation of 5-LO metabolites (1). Therefore, the priming activity of LPS in human PMNL for an enhanced capacity for 5-LO activation likely involves the acquired capacity of the cell to translocate the 5-LO enzyme to the nucleus in response to receptor-mediated stimuli like fMLP demonstrated herein.
In conclusion, since freshly isolated PMNL exhibit a weak capacity for several functional responses after stimulation by soluble agonists that act via receptor-dependent mechanisms, it seems reasonable to speculate that the more quiescent circulating neutrophils are primed by agents such as TNF-, GM-CSF, and LPS present at inflammatory sites, and that priming likely plays an important role in the potentiation of functional responses of cells recruited from the circulation (5, 58–60). In the case of leukotriene biosynthesis, we have shown here that LPS simultaneously increases the capacities of cells to release AA from cellular phospholipids and to transform this fatty acid to the biologically active leukotrienes. Equally important is the ability to eventually resolve or dampen the activity of PMNL at inflammatory sites. As shown here, the enhancement of cell functional responses by LPS is transient and gradually resolves resulting in a quiescent phenotype resembling that of the circulating PMNL. Other have recently shown that LPS will eventually induce the expression of cyclooxygenase-2 in human PMNL and the capacity to synthesize prostaglandin E2, which has inhibitory effects on some PMNL functions (61, 62); ultimately, these cells will be eliminated by apoptosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: PMNL, polymorphonuclear leukocytes; LTA, leukotriene A; AA, arachidonic acid; 5-LO, 5-lipoxygenase; fMLP, formyl-methionyl-leucyl-phenylalanine; PAF, platelet-activating factor; LPS, lipopolysaccharides; FLAP, five lipoxygenase-activating protein; cPLA, cytosolic phospholipase; sPLA, secreted PLA; GM-CSF, granulocyte-macrophage colony-stimulating factor; TNF, tumor necrosis factor; PGB, prostaglandin B; PMSF, phenylmethanesulfonyl fluoride; MAFP, methyl arachidonyl fluorophosphate; 15-HpETE, 15-hydroperoxyeicosatetraenoic acid; ECL, enhanced chemiluminescence; RP, reverse phase; HPLC, high-performance liquid chromagraphy.
Received for publication March 5, 1998.
Revision received April 28, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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P. Pacheco, F. A. Bozza, R. N. Gomes, M. Bozza, P. F. Weller, H. C. Castro-Faria-Neto, and P. T. Bozza Lipopolysaccharide-Induced Leukocyte Lipid Body Formation In Vivo: Innate Immunity Elicited Intracellular Loci Involved in Eicosanoid Metabolism J. Immunol., December 1, 2002; 169(11): 6498 - 6506. [Abstract] [Full Text] [PDF]
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 J. Bylund, A. Karlsson, F. Boulay, and C. Dahlgren Lipopolysaccharide-Induced Granule Mobilization and Priming of the Neutrophil Response to Helicobacter pylori Peptide Hp(2-20), Which Activates Formyl Peptide Receptor-Like 1 Infect. Immun., June 1, 2002; 70(6): 2908 - 2914. [Abstract] [Full Text] [PDF]
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 N. Degousee, F. Ghomashchi, E. Stefanski, A. Singer, B. P. Smart, N. Borregaard, R. Reithmeier, T. F. Lindsay, C. Lichtenberger, W. Reinisch, et al. Groups IV, V, and X Phospholipases A2s in Human Neutrophils. ROLE IN EICOSANOID PRODUCTION AND GRAM-NEGATIVE BACTERIAL PHOSPHOLIPID HYDROLYSIS J. Biol. Chem., February 8, 2002; 277(7): 5061 - 5073. [Abstract] [Full Text] [PDF]
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 O. Werz, E. Burkert, B. Samuelsson, O. Radmark, and D. Steinhilber Activation of 5-lipoxygenase by cell stress is calcium independent in human polymorphonuclear leukocytes Blood, February 1, 2002; 99(3): 1044 - 1052. [Abstract] [Full Text] [PDF]
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O. Werz, J. Klemm, B. Samuelsson, and O. Radmark Phorbol ester up-regulates capacities for nuclear translocation and phosphorylation of 5-lipoxygenase in Mono Mac 6 cells and human polymorphonuclear leukocytes Blood, April 15, 2001; 97(8): 2487 - 2495. [Abstract] [Full Text] [PDF]
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J. Almkvist, J. Faldt, C. Dahlgren, H. Leffler, and A. Karlsson Lipopolysaccharide-Induced Gelatinase Granule Mobilization Primes Neutrophils for Activation by Galectin-3 and Formylmethionyl-Leu-Phe Infect. Immun., February 1, 2001; 69(2): 832 - 837. [Abstract] [Full Text] [PDF]
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J. Marshall, E. Krump, T. Lindsay, G. Downey, D. A. Ford, P. Zhu, P. Walker, and B. Rubin Involvement of Cytosolic Phospholipase A2 and Secretory Phospholipase A2 in Arachidonic Acid Release from Human Neutrophils J. Immunol., February 15, 2000; 164(4): 2084 - 2091. [Abstract] [Full Text] [PDF]
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 N. FLAMAND, S. BOUDREAULT, S. PICARD, M. AUSTIN, M. E. SURETTE, H. PLANTE, E. KRUMP, M.-J. VALLEE, C. GILBERT, P. NACCACHE, et al. Adenosine, a Potent Natural Suppressor of Arachidonic Acid Release and Leukotriene Biosynthesis in Human Neutrophils Am. J. Respir. Crit. Care Med., February 1, 2000; 161(2): S88 - 94. [Full Text] [PDF]
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 M. E. Surette, E. Krump, S. Picard, and P. Borgeat Activation of Leukotriene Synthesis in Human Neutrophils by Exogenous Arachidonic Acid: Inhibition by Adenosine A2a Receptor Agonists and Crucial Role of Autocrine Activation by Leukotriene B4 Mol. Pharmacol., November 1, 1999; 56(5): 1055 - 1062. [Abstract] [Full Text]
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S. Kastenbauer and H. W. L. Ziegler-Heitbrock NF-kappa B1 (p50) Is Upregulated in Lipopolysaccharide Tolerance and Can Block Tumor Necrosis Factor Gene Expression Infect. Immun., April 1, 1999; 67(4): 1553 - 1559. [Abstract] [Full Text] [PDF]
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E. Boilard and M. E. Surette Anti-CD3 and Concanavalin A-induced Human T Cell Proliferation Is Associated with an Increased Rate of Arachidonate-Phospholipid Remodeling. LACK OF INVOLVEMENT OF GROUP IV AND GROUP VI PHOSPHOLIPASE A2 IN REMODELING AND INCREASED SUSCEPTIBILITY OF PROLIFERATING T CELLS TO CoA-INDEPENDENT TRANSACYLASE INHIBITOR-INDUCED APOPTOSIS J. Biol. Chem., May 11, 2001; 276(20): 17568 - 17575. [Abstract] [Full Text] [PDF]
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