The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhu, X.
Right arrow Articles by Leff, A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhu, X.
Right arrow Articles by Leff, A. R.
The Journal of Immunology, 2001, 167: 461-468.
Copyright © 2001 by The American Association of Immunologists

Role of Mitogen-Activated Protein Kinase-Mediated Cytosolic Phospholipase A2 Activation in Arachidonic Acid Metabolism in Human Eosinophils1

Xiangdong Zhu*, Hiroyuki Sano*, Kwang Pyo Kim{dagger}, Akiko Sano*, Evan Boetticher*, Nilda M. Muñoz*, Wonhwa Cho{dagger} and Alan R. Leff2,*

* Section of Pulmonary and Critical Care Medicine, Department of Medicine and Department of Pharmacological and Physiological Sciences, Pediatrics, Anesthesia, and Critical Care, and Committees on Clinical Pharmacology and Cell Physiology, Division of Biological Sciences, University of Chicago, Chicago, IL 60637; and {dagger} Department of Chemistry, University of Illinois, Chicago, IL 60607


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The objective of this investigation was to determine the role of secretory and cytosolic isoforms of phospholipase A2 (PLA2) in the induction of arachidonic acid (AA) and leukotriene synthesis in human eosinophils and the mechanism of PLA2 activation by mitogen-activated protein kinase (MAPK) isoforms in this process. Pharmacological activation of eosinophils with fMLP caused increased AA release in a concentration (EC50 = 8.5 nM)- and time-dependent (t1/2 = 3.5 min) manner. Both fMLP-induced AA release and leukotriene C4 (LTC4) secretion were inhibited concentration dependently by arachidonic trifluoromethyl ketone, a cytosolic PLA2 (cPLA2) inhibitor; however, inhibition of neither the 14-kDa secretory phospholipase A2 by 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid nor cytosolic Ca2+-independent phospholipase A2 inhibition by bromoenol lactone blocked hydrolysis of AA or subsequent leukotriene synthesis. Pretreatment of eosinophils with a mitogen-activated protein/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, U0126, or a p38 MAPK inhibitor, SB203580, suppressed both AA production and LTC4 release. fMLP induced phosphorylation of MAPK isoforms, ERK1/2 and p38, which were evident after 30 s, maximal at 1–5 min, and declined thereafter. fMLP stimulation also increased cPLA2 activity in eosinophils, which was inhibited completely by 30 µM arachidonic trifluoromethyl ketone. Preincubation of eosinophils with U0126 or SB203580 blocked fMLP-enhanced cPLA2 activity. Furthermore, inhibition of Ras, an upstream GTP-binding protein of ERK, also suppressed fMLP-stimulated AA release. These findings demonstrate that cPLA2 activation causes AA hydrolysis and LTC4 secretion. We also find that cPLA2 activation caused by fMLP occurs subsequent to and is dependent upon ERK1/2 and p38 MAPK activation. Other PLA2 isoforms native to human eosinophils possess no significant activity in the stimulated production of AA or LTC4.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Regulation of leukotriene synthesis in human eosinophils depends upon initial hydrolysis of membrane phospholipid into arachidonic acid (AA)3 by phospholipase A2 (PLA2) (1). There are several forms of PLA2, including low molecule mass secretory PLA2 (sPLA2), an 85-kDa cytosolic PLA2 (cPLA2), and Ca2+-independent PLA2 (iPLA2) (2, 3). While several PLA2 isoforms exist within eosinophils, the precise regulatory role of each has not been established (4, 5, 6). The 85-kDa cPLA2 has received specific attention because it is AA specific and appears to represent the enzyme that is distinctively regulated by cell-signaling mechanisms downstream of surface membrane receptor (7). A model of dual regulation of cPLA2 has been proposed (8). Agents causing increase in cytosolic Ca2+ concentration have been associated with translocation of cPLA2 from cytosol to an intracellular membrane (9), in which cPLA2 can bind via its N-terminal Ca2+-dependent lipid-binding domain. Translocation also facilitates access of cPLA2 to phospholipid substrate and, subsequently, access of free AA to metabolism by 5-lipoxygenase, which is thought to be a nuclear enzyme (10).

The enzymatic activity of cPLA2 is increased through phosphorylation by mitogen-activated protein kinase (MAPK) (11, 12, 13). There are at least three parallel MAPK pathways, the p44/p42 or extracellular signal-regulated protein kinase (ERK) 1/2 pathway, the c-Jun N-terminal kinase (JNK) pathway, and the p38 kinase pathway, which may be activated by diverse stimuli including stress, such as hyperosmolality (14, 15, 16, 17). These subgroups are distinguished by both the sequence of the tripeptide dual phosphorylation motif that is required for MAPK activation and the distinct subgroups of mitogen-activated protein/ERK kinases that activate the ERK group (MEK) and the JNK and p38 groups (MAPK kinase of the stress group). The specific MAPK isoform involved in cPLA2 activation is controversial. Several studies have shown that cPLA2 activity is regulated by phosphorylation via ERK1/2 activation (12, 13, 18, 19, 20). However, in thrombin-stimulated platelets and TNF-{alpha}-stimulated human neutrophils, p38 MAPK activation was attributed to cPLA2 activation (21, 22). In thrombin-stimulated astrocytes, JNK has been implied in cPLA2 activation (23). The role of the specific MAPK isoform in cPLA2 activation in human eosinophils has not been determined.

fMLP, a tripeptide purified from bacteria, has a variety of biological effects on human eosinophils. These include degranulation, adhesion, chemoattraction, superoxide synthesis, and leukotriene synthesis and release (24, 25, 26, 27). fMLP acts through a specific G protein-coupled cell surface receptor. This promotes a rapid and transient increase in intracellular Ca2+ in human eosinophils (28). Furthermore, many of the functional effects elicited by fMLP are mediated by a pertussis toxin-sensitive mechanism, indicating the involvement of one of more members of the Gi or Go family of heterotrimeric GTP-binding proteins (29, 30). However, the signaling pathway leading to AA metabolism after fMLP receptor occupation has not been defined in human eosinophils.

The objective of this study was to characterize the isoform of PLA2 involved in arachidonic hydrolysis during activation of human eosinophils. Studies also were performed to determine the contribution of MAPK activation in AA hydrolysis in fMLP-stimulated human eosinophils. We found that both ERK1/2 and p38 MAPK are involved in cPLA2 activation, AA release, and subsequent leukotriene C4 (LTC4) synthesis. We found further that other PLA2 isoforms native to human eosinophils possess little or no activity in stimulated production of AA or its metabolites.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

The cPLA2 inhibitor arachidonic trifluoromethyl ketone (AACOCF3) and iPLA2 inhibitor bromoenol lactone (BEL) were purchased from Biomol (Plymouth Meeting, PA). The sPLA2 inhibitor, 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane phosphonic acid (LY311727), was kindly donated by N. Roehm (Eli Lilly, Indianapolis, IN). The p38 inhibitor SB203580 was purchased from Upstate Biotechnology (Lake Placid, NY). Eosinophil isolation materials were obtained from Miltenyi Biotec (Sunnyvale, CA). fMLP was purchased from Sigma (St. Louis, MO). SB202474 and Ras farnesyltransferase inhibitors farnesyl protein transferase (FPT) inhibitor III and manumycin A were purchased from Calbiochem (San Diego, CA). The purified cPLA2 and polyclonal anti-cPLA2 antiserum were obtained as previously described (4). [5,6,8,9,11,12,14,15-3H]AA (sp. act., 100 Ci/mmol) and 1-palmitoyl-2-[14C]arachidonyl phosphatidylcholine (PAPC) were purchased from New England Nuclear (Boston, MA). Anti-phospho-ERK1/2 Ab and MEK inhibitor, U0126, were purchased from Promega (Madison, WI). Anti-ERK1/2, anti-phospho-p38 MAPK, anti-phospho JNK, anti-p38 MAPK, p38 kinase activity assay kit, and anti-JNK Abs were purchased from New England Biolabs (Beverly, MA). Goat anti-rabbit Ig conjugated with HRP was purchased from Amersham (Arlington Heights, IL).

Isolation of human eosinophils

Eosinophils were isolated by a method modified from Hansel et al. (31). The method is based on Percoll centrifugation (density 1.089 g/ml) to isolate granulocytes, hypotonic lysis of RBCs, and, finally, immunomagnetic depletion of neutrophils by the magnetic cell separation system using anti-CD16-coated MACS particles. Eosinophil purity of >=98% was routinely obtained, as assessed by Wright-Giemsa staining. Cells were kept on ice until use.

Immunoblot analysis of MAPK

Eosinophils (2–3 x 106/group) were preincubated with cytochalasin B for 2 min, and then stimulated with fMLP for various times, and the reaction was stopped by centrifugation at 12,000 x g for 10 s. The pellets then were lysed in 80 µl 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 PMSF, 2 mM Na3VO4, and 0.5% deoxycholic acid. After 10 min on ice, the sample was centrifuged at 12,000 x g for 20 min to remove nuclear and cellular debris. The supernatants then were mixed with 14 µl of 6x sample buffer and boiled for 5 min. The samples were collected and saved at -70°C.

Samples were subjected to SDS-PAGE, using 10% acrylamide gels under reducing condition (15 mA/gel). Electrotransfer of proteins from the gels to polyvinylidene fluoride membrane was achieved using a semidry system (400 mA, 60 min). The membrane was blocked with 1% BSA for 60 min, then incubated with 1/5000 anti-phosphorylation-specific ERK1/2 Ab, 1/1000 anti-ERK1/2 Ab, 1/1000 anti-phosphorylation-specific p38 MAPK, 1/1000 anti-p38 Ab, 1/1000 anti-phosphorylation-specific JNK, or 1/1000 anti-JNK Ab diluted in TBST overnight. The membranes then were washed three times for 20 min with TBST. Goat anti-rabbit IgG conjugated with HRP was diluted 1/3000 in TBST and incubated with polyvinylidene fluoride membrane for 60 min. The membrane was again washed three times with TBST and assayed by an ECL chemiluminescence system (Amersham).

Assay of p38 kinase activity

p38 kinase activity was assayed with a p38 MAPK assay kit following the manufacturer’s instructions (New England Biolabs). Briefly, eosinophils (2–3 x 106/group) were preincubated with various concentrations of SB203580 or SB202474 for 30 min, treated with cytochalasin B for 2 min, and then stimulated with fMLP for 1 min. The reaction was stopped by centrifugation, and the pellets were solubilized in 200 µl lysis buffer, as above. After removing nuclear and cellular debris, cell lysates were incubated overnight with 1 µg anti-phospho (Thr180/Tyr182) p38 MAPK Ab bound to agarose hydrazide beads. The immune complexes then were washed twice with 500 µl lysis buffer and twice with 500 µl kinase buffer (25 mM Tris, pH 7.5, 2.5 mM {beta}-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2), and resuspended in 50 µl kinase buffer containing 100 µM ATP and 1 µg substrate GST/activating transcription factor (ATF)-2. The kinase reactions were conducted at 30°C for 30 min and terminated by the addition of 10 µl 6x Laemmli sample buffer before SDS-PAGE. Phosphorylation of GST/ATF-2 substrate was detected by immunoblotting with anti-phospho (Thr71) ATF-2 Ab.

Determination of cPLA2 enzyme activity

cPLA2 activity assay was modified from Kim et al. (32). Briefly, 2 x 106 cytochalasin B-pretreated eosinophils were stimulated with or without 1 µM fMLP for various times. The reaction was stopped by centrifugation, and the pellets were resuspended in 70 µl sonication buffer (20 mM Tris, pH 8, 2.5 mM EDTA, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM PMSF, 2 mM Na3VO4, 50 mM NaF, and 5 µg/ml pepstatin) and sonicated briefly (4 x 10 s, at a power setting of 3). Lysates were pretreated with 5 mM DTT on ice for 5 min to inactivate sPLA2, and 10 µl of 50 mM CaCl2 was then added to each sample. A total of 10 µl substrate ([14C]PAPC) was dried under a stream of N2 and resuspended in 200 µl 10% ethanol in H2O with vigorous vortex mixing. The reaction was initiated by adding 10 µl portion of the substrate (final concentration 9 µM) to cell lysate. The reaction was conducted for 30 min at 37°C and was stopped by adding 560 µl Dole’s reagent (heptane-isopropyl alcohol-1 N H2SO4, 400:390:10 by volume), followed by 110 µl H2O, vortexed for 20 s, and then centrifuged at 12,000 x g. Upper layer (180 µl) was transferred to 800 µl hexane containing 25 mg silica gel. A total of 750 µl of samples was then mixed with 2 ml scintillation fluids, and the radioactivity was counted in a liquidscintillation counter. cPLA2 activity was expressed as percentage of nonstimulated control ((cpm of activated eosinophils/cpm of nonstimulated eosinophils) x 100).

Measurement of AA release

Eosinophils were incubated in RPMI media containing 5% FBS and 0.5 µCi [3H]AA. After a 2-h incubation period, labeled medium was aspirated, and unincorporated [3H]AA was washed away by HBSS containing 0.2% BSA. Uptake of [3H]AA by eosinophils occurred in a time-dependent manner, reaching its maximum (61 ± 2.3% of the total added [3H]AA) after 30-min incubation at 37°C. Maximal incorporation remained constant >=2-h incubation. Aliquots of 106 eosinophils were preincubated with or without U0126, SB203580, AACOCF3, BEL, LY311727 for 30 min, or FPT inhibitor III, manumycin A for 60 min. Cells were subsequently incubated with 5 µg/ml cytochalasin B for 2 min before stimulation by fMLP for additional 10 min at 37°C. The addition of cytochalasin B was used to promote AA metabolism, as described previously (24). The reactions were terminated by centrifugation at 12,000 x g for 1 min. Supernatants were collected, and pellets were lysed in 1% Triton X-100. [3H]AA release was measured by scintillation counting and expressed as percentage of total AA incorporation (100 x cp. of supernatant/(cpm of supernatant + cpm of pellet)).

LTC4 assay

Aliquots of 250,000 eosinophils were preincubated with various concentrations of AACOCF3, LY311727, BEL, U0126, SB203580, or SB202474 for 30 min, and then incubated with cytochalasin B for 2 min before being stimulated by fMLP for additional 10 min at 37°C in a final volume of 250 µl HBSS. The reactions were terminated by centrifugation at 12,000 x g for 1 min. Aliquots of supernatants were assayed with a commercial enzyme immunoassay kit, as described previously (24).

Statistical analysis

All data are expressed as mean ± SEM. Differences between groups were assessed by paired t test. Where more than two groups were compared, differences among groups were assessed by one-way ANOVA. Where differences were found, comparisons among groups were made by Fisher’s least-protected difference test. Statistical significance was claimed where p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of fMLP on AA release in eosinophils

FMLP caused [3H]AA release from purified eosinophils in a concentration-dependent manner with an EC50 value of 8.5 ± 1 nM (Fig. 1GoA). Nonstimulated eosinophils release minimal amount of [3H]AA during 10-min incubation period. A significant increase in [3H]AA release was observed at 10 nM fMLP (4.3 ± 0.4% vs 0.7 ± 0.03% of total incorporation for nonstimulated control, p < 0.001), and increasing the fMLP concentrations provoked a corresponding increase in AA release, reaching maximum of 7.3 ± 0.4% at 1 µM (Fig. 1GoA).



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 1. Concentration response and time course of fMLP-stimulated AA release. A, Concentration-dependent effect of fMLP on AA release. [3H]AA-labeled eosinophils were preincubated with cytochalasin B for 2 min at 37°C before stimulation by various concentrations of fMLP for 10 min. Results are presented as the mean ± SEM from five separate experiments. B, Time course of fMLP-stimulated AA release. Eosinophils were preincubated with cytochalasin B for 2 min, and then stimulated by 1 µM fMLP for the indicated times. Results are presented as the mean ± SEM from five separate experiments.

 
fMLP caused [3H]AA release in a time-dependent manner with a t1/2 of 3.5 ± 0.5 min (Fig. 1GoB). With 1 µM fMLP, 63% of maximal AA release was elicited in 5 min, and at 10 min AA release reached its plateau value of 7.1 ± 0.8% (p < 0.001 vs 0.6 ± 0.03% for control). Similar dose-response curve and time course were observed in fMLP-induced LTC4 production (data not shown).

Effects of inhibitors of sPLA2, cPLA2, or iPLA2 on fMLP-induced [3H]AA release and LTC4 production

To determine which of the three PLA2 subtypes was responsible for the production of AA and LTC4 in fMLP-stimulated eosinophils, the effect of selective inhibitory agents on AA metabolism was assessed. Eosinophils were preincubated with AACOCF3, a cPLA2 and iPLA2 inhibitor (33, 34); LY311727, a sPLA2 inhibitor (35, 36); or BEL, an iPLA2 inhibitor (34), before stimulation by 1 µM fMLP. Nonstimulated eosinophils released minimal amounts of AA and undetectable amount of LTC4. AACOCF3 inhibited both fMLP-induced [3H]AA release (Fig. 2GoA) and LTC4 production (Fig. 2GoB) in a concentration-dependent manner. The fMLP-stimulated net AA release (background subtracted) was decreased significantly from 6.6 ± 0.8% to 2.7 ± 0.7% (p < 0.05) at 10 µM AACOCF3, and was further blocked to 1.5 ± 0.4% at 30 µM AACOCF3 (p < 0.01). Similarly, LTC4 release was decreased from 1120 ± 97 pg/ml to 429.4 ± 235.4 pg/ml (p < 0.05) with 10 µM AACOCF3, and was further blocked to 50 ± 10.4 pg/ml (p < 0.001) with 30 µM AACOCF3. In additional studies, the blocking effects of the cPLA2 inhibitor, surfactant, were examined (32). At 10 µM, surfactant significantly inhibited fMLP-stimulated AA and LTC4 release (data not shown). By contrast, neither the sPLA2 inhibitor, LY311727, nor the iPLA2 inhibitor, BEL, had any inhibitory effect on the stimulated AA or LTC4 production (Fig. 2Go, A and B), demonstrating that cPLA2, but neither sPLA2 nor iPLA2, is involved in the fMLP-induced AA metabolism in human eosinophils.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 2. Effects of sPLA2, cPLA2, and iPLA2 inhibition on fMLP-stimulated AA release and LTC4 production. [3H]AA-labeled eosinophils (A) or nonlabeled eosinophils (B) were preincubated with LY311727,AACOCF3, or BEL for 30 min at 37°C, and then incubated with cytochalasin B for 2 min before stimulation by 1 µM fMLP for another 10 min. [3H]AA release (A) was measured by scintillation counting and was calculated by subtraction of background release. LTC4 production (B)was measured by enzyme immunoassay. Results are presented as the mean ± SEM from four separate experiments.

 
FMLP-induced activation of cPLA2 in eosinophils

To demonstrate further the role of cPLA2 in fMLP-induced AA release, we next examined whether fMLP induced cPLA2 activation. fMLP increased cPLA2 activity by 23 ± 6.9% after 1 min, and was maximal after 5 min (60.7 ± 8.8%, p < 0.01), decreasing slightly thereafter (Fig. 3GoA). cPLA2 activity in fMLP-stimulated eosinophil lists was inhibited by AACOCF3 in a concentration-dependent manner (Fig. 3GoB). cPLA2 activity decreased to 30 ± 4.6% of buffer-treated control with 10 µM AACOCF3 and 10.9 ± 1.9% with 30 µM AACOCF3 (p < 0.01 vs control for both comparisons).



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 3. cPLA2 activity in fMLP-stimulated eosinophil lysates. A, Time-dependent effect of fMLP on cPLA2 activity. Eosinophils were preincubated with cytochalasin B and then stimulated with 1 µM fMLP for indicated times. cPLA2 activity in the eosinophil lysates was measured, as described in Materials and Methods, using substrate [14C]PAPC, and expressed as percentage of nonstimulated eosinophils in three separate experiments. B, Effect of AACOCF3 on cPLA2 activity. FMLP/cytochalasin B-stimulated eosinophil lysates were incubated with the indicated concentrations of AACOCF3 for 10 min at 37°C and then assayed for cPLA2 activity. Data are normalized as percentage of buffer-treated controls from three independent experiments.

 
FMLP-induced activation of MAPK isoforms in eosinophils

To assess the involvement of MAPKs in fMLP-induced AA release, we next investigated the effect of fMLP on ERK1/2, p38, and JNK activation in eosinophils. Phosphorylation of ERK1/2, p38, and JNKs is commonly used as an indicator of activation. As shown in Fig. 4GoA, fMLP caused time-dependent ERK1/2 phosphorylation, which was observed within 0.5 min, peaked at 1–5 min, and declined thereafter (top panel). The phosphorylation was not due to differences in ERK1/2 content in each treatment, as equal amounts of ERK1/2 were present for all treatment groups (bottom panel).



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 4. MAPK phosphorylation in fMLP-stimulated eosinophils. Time-dependent effects of fMLP on ERK1/2 (A) and p38 (B) phosphorylation. Cytochalasin B-pretreated eosinophils were incubated with 1 µM fMLP for indicated times. Eosinophils were lysed, and the lysates were mixed with sample buffer and loaded on 10% SDS-PAGE, followed by immunoblotting with anti-phosphorylation-specific ERK1/2 (A, top panel), anti-ERK1/2 (A, bottom panel), anti-phosphorylation-specific p38 Ab (B, top panel), or anti-p38 (B, bottom panel), as described in Materials and Methods. These results are representative of three experiments. C, Effects of U0126 on fMLP-stimulated ERK1/2 phosphorylation. Eosinophils were preincubated with different concentrations of U0126 for 20 min, incubated with cytochalasin B for 2 min, and stimulated with 1 µM fMLP for 2 min. ERK1/2 phosphorylation (top panel) and total ERK1/2 (phosphorylated + nonphosphorylated) (bottom panel) were measured as in A (n = 3). D, Effects of SB203580 and SB202474 on fMLP-stimulated p38 MAPK activity. Eosinophils were preincubated with different concentrations of either compound for 30 min, incubated with cytochalasin B for 2 min, and stimulated with fMLP for 1 min. p38 kinase activity was measured by phosphorylation of substrate GST/ATF-2, as described in Materials and Methods. The result shown is representative of three different experiments. Con, Control.

 
p38 MAPK phosphorylation followed the same kinetics, except for the presence of minimal constitutive phosphorylation in unstimulated eosinophils (Fig. 4GoB, top panel). Differences in phosphorylation were not due to differences in p38 content among treatment groups, as demonstrated by the equal loading of p38 for all treatment groups (Fig. 4GoB, bottom panel). No JNK phosphorylation was observed in fMLP-stimulated eosinophils (data not shown).

FMLP-induced ERK1/2 phosphorylation was inhibited concentration dependently by U0126, an inhibitor of ERK1/2 activation secondary to its inhibition of upstream MAPK kinase (MEK) (37, 38). ERK1/2 phosphorylation was partially blocked at 3 µM U0126, and was completely blocked by 10 µM U0126 (Fig. 4GoC).

Stimulation with fMLP also caused an increase in p38 activity (Fig. 4GoD), which was inhibited concentration dependently by SB203580 (39), a specific p38 MAPK inhibitor. Inhibition was observed at 10 µM and was complete at 30 µM SB203580. SB202474, an inactive analogue of SB203580 (39), did not inhibit p38 activity at 3–10 µM. However, at 30 µM, it also suppressed fMLP-stimulated p38 activity in eosinophils.

Effects of MAPK inhibitors on fMLP-induced [3H]AA and LTC4 productions

The involvement of MAPK in agonist-induced AA release has been reported in platelet as well as in other cells (21, 40, 41). In these studies, pharmacological inhibitors of MAPK were employed. To address the question of whether MAPK isoforms were involved in the events leading to fMLP-stimulated AA release, we measured the effect of the MEK inhibitor, U0126, and the specific p38 MAPK inhibitor, SB203580, on fMLP-stimulated AA and LTC4 release. As shown in Fig. 5Go, both U0126 and SB203580 inhibited fMLP-induced AA release (Fig. 5GoA) and LTC4 production (Fig. 5GoB) in a concentration-dependent manner. fMLP-stimulated AA release was decreased from 6.6 ± 0.8% to 1 ± 0.07% with 10 µM U0126, and to 1.4 ± 0.3% with 30 µM SB203580 (p < 0.01 for both comparisons). Similarly, fMLP-stimulated LTC4 release was decreased from 1280.7 ± 97.1 pg/ml to 21.7 ± 3.1 pg/ml with 10 µM U0126, and to 13.6 ± 0.7 pg/ml with 30 µM SB203580 (p < 0.001 for both comparisons). These data demonstrate that both ERK1/2 and p38 MAPK are involved in fMLP-induced AA release in human eosinophils. SB202474 did not inhibit fMLP-stimulated LTC4 release at <=10 µM. However, at 30 µM, the same concentration causing inhibition of p38 kinase activity (see Fig. 4GoD), SB202474 partially inhibited LTC4 release (p < 0.05 vs fMLP) (Fig. 5GoB).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 5. Effect of ERK1/2 or p38 MAPK inhibition on fMLP-stimulated AA release and LTC4 production. [3H]AA-labeled eosinophils (A) or nonlabeled eosinophils (B) were preincubated with U0126, SB203580, or SB202474 for 30 min, and then incubated with cytochalasin B for 2 min before stimulation by fMLP for another 10 min. [3H]AA release (A) was measured by scintillation counting, and was calculated by subtraction of background (n = 3). LTC4 production (B) was measured by enzyme immunoassay (n = 3–7). Results are presented as the mean ± SEM.

 
Reversal of inhibition of LTC4 secretion by AA

To determine the specificity of MAPK and cPLA2 inhibitors in blocking eosinophil LTC4 secretion, experiments were generated measuring LTC4 secretion in activated eosinophils after treatment with exogenous AA. Treatment of eosinophils with AA reversed substantially the inhibitory effects of AACOCF3, U0126, or SB203580 on secretion of LTC4 (Fig. 6Go). In three experiments, activation of eosinophils with fMLP caused 771 ± 136.4 pg/ml LTC4 secretion after 10 min, vs 2.1 ± 0.8 pg/ml for nonactivated eosinophils (p < 0.01). Incubation with 10 µM U0126, 30 µM SB203580, or 30 µM AACOCF3 before activation with 1 µM fMLP almost completely inhibited LTC4 secretion (p < 0.01). In eosinophils treated with either inhibitor, addition of 10 µM AA restored LTC4 secretion to 649.7 ± 118.1 pg/ml for SB203580-treated cells, and 704.2 ± 181.7 pg/ml for AACOCF3-treated cells (p = NS vs fMLP alone). Addition of AA substantially increased LTC4 secretion for U0126-treated cells to 331.6 ± 90.1 pg/ml (p < 0.01); however, this was still less than fMLP-treated cells that received no inhibitor (p < 0.05). The incomplete restoration of U0126-inhibited LTC4 release may be explained by the fact that MEK is also required for 5-lipoxygenase activation (42). Treatment with AA did not alter LTC4 secretion in nonactivated eosinophils (data not shown). These results demonstrated MAPK and cPLA2 inhibitors specifically blocked AA mobilization from fMLP-stimulated eosinophils.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 6. Reversal of AACOCF3-, U0126-, or SB203580-induced inhibition of eosinophil LTC4 release by AA. Eosinophils were incubated with either inhibitor for 30 min, and further incubated with or without 10 µM AA for another 10 min, followed by stimulation with fMLP + cytochalasin B. LTC4 production was measured by enzyme immunoassay, and results are presented as the mean ± SEM from three separate experiments. Con, control.

 
Effects of MAPK inhibition on fMLP-induced cPLA2 activation

To evaluate the possible biological significance of MAPK in causing fMLP-induced cPLA2 activation, we tested the effects of U0126 and SB203580 on fMLP-stimulated cPLA2 activity in eosinophils. Eosinophils were pretreated for 30 min with 30 µM SB203580 or 10 µM U0126, followed by stimulation with fMLP for 5 min. Inhibitors were present throughout the activation period. cPLA2 activity increased from 0.5 ± 0.07 pM/106 cells/30 min for nonstimulated eosinophils to 1.1 ± 0.4 pM/106 cells/30 min after fMLP stimulation (p < 0.01). This increased activity was almost completely blocked by the MEK inhibitor, U0126, or the p38 inhibitor, SB203580 (p < 0.01 for both comparisons vs fMLP only, Fig. 7Go). These observations suggest that both ERK1/2 and p38 MAPKs are substantially involved in fMLP-stimulated cPLA2 activation.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 7. Effect of MAPK inhibition on cPLA2 activation. Eosinophils were preincubated with 10 µM U0126 or 30 µM SB203580 for 30 min, and treated with cytochalasin B for 2 min before stimulation by fMLP for 2 min. cPLA2 activity was measured using [14C]PAPC as substrate. Results are presented as the mean ± SEM from three separate experiments. Con, control.

 
Effects of Ras farnesylation inhibition on fMLP-stimulated AA release

In three additional experiments, the role of the small GTP-binding protein, Ras, which is a known upstream kinase for ERK1/2 (43), was investigated by using two structurally unrelated Ras farnesyltransferase inhibitors, FPT inhibitor III and manumycin (44, 45). Activation of eosinophils with fMLP caused 6.9 ± 0.4% [3H]AA release after 10 min, vs 0.7 ± 0.03% release for nonactivated eosinophils (p < 0.01). Incubation with 100 µM FPT inhibitor III or 10 µM manumycin before activation with 1 µM fMLP inhibited [3H]AA release to <2% (Fig. 8Go, p < 0.01 for both groups).



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 8. Effect of Ras farnesylation inhibition on fMLP-stimulated AA release. [3H]AA-labeled eosinophils were preincubated with 100 µM manumycin A or 10 µM FPT inhibitor III for 60 min at 37°C, and then incubated with cytochalasin B for 2 min before stimulation by 1 µM fMLP for another 10 min. [3H]AA release was measured by scintillation counting, and was calculated by subtraction of background. Results are presented as the mean ± SEM from four separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The objective of this investigation was to determine the relative contribution of endogenous isoforms of PLA2 in the hydrolysis of AA and subsequent downstream synthesis and secretion of LTC4 during metabolic activation of human eosinophils. Prior investigations have suggested that the 85-kDa cPLA2 plays a major role in AA hydrolysis and downstream eicosanoid metabolism (46, 47), and that this occurs as the result of transport of cPLA2 to nuclear membrane in its phosphorylated state (48). Other investigations, however, have identified a substantive role for the 14-kDa sPLA2 and/or the 80-kDa calcium-dependent iPLA2 (49, 50). In this investigation, we examine the potential roles for all PLA2 isoforms native to eosinophils as well as that of cPLA2. Multiple specific blockers of each isoform were used, and cross-reactivity was excluded between sPLA2, iPLA2, and cPLA2. We found that cPLA2 plays a critical role in activated hydrolysis of AA and secretion of LTC4 by human eosinophils.

We also examined the relationship between the upstream phosphorylation of ERK1/2 and p38 MAPKs on the subsequent cPLA2 activation, which is required for catalysis of phospholipid. Our investigations demonstrated that cPLA2 inhibition prevents AA hydrolysis and subsequent secretion of LTC4 for isolated human eosinophils activated by fMLP. In these studies, fMLP was selected because it is a widely studied activator of eosinophil activity that causes both cell degranulation (26) and eosinophil secretion of LTC4 (24). Because the physiological event(s) causing activation of eosinophil secretion in human airways has not yet been identified, fMLP, which binds specifically to a G protein-coupled cell surface receptor, has been used widely to replicate eosinophil activation (27).

Eosinophils possess both the sPLA2 and cPLA2 (4, 5), and possibly iPLA2 (6). The role of specific PLA2 subtypes involved in receptor-mediated AA mobilization has not been definitively elucidated. Our data demonstrated that endogenous iPLA2 and sPLA2 play no significant role in fMLP-stimulated eosinophils. BEL is a specific inhibitor of iPLA2, which does not inhibit sPLA2 or cPLA2, and has been shown to block enzyme activity in A10 smooth muscle cells and P388D1 cells at concentrations <=1–5 µM (51, 52). Preincubation of eosinophils with BEL at <=10 µM had no effect on AA or LTC4 release (Fig. 2Go); hence, iPLA2 has no effect in fMLP-induced AA release from human eosinophils. LY311727 is a structure-based sPLA2 inhibitor, and has been shown to suppress the catalytic activity of both group IIa and group V sPLA2 with an IC50 of 23 and 36 nM, respectively (36, 53). Our data demonstrate that 10 µM LY311727 had no inhibitory effect on fMLP-stimulated AA or LTC4 release (Fig. 2Go). This demonstrates that endogenous 14-kDa sPLA2 activity is not required in fMLP-induced AA hydrolysis or metabolism. However, this does not exclude the antigenic properties of sPLA2 in AA release, as suggested by others (54). Indeed, the minor role of other PLA2 in AA release is suggested in Fig. 5Go, in which the IC50 for AA is greater than the IC50 for LTC4 for both U0126 and SB203580.

In these investigations, we found that the cPLA2 inhibitor, AACOCF3, substantially blocked fMLP-stimulated AA release and subsequent LTC4 production (Fig. 2Go). AACOCF3 also inhibits iPLA2 in vitro (34), but studies with BEL indicated that iPLA2 does not participate in eosinophil AA release. Another cPLA2 inhibitor, surfactin (32, 55), also suppressed fMLP-induced eosinophil AA and LTC4 production (data not shown). We also found that cPLA2 activation induced by fMLP follows the same kinetics as that for AA release (Figs. 1GoB and 3). Our data therefore suggest that fMLP-induced AA hydrolysis and LTC4 synthesis occur mainly through activation of cPLA2.

ERK1/2 are associated with the activation of cPLA2 in many cell systems (11, 12, 19, 20); however, different G protein-coupled receptors can activate cPLA2 through ERK-independent pathways (21, 22, 23). The data presented in this study show that fMLP stimulation of eosinophils activates both ERK1/2 and p38 to activate cPLA2. In our studies, ERK1/2 phosphorylation preceded cPLA2 activation (Figs. 3Go and 4Go), and pretreatment of eosinophils with the MEK inhibitor, U0126, inhibited cPLA2 activity (Fig. 7Go) and AA release (Fig. 5Go) caused by fMLP. Pretreatment of eosinophils with 30 µM SB203580 also prevented fMLP-induced cPLA2 activity (Fig. 7Go) and AA release (Fig. 5Go). Thus, our experiments provide direct evidence that both ERK and p38 MAPK are required for cPLA2 activation and AA release in fMLP-stimulated eosinophils. These results are consistent with recent findings in macrophages and Fc{gamma}RIIa- or Fc{gamma}RIIIB-stimulated neutrophils (56, 57). However, our data were in contrast with those of Syrbu et al. (41), who found in human neutrophils that fMLP-mediated ERK1/2 and p38 are not involved in cPLA2 phosphorylation. Taken together, the findings from those studies and ours suggest that cell-specific pathways most likely are involved in the regulation of eicosanoid synthesis in different inflammatory cells.

Our study showed that relatively high concentrations of SB203580 (10–30 µM) are required to inhibit p38 activity and AA metabolism in fMLP-stimulated eosinophils. Similar concentrations also are required to inhibit p38 activity. This contrasts with the submicromolar concentration of SB203580 that is required to inhibit cytokine production in monocytes (39). However, our study is consistent with prior investigations, which have required high concentrations of SB203580 to suppress fMLP-induced respiratory burst activity and degranulation of neutrophils (58, 59). It should be noted that SB203580 (>5 µM) has recently been reported to affect 3-phosphoinositide-dependent protein kinase 1 activity, which is upstream kinase for protein kinase B (60). However, in fMLP-stimulated eosinophils, no protein kinase B phosphorylation was observed. This excluded the possibility of SB203580 acting on 3-phosphoinositide-dependent protein kinase 1. The concentrations of SB203580 required to block p38 activity in our study, as measured by blockade of ATF-2 phosphorylation, suggests that p38 is involved in cPLA2 activation during AA hydrolysis. Nevertheless, the possibility that SB203580 targets an enzyme other than p38 MAPK cannot be excluded. This is suggested by the ability of the inactive p38 MAPK inhibitor, SB202474, to block AA metabolism at concentrations greater than required for the active inhibitor.

We conclude that fMLP induces AA release from human eosinophils through the activation of cPLA2. ERK1/2 and p38 MAPK regulate cPLA2 activation, which is essential for AA hydrolysis and subsequent LTC4 secretion. Our data demonstrate no significant role for sPLA2 or iPLA2, the other endogenous phospholipases, in either the regulation phospholipid hydrolysis in the production of AA or in the generation of cysteinyl leukotriene, as modeled by this system of pharmacological activation of human eosinophils.


   Acknowledgments
 
We are grateful for the generous gift of Drs. Ruth Kramer and Neal Roehm (for purified cPLA2, sPLA2 inhibitor, LY311727, and polyclonal rabbit anti-cPLA2; Eli Lilly) and the Immunology Department of Genetics Institute (Andover, MA) for the monoclonal mouse anti-cPLA2.


   Footnotes
 
1 This work was supported by National Heart, Lung, and Blood Institute Grant HL-45368 and Training Fellowship Grant T32-HL07605 (to X.Z.), by National Heart, Lung, and Blood Institute Specialized Centers of Research Grant HL-56399, and by the Glaxo Wellcome Center Excellence in Asthma Award. Back

2 Address correspondence and reprint requests to Dr. Alan R. Leff, Section of Pulmonary and Critical Care Medicine, Department of Medicine, MC6076, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail address: aleff{at}medicine.bsd.uchicago.edu Back

3 Abbreviations used in this paper: AA, arachidonic acid; AACOCF3, arachidonic trifluoromethyl ketone; ATF, activating transcription factor; BEL, bromoenol lactone; cPLA2, cytosolic group IV PLA2; ERK, extracellular signal-regulated protein kinase; FPT, farnesyl protein transferase; iPLA2, cytosolic Ca2+-independent PLA2; JNK, c-Jun N-terminal kinase; LTC4, leukotriene C4; LY311727, 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein/ERK kinase; PAPC, 1-palmitoyl-2-[14C]arachidonylphosphatidylcholine; PLA2, phospholipase A2; sPLA2, secretory PLA2. Back

Received for publication September 14, 2000. Accepted for publication April 17, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dennis, E. A.. 1983. Phospholipases. P. Boyer, ed. The Enzyme 307. Academic Press, New York.
  2. Dennis, E. A.. 1994. Diversity of group types, regulation, and function of phospholipase A2. J. Biol. Chem. 269:13057.[Free Full Text]
  3. Mayer, R. J., L. A. Marshall. 1993. New insights on mammalian phospholipase A2(s); comparison of arachidonoyl-selective and -nonselective enzymes. FASEB J. 7:339.[Abstract]
  4. Zhu, X., N. M. Munoz, N. Rubio, A. Herrnreiter, D. Mayer, I. Douglas, A. R. Leff. 1996. Quantitation of the cytosolic phospholipase A2 (type IV) in isolated human peripheral blood eosinophils by sandwich-ELISA. J. Immunol. Methods 199:119.[Medline]
  5. Blom, M., A. T. J. Tool, P. C. Wever, G. J. Wolbink, M. C. Brouwer, J. Calafat, A. Egesten, E. F. Knol, E. E. Hack, D. Roos, A. J. Verhoeven. 1998. Human eosinophils express, relatively to other circulating leukocytes, large amounts of secretory 14-kD phospholipase A2. Blood 91:3037.[Abstract/Free Full Text]
  6. Lynch, O., M. A. Giembyz, P. J. Barnes, M. A. Lindsay. 1998. Identification and characterization of two phospholipase A2 enzymes activated following LTB4 induced stimulation of eosinophils. Am. J. Respir. Crit. Care Med. 157:A600. (Abstr.).
  7. Clark, J., L. Lin, R. Kriz, C. Ramesha, L. Sultzman, A. Lin, N. Milona, J. Knopf. 1991. A novel arachidonic acid-selective cytosolic PLA2 contains a calcium-dependent translocation domain with homology to PKC and GAP. Cell 65:1043.[Medline]
  8. Leslie, C. C.. 1997. Properties and regulation of cytosolic phospholipase A2. J. Biol. Chem. 272:16709.[Free Full Text]
  9. Peters-Golden, M., K. Song, T. Marshall, T. Brock. 1996. Translocation of cytosolic phospholipase A2 to the nuclear envelope elicits topographically localized phospholipid hydrolysis. Biochem. J. 318:797.
  10. Woods, J. W., M. J. Coffey, T. G. Brock, I. I. Singer, M. Peters-Golden. 1995. 5-lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocated to the nuclear envelope upon cell activation. J. Clin. Invest. 95:2035.
  11. Durstin, M., S. Durstin, T. F. P. Molski, E. L. Becker, R. I. Sha’afi. 1994. Cytoplasmic phospholipase A2 translocates to membrane fraction in human neutrophils activated by stimuli that phosphorylate mitogen-activated protein kinase. Proc. Natl. Acad. Sci. USA 91:3142.[Abstract/Free Full Text]
  12. Nemenoff, R. A., S. Winitz, X. Qiann, V. Van Pattern, G. L. Johnson, L. E. Heasley. 1993. Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C. J. Biol. Chem. 268:1960.[Abstract/Free Full Text]
  13. Lin, L. L., M. Wartmann, A. Y. Lin, J. L. Knopf, R. J. Davis. 1993. cPLA2 is phosphorylated and activated by MAP kinase. Cell 72:269.[Medline]
  14. Cano, E., L. C. Mahadevan. 1995. Parallel signal processing among mammalian MAPKs. Trends Biochem. Sci. 20:117.[Medline]
  15. Han, J., J. D. Lee, L. Bibbs, R. J. Ulevitch. 1994. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265:808.[Abstract/Free Full Text]
  16. Rouse, J., P. Cohen, S. Trigon, M. Morange, A. Alonso-Liamazares, D. Zamanillo, T. Hunt, A. R. Nebreda. 1994. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78:1027.[Medline]
  17. Seger, R., E. G. Krebs. 1995. The MAPK signaling cascade. FASEB J. 9:726.[Abstract]
  18. Qiu, Z. H., M. A. Gijon, M. S. de Carvalho, D. M. Spencer, C. C. Leslie. 1998. The role of calcium and phosphorylation of cytosolic phospholipase A2 in regulating arachidonic acid release in macrophages. J. Biol. Chem. 273:8203.[Abstract/Free Full Text]
  19. Xing, M. Z., P. A. Insel. 1996. Protein kinase C-dependent activation of cytosolic phospholipase A2 and mitogen-activated protein kinase by {alpha}1-adrenergic receptors in Madin-Darby canine kidney cells. J. Clin. Invest. 97:1302.[Medline]
  20. Qiu, Z. H., C. C. Leslie. 1994. Protein kinase C-dependent and -independent pathways of mitogen-activated protein kinase activation in macrophages by stimuli that activate phospholipase A2. J. Biol. Chem. 269:19480.[Abstract/Free Full Text]
  21. Kramer, R. M., E. F. Roberts, S. L. Um, A. G. Borsch-Haubold, S. P. Watson, M. J. Fisher, J. A. Jakubowski. 1996. P38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. J. Biol. Chem. 271:27723.[Abstract/Free Full Text]
  22. Waterman, W. H., R. I. Sha’afi. 1995. A mitogen-activated protein kinase independent pathway involved in the phosphorylation and activation of cytosolic phospholipase A2 in human neutrophils stimulated with tumor necrosis factor-{alpha}. Biochem. Biophys. Res. Commun. 209:271.[Medline]
  23. Hernandez, M., Y. Bayon, M. Sanchez Crespo, M. L. Nieto. 1997. Thrombin produces phosphorylation of cytosolic phospholipase A2 by a mitogen-activated protein kinase-independent mechanism in the human astrocytoma cell line 1321N1. Biochem. J. 328:263.
  24. White, S. R., M. E. Strek, G. V. P. Kulp, S. M. Spaethe, R. A. Burch, S. P. Neeley, A. R. Leff. 1993. Regulation of human eosinophil degranulation and activation by endogenous phospholipase A2. J. Clin. Invest. 91:2118.
  25. Neeley, S. P., K. J. Hamann, S. R. White, S. L. Baranowski, R. A. Burch, A. R. Leff. 1993. Selective regulation of expression of surface adhesion molecules Mac-1, L-selectin, and VLA-4 on human eosinophils and neutrophils. Am. J. Respir. Cell Mol. Biol. 8:633.
  26. Takafuji, S., K. Tadokoro, K. Ito, C. A. Dahinden. 1995. Effects of physiologic soluble agonists on leukotriene C4 production and degranulation by human eosinophils. Int. Arch. Allergy Immunol. 108:(Suppl. 1):36.
  27. Giembycz, M. A., M. A. Lindsay. 1999. Pharmacology of the eosinophils. Pharmacol. Rev. 51:213.[Abstract/Free Full Text]
  28. Kita, H., R. I. Abu Ghazaleh, G. J. Gleich, R. T. Abraham. 1991. Role of pertussis toxin-sensitive G proteins in stimulus-dependent human eosinophils degranulation. J. Immunol. 147:3466.[Abstract]
  29. Yazdanbakhsh, M., C. M. Eckmann, L. Koenderman, A. J. Verhoeven, D. Roos. 1987. Eosinophils do respond to fMLP. Blood 70:379.[Abstract/Free Full Text]
  30. Kerner, P., M. P. Wymann, V. von Tsharner, D. A. Deranleau, P. C. Tai, C. J. Spry, A. A. Dahinden, M. Baggiolini. 1991. Shape changes, exocytosis, and cytosolic free calcium changes in stimulated human eosinophils. J. Clin. Invest. 87:2012.
  31. Hansel, T. T., I. J. M. De Vries, T. Iff, S. Rihs, M. Wandzilak, S. Bets, K. Blaser, C. Walker. 1991. An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils. J. Immunol. Methods 145:105.[Medline]
  32. Kim, K., S. Y. Jung, D. K. Lee, J.-K. Jung, J. K. Park, D. K. Kim, C.-H. Lee. 1998. Suppression of inflammatory responses by Surfactin, a selective inhibitor of platelet cytosolic phospholipase A2. Biochem. Pharmacol. 55:975.[Medline]
  33. Street, I. P., H.-K. Lin, F. Laliberte, F. Ghomashchi, Z. Wang, H. Perrier, N. M. Tremblay, A. Huang, P. K. Weech, M. H. Gelb. 1993. Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2. Biochemistry 32:5935.[Medline]
  34. Ackernann, E. J., K. Conde-Frieboes, E. A. Dennis. 1995. Inhibition of macrophage Ca2+-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones. J. Biol. Chem. 270:445.[Abstract/Free Full Text]
  35. Schevitz, R. W., N. J. Bach, D. G. Carlson, N. Y. Chirgadze, D. K. Clawson, R. D. Dillard, S. E. Draheim, L. W. Hartley, N. D. Jones, E. D. Mihelich, et al 1995. Structure-based design of the first potent and selective inhibitor of human non-pancreatic secretory phospholipase A2. Nat. Struct. Biol. 2:458.[Medline]
  36. Chen, Y., E. A. Dennis. 1998. Expression and characterization of human group V phospholipase A2. Biochim. Biophys. Acta 1394:57.[Medline]
  37. Scherle, P. A., E. A. Jones, M. F. Favata, A. J. Daulerio, M. B. Covington, S. A. Nurnberg, R. L. Magolda, J. M. Trzaskos. 1998. Inhibition of MAP kinase kinase prevents cytokine and prostaglandin E2 production in lipopolysaccharide-stimulated monocytes. J. Immunol. 161:5681.[Abstract/Free Full Text]
  38. Favata, M. F., K. Y. Horiuchi, E. J. Manos, A. J. Daulerio, D. A. Stradley, W. S. Feeser, D. E. Van Dyk, W. J. Pitts, R. A. Earl, F. Hobbs, et al 1998. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273:18623.[Abstract/Free Full Text]
  39. Lee, J. C., J. T. Laydon, P. C. McDonnell, T. F. Gallagher, S. Kumar, D. Green, D. McNulty, M. J. Blumenthal, J. R. Heys, S. W. Landvatter, et al 1994. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:739.[Medline]
  40. Miura, K., J. T. Schroeder, W. C. Hubbard, Jr D. W. MacGlashan. 1999. Extracellular signal-regulated kinases regulate leukotriene C4 generation, but not histamine release or IL-4 production from human basophils. J. Immunol. 162:4198.[Abstract/Free Full Text]
  41. Syrbu, S. I., W. H. Waterman, T. F. P. Molski, D. Nagarkatti, J.-J. Hajjar, R. I. Sha’afi. 1999. Phosphorylation of cytosolic phospholipase A2 and the release of arachidonic acid in human neutrophils. J. Immunol. 162:2334.[Abstract/Free Full Text]
  42. Lepley, R. A., F. A. Fitzpatrick. 1996. Inhibition of mitogen-activated protein kinase kinase blocks activation and redistribution of 5-lipoxygenase in HL-60 cells. Arch. Biochem. Biophys. 331:141.[Medline]
  43. Worthen, G. S., N. Avdi, A. M. Buhl, N. Suzuki, G. L. Johnson. 1994. FMLP activates Ras and Raf in human neutrophils: potential role in activation of MAP kinase. J. Clin. Invest. 94:815.
  44. Patel, D. V., R. J. Schmidt, S. A. Biller, E. M. Gordon, S. S. Robinson, V. Manne. 1995. Farnesyl diphosphate-based inhibitors of Ras farnesyl protein transferase. J. Med. Chem. 38:2906.[Medline]
  45. Hara, M., K. Akasaka, S. Akinaga, M. Okabe, H. Nakano, R. Gomez, D. Wood, M. Uh, F. Tamanoi. 1993. Identification of Ras farnesyltransferase inhibitors by microbial screening. Proc. Natl. Acad. Sci. USA 90:2281.[Abstract/Free Full Text]
  46. Uozumi, N., K. Kume, T. Nagase, N. Nakatani, S. Ishii, F. Tashiro, Y. Komagata, K. Maki, K. Ikuta, Y. Ouchi, et al 1997. Role of cytosolic phospholipase A2 in allergic response and parturition. Nature 390:618.[Medline]
  47. Bonventre, J. V., A. Huang, M. R. Taheri, E. O’Leary, E. Li, M. A. Moskowitz, A. Sapirstein. 1997. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature 390:622.[Medline]
  48. Pouliot, M., P. P. McDonald, E. Krump, J. A. Mancini, S. R. McColl, P. K. Weech, P. Borgeat. 1996. Colocalization of cytosolic phospholipase A2, 5-lipoxygenase, and 5-lipoxygenase-activating protein at the nuclear membrane of A23187-stimulated human neutrophils. Eur. J. Biochem. 238:250.[Medline]
  49. Hundley, T. R., L. A. Marshall, W. C. Hubbard, Jr D. W. MacGlashan. 1998. Characteristics of arachidonic acid generation in human basophils: relationship between the effects of inhibitors of secretory phospholipase A2 activity and leukotriene C4 release. J. Pharmacol. Exp. Ther. 284:847.[Abstract/Free Full Text]
  50. Akiba, S., S. Mizunaga, K. Kume, M. Hayama, T. Sato. 1999. Involvement of group VI Ca2+-independent phospholipase A2 in protein kinase C-dependent arachidonic acid liberation in zymosan-stimulated macrophage-like P388D1 cells. J. Biol. Chem. 274:19906.[Abstract/Free Full Text]
  51. Hazen, S. L., R. W. Gross. 1991. Human myocardial cytosolic Ca2+-independent phospholipase A2 is modulated by ATP: concordant ATP-induced alterations in enzyme kinetics and mechanism-based inhibition. Biochem. J. 280:581.
  52. Balsinde, J., E. A. Dennis. 1996. Distinct roles in signal transduction for each of the phospholipase A2 enzymes present in P388D1 macrophages. J. Biol. Chem. 271:6758.[Abstract/Free Full Text]
  53. Fleisch, J. H., C. T. Armstrong, C. R. Roman, E. D. Mihelich, S. M. Spaethe, W. T. Jackson, J. L. Bobbitt, S. Draheim, N. J. Bach, R. D. Dillard, et al 1996. Recombinant human secretory phospholipase A2 released thromboxane from guinea pig bronchoalveolar lavage cells: in vitro and ex vivo evaluation of a novel secretory phospholipase A2 inhibitor. J. Pharmacol. Exp. Ther. 278:252.[Abstract/Free Full Text]
  54. Hernandez, M., S. L. Burillo, M. S. Crespo, M. L. Nieto. 1998. Secretory phospholipase A2 activates the cascade of mitogen-activated protein kinases and cytosolic phospholipase A2 in the human astrocytoma cell line 1321N1. J. Biol. Chem. 273:606.[Abstract/Free Full Text]
  55. Zhu, X., N. M. Munoz, K. P. Kim, H. Sano, W. H. Cho, A. R. Leff. 1999. Cytosolic phospholipase A2 activation is essential for {beta}1 and {beta}2 integrin-dependent adhesion of human eosinophils. J. Immunol. 163:3423.[Abstract/Free Full Text]
  56. Gijon, M. A., D. M. Spencer, A. R. Siddiqi, J. V. Bonventre, C. C. Leslie. 2000. Cytosolic phospholipase A2 is required for macrophage arachidonic acid release by agonists that do and do not mobilize calcium. J. Biol. Chem. 275:20146.[Abstract/Free Full Text]
  57. Hazan-Halevy, I., R. Seger, R. Levy. 2000. The requirement of both extracellular regulated kinase and p38 mitogen-activated protein kinase for stimulation of cytosolic phospholipase A2 activity by either Fc{gamma}RIIA or Fc{gamma}RIIIB in human neutrophils. J. Biol. Chem. 275:12416.[Abstract/Free Full Text]
  58. McLeish, K. R., C. Knall, R. A. Ward, P. Gerwins, P. Y. Coxon, J. B. Klein, G. L. Johnson. 1998. Activation of mitogen-activated protein kinase cascades during priming of human neutrophils by TNF-{alpha} and GM-CSF. J. Leukocyte Biol. 64:537.[Abstract]
  59. Mocsai, A., Z. Jakus, T. Vantus, G. Berton, C. A. Lowell, E. Ligeti. 2000. Kinase pathways in chemoattractant-induced degranulation of neutrophils: the role of p38 mitogen-activated protein kinase activated by Src family kinases. J. Immunol. 164:4321.[Abstract/Free Full Text]
  60. Lali, F. V., A. E. Hunt, S. J. Turner, B. M. J. Foxwell. 2000. The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J. Biol. Chem. 275:7395.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
T. Miyahara, K. Hamanaka, D. S. Weber, M. Anghelescu, J. R. Frost, J. A. King, and J. C. Parker
Cytosolic phospholipase A2 and arachidonic acid metabolites modulate ventilator-induced permeability increases in isolated mouse lungs
J Appl Physiol, February 1, 2008; 104(2): 354 - 362.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
W.-C. Chang, C. Nelson, and A. B. Parekh
Ca2+ influx through CRAC channels activates cytosolic phospholipase A2, leukotriene C4 secretion, and expression of c-fos through ERK-dependent and -independent pathways in mast cells
FASEB J, November 1, 2006; 20(13): 2381 - 2383.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
W. Cao, L. Cheng, J. Behar, P. Biancani, and K. M. Harnett
IL-1beta signaling in cat lower esophageal sphincter circular muscle
Am J Physiol Gastrointest Liver Physiol, October 1, 2006; 291(4): G672 - G680.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
I. B. Copland, D. Reynaud, C. Pace-Asciak, and M. Post
Mechanotransduction of stretch-induced prostanoid release by fetal lung epithelial cells
Am J Physiol Lung Cell Mol Physiol, September 1, 2006; 291(3): L487 - L495.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
S. Yoshikawa, T. Miyahara, S. D. Reynolds, B. R. Stripp, M. Anghelescu, F. G. Eyal, and J. C. Parker
Clara cell secretory protein and phospholipase A2 activity modulate acute ventilator-induced lung injury in mice
J Appl Physiol, April 1, 2005; 98(4): 1264 - 1271.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Myou, A. R. Leff, S. Myo, E. Boetticher, A. Y. Meliton, A. T. Lambertino, J. Liu, C. Xu, N. M. Munoz, and X. Zhu
Activation of Group IV Cytosolic Phospholipase A2 in Human Eosinophils by Phosphoinositide 3-Kinase Through a Mitogen-Activated Protein Kinase-Independent Pathway
J. Immunol., October 15, 2003; 171(8): 4399 - 4405.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. M. Munoz, Y. J. Kim, A. Y. Meliton, K. P. Kim, S.-K. Han, E. Boetticher, E. O'Leary, S. Myou, X. Zhu, J. V. Bonventre, et al.
Human Group V Phospholipase A2 Induces Group IVA Phospholipase A2-independent Cysteinyl Leukotriene Synthesis in Human Eosinophils
J. Biol. Chem., October 3, 2003; 278(40): 38813 - 38820.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Sakamoto, T. Tosaki, and Y. Nakagawa
Overexpression of Phospholipid Hydroperoxide Glutathione Peroxidase Modulates Acetyl-CoA, 1-O-Alkyl-2-lyso-sn-glycero-3-phosphocholine Acetyltransferase Activity
J. Biol. Chem., December 20, 2002; 277(52): 50431 - 50438.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
X. Zhu, B. Jacobs, E. Boetticher, S. Myou, A. Meliton, H. Sano, A. T. Lambertino, N. M. Munoz, and A. R. Leff
IL-5-induced integrin adhesion of human eosinophils caused by ERK1/2-mediated activation of cPLA2
J. Leukoc. Biol., November 1, 2002; 72(5): 1046 - 1053.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhu, X.
Right arrow Articles by Leff, A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhu, X.
Right arrow Articles by Leff, A. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS