Background
IL-8/CXCL8 is a selective and potent neutrophil chemoattractant. Previous studies have shown that upstream activation of PI3K, ERK-1/2, or p38 MAPK [
1‐
7] pathways caused by IL-8/CXCL8 regulates the induction of transendothelial PMN migration. However, the signaling mechanism downstream of these kinases in causing migration of PMNs has not been established previously and critical intermediate steps regulating neutrophil migration remain unknown.
Phospholipase A
2s (PLA
2) are esterases that cleave glycerophospholipids at the
sn-2 ester bond, releasing a fatty acid and a lysophospholipid [
8‐
11]. PLA
2s are divided into five different groups; a) secretory PLA
2 [
12,
13], b) cytosolic gIVPLA
2 (gIVPLA
2) [
14], c) Ca
2+-independent PLA
2, [
15,
16] d) platelet-activating factor hydrolyses, [
17,
18] and e) lysosomal PLA
2 [
19]. Among these groups, gIVaPLA
2 is thought to be not only a rate-limiting enzyme in eicosanoid biosynthesis [
20] but also to be essential in maintenance of β
2-integrin adhesion in granulocytes [
21,
22]. We have shown previously that ERK-1/2 and Akt/PKB phosphorylation activated gIVaPLA
2 to cause β
2-integrin adhesion of granulocytes to ICAM-1[
23]. We also have shown that phosphorylation to activate gIVaPLA
2 results from upstream phosphorylation of these kinase and that maintenance of this phosphorylated state regulates the process of β
2-integrin adhesion [
24,
25].
Because MAP kinase and PI3K also regulate gIVaPLA2 phosphorylation, we postulated that activation of gIVaPLA2 might regulate neutrophil migration. The objective of this study was to examine specifically the functional role of gIVaPLA2 in PMN migration caused by IL-8/CXCL8. IL-8/CXCL8 was applied in concentration causing upstream phosphorylation of ERK-1/2, p38 MAPK and Akt/PKB. We found that inhibition of gIVaPLA2 activity blocked substantially the transmigration toward IL-8/CXCL8 in a transwell chamber. This study is the first demonstration that activation of gIVaPLA2 is a critical regulatory step subsequent to upstream activation of signaling kinases in eliciting PMN migration.
Methods
Antibodies and reagents
IL-8/CXCL8 was purchased from Peprotech (Rocky Hill, NJ) while bovine serum albumin fraction V and human polymophonuclear leukocytes (PMNs) isolation materials were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Anti-phosphorylated gIVaPLA2 Ab (Ser505) was purchased from Cell Signaling Technology (Beverly, MA). Mouse IgG was purchased from BD Biosciences (Mountain View, CA). Polystyrene 96-well microtiter plates were obtained from Neuro Probe (Gaithersburg, MD). Rhodamine-phalloidin was obtained from Sigma-Aldrich Chemical (St. Louis, MO).
Isolation of human PMNs
Venous blood from normal human subjects (20-45 years old) was collected in heparin-containing tubes, and PMNs were isolated by Ficoll-Paque sedimentation as described previously [
26,
27]. Purity of PMN on H and E-stained cytoslides was ~90-95%. Informed written consent was obtained from all volunteers in this study.
Transwell migration assay
PMN migration in transwell microplates was assessed using the standard methods as described previously [
28]. Preliminary experiments have established that the number of cells (4 × 10
4 cells) used allow the optimal % cell migration without clogging the pores of transwell filter of the upper chamber. Cells then were preincubated with HBSS, 3 μM - 30 μM arachidonyl trifluoromethylketone [TFMK; inhibitor of gIVaPLA
2 [
29], or 10
-9 M -10
-6 M pyrrophenone [inhibitor of gIVaPLA
2 [
30] for 30 min at 37°C. Treated cells in 50 μl HBSS were transferred onto 5 μm-pore transwell filters positioned on top of the migration chamber. HBSS or 10 ng/ml to 1000 ng/ml IL-8/CXCL8 was loaded in the bottom chamber (final volume = 310 μl), and the transwell microplates were incubated for 60 min and 90 min at 37°C. The migrated PMNs were treated with 100 μl of HBSS + 10% FBS buffer and 100 μl developing solution [8 ml 100 nM NaH
2PO
4 (pH = 5.5), 1000 μl 10% hexadecyltrimethylammonium bromide (HTAB), 3 μl 30% hydrogen peroxide, 1000 μl 10%
o-dianisidine dihydrochloride]. The reaction was terminated by addition of 50 μl sulfuric acid and myeloperoxidase (MPO) activity was measured at 405 nm in a Thermomax microplate reader (Molecular Devices, Menlo Park, CA). The fraction of migrated PMNs present in the lower chamber was measured as total MPO content. Data were expressed as % cell migration. Maximal, no-toxic inhibitory concentration of TFMK and pyrrophenone were established in initial studies demonstrating blockade of gIVaPLA
2 activity [see also Results].
In separate studies, morphological changes of the non-migrated cells (top chamber) and migrated cells (bottom chamber) toward IL-8/CXCL8 were examined. The effect of 30 μM TFMK or 10-6 M pyrrophenone on cell deformability caused by IL-8/CXCL8 also was examined using confocal microscopy.
Immunoblotting analysis
PMNs (106 cells/group) were activated with HBSS and 100 ng/ml IL-8/CXCL8 at different time intervals, and phosphorylation of ERK-1/2, p38 MAPK, and Akt/PKB were analyzed using Western blot. The pellet was lysed in disruption 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. Samples were loaded to SDS-PAGE using 8% (gIVaPLA2) or 10% (ERK-1/2, p38 MAPK, PI3K) acrylamide gels under reducing conditions. The membrane was blocked with 1% BSA in TBS-T buffer and phosphorylated Ab against ERK-1/2 (1:1000; Cell Signaling Technology; Beverly, MA), p38 MAPK and Akt/PKB (1:1000; Cell Signaling Technology; Beverly, MA), or Ser505 gIVaPLA2 (1:1000) was added followed by relevant secondary Ab conjugated with HRP. Protein of interest was analyzed by an enhanced chemiluminescence system (Amersham, Arlington Heights, IL).
Measurement of arachidonic acid (AA) release
Isolated PMNs were incubated in RPMI medium containing 5% FBS and 0.5 μCi [
3H]AA. Labeled PMNs were incubated for 2 h and unincorporated [
3H]AA was washed away by HBSS containing 0.2% BSA. Thereafter, treated PMNs were activated with saline, 100 ng/ml IL-8/CXCL8 or 1 μM FMLP (+ 5 μg/ml cytochalasin B). The reaction was terminated by centrifugation at 12,000 ×
g for 1 min. Supernatant were collected, and pellets were lysed in 1% Triton X-100. [
3H]AA release was measured by scintillation counting and expressed as counts per min (cpm) [
21,
24].
Measurement of LTB4 secretion
Aliquots of 250,000 were activated with saline, 1-1000 ng/ml IL-8/CXCL8, or 1 μM FMLP (+ 5 μg/ml cytochalasin B) for 15 min at 37°C in a final volume of 250 μl HBSS. The reaction was terminated by centrifugation at 12,000 ×
g for 1 min. Aliquots of supernatants were assayed with a commercial EIA kit as previously described [
24,
31].
GIVaPLA2 activity assay
GIVaPLA
2 activity assay was determined in aliquots of 2 × 10
6 cells incubated for 15 min at 37°C with 3 μM - 30 μM TFMK or 10
-10 M - 10
-6 M pyrrophenone. Activity was measured at optimal time (30 min) after 100 ng/ml IL-8/CXCL8 [see Results]. This time and concentration were shown in initial studies to cause phosphorylation of gIVaPLA
2 in PMNs. The cell pellets were resuspended in disruption buffer (see above) and immediately sonicated followed by addition of specific substrate ([
14C]-PAPC) for gIVaPLA
2[
24,
31]. To measure precisely the total gIVaPLA
2 activity, 5 mM dithiotrietol was added to cell lysate to inactivate, if any, the remaining 10-14 kDa secretory PLA
2 enzymes that could interfere with the assay. Thirty min later, the reaction was terminated by adding 560 μl of Dole's reagent (heptane:isopropyl alcohol:1 N H
2SO
4; 400:390:10 by vol), and the radioactivity was measured in a liquid scintillation counter and expressed as picomoles/30 min/10
6 cells [
31].
Subfractionation
Freshly isolated PMNs were preincubated with either saline, 100 ng/ml IL-8/CXCL8, or 10-6 M FMLP for 15 min at 37°C. After washing with PBS, treated cells were centrifuged for 1 min at 400 × g. The pellet were lysed in 50 μl disruption buffer (see above) and put on ice for 10 min. The disrupted pellets, which are mainly nuclear component of the cells, were centrifuged at 500 × g for 1 min. A total of 50 μl of boiling buffer was added to the pellets and boiled for 5 min. The supernatants were centrifuged again at 100,000 × g for 1 h. Eight μl of loading buffer was added to the collected supernatant, which is the cytoplasm fraction, and was boiled for 5 min. Samples were loaded onto SDS-PAGE and membrane was probed with pAb against cPLA2. The translocation of cytosolic gIVaPLA2 to the nuclear component of the cells was detected by an enhanced chemiluminescence (Amersham, Arlington Heights, IL).
Change in cell shape and F-actin polymerization
Change in cell shape and F-actin polymerization were examined in migrated cells. HBSS or rhodamine-phalloidin [
32] was added to the paraformadehyde-fixed cytoslides containing samples and changes in cell shape and F-actin polymerization were analyzed by confocal microscopy.
Statistical analysis
Experimental data are expressed as mean ± SEM in each group. Student's t-test was used for comparison between two-paired groups. Where multiple comparisons were made, differences on concentration-response curves for the same agonist or inhibitor were compared after Bonferonni correction. Variation between more than two groups was tested using one-way ANOVA followed by Fisher' least protected difference test. Statistical significance was claimed when P < 0.05.
Discussion
The objective of this study was to examine the functional role of gIVaPLA
2 in the regulation of PMNs migration caused by IL-8/CXCL8. Prior studies have reported the signaling role of upstream kinases, ERK-1/2, p38 MAPK, and PI3K [
1,
5,
6], in the initiation of cell migration; however, the downstream regulation of PMN migration elicited by IL-8/CXCL8 has not been elucidated previously.
We used a transwell-migration chamber [
28] and determined whether inhibition of activated gIVaPLA
2 by TFMK or pyrrophenone blocked PMN migration caused by IL-8/CXCL8. We also examined the functional role of gIVaPLA
2 in causing in PMN elongation and F-actin polymerization, which both are necessary for PMN migration [
34,
35]. While the specific mechanism causing the PMN change in cell shape was not elucidated fully in these studies, we found that inhibition of gIVaPLA
2 is sufficient to block change in cell shape caused by IL-8/CXCL8 even in the presence of F-actin polymerization (Figure
6).
Studies were designed using IL-8/CXCL8, a potent chemoattractant of PMNs. Prior studies have suggested that IL-8/CXCL8 is rather weak stimulator of human PMNs in comparison to rodent models [
36]. Activation of PMNs with IL-8/CXCL8 did not elicit arachidonic acid or LTB
4 secretion in human PMNs. Thus, our findings suggest that IL-8/CXCL8 caused transmigration of PMNs by a process that does not involve activation of arachidonate synthesis.
Cytosolic gIVaPLA
2 is a critical messenger protein for cellular adhesion [
21,
22,
27]. We have shown recently that neutrophil or eosinophil binding to ICAM-1 is mediated through activation of ERK-1/2 and subsequent phosphorylation of gIVaPLA
2 [
22‐
24,
27]. In all prior cases, we have found that stimuli that upregulate cell adhesion CD11b expression also induce the activation of gIVaPLA
2 [
22,
24,
25,
27]. However, the role of gIVaPLA
2 to mediate PMN migration has not been previously reported.
Initial experiments were performed to confirm that upstream kinases, ERK-1/2, p38 MAPK, and PI3K, were activated by the concentration of IL-8/CXCL8 used in these studies (Figure
2). Immunoblotting analysis demonstrates that IL-8/CXCL8 elicited rapid phosphorylation of ERK-1/2, p38 MAPK, and Akt/PKB (Figure
2), confirming that these kinases were activated in these experiments; however, the downstream signaling pathway for cell migration has not been characterized. In this study, we used two-unrelated pharmacological inhibitors of gIVaPLA
2, TFMK and pyrrophenone, to elucidate the role of gIVaPLA
2 in cell migration. Transmigration of PMNs was blocked substantially in the presence of upstream phosphorylation of ERK-1/2, p38 MAPK and Akt/PKB (a target protein of PI3K) using inhibitors of activated gIVaPLA
2. Accordingly, these data indicate a downstream regulatory role for gIVaPLA
2 in
in vitro PMN migration subsequent to activation of upstream kinases by IL-8/CXCL8.
Immunoblotting analysis demonstrated that IL-8/CXCL8 caused phosphorylation and translocation of cytosolic gIVaPLA
2 to the nuclear component of PMNs [
27]. It has been shown that gIVaPLA
2 inhibition effectively blocked cell adhesion and secreted mediators after cell activation [
21,
24,
31]. We have demonstrated that inhibition of gIVaPLA
2 blocked both gIVaPLA
2 enzymatic activity (Figure
4) and cell migration (Figure
5) elicited by IL-8/CXCL8 in concentration dependent manner. These data thus imply that activated gIVaPLA
2 is an essential intermediate step in PMN migration in vitro.
Prior studies have demonstrated that interference with F-actin rearrangement could contribute to decrease cell migration [
37]. We observed that inhibition of gIVaPLA
2 with TFMK or pyrrophenone did not prevent the rearrangement of F-actin assembly elicited by IL-8/CXCL8. F-actin polymerization still was evident around the edges of inner cell membrane (Figure
6g-h). These findings suggest that while IL-8/CXCL8 caused change in cell shape, gIVaPLA
2 does not directly regulate the rearrangement of the actin cytoskeleton in PMNs.
It is important to note some limitations to our
in vitro models of PMN migration. We used transwell chamber
in vitro to study transmigration of human PMNs. Migration
in vitro occurred in the absence of β
2-integrin ligation, which is the first step (adhesion) in cell migration
in vivo [
38].
In vivo conditions are a more complex environment, and it is not possible to extrapolate these data directly to the human situation. Studies
in vivo, however, do not allow for stimulus isolation to specify mechanisms and sequence of cell migration. In these studies, maximal migration of PMNs from the upper chamber to the lower chamber containing IL-8/CXCL8 was ~50%. This is comparable to other chemoattractants, i.e., FMLP, C5a and LTB
4 [
39]. The initial number of cells (5 × 10
4 cells) was constant in all studies, and was sufficient to cover the area of a chamber (96-well chamber) for optimal PMN migration.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
NMM and ARL equally contributed to the concept and design of the study, and to the manuscript writing. NMM and AYM performed the data analysis. AYM and LNM performed the assays for gIVaPLA2 enzyme activity, PMN migration, F-actin polymerization, and change in cell shape. CO and DB performed the isolation of PMNs and western blot analysis. XZ and SD participated in the migration assay. All authors read and approved the final manuscript.