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Am J Physiol Lung Cell Mol Physiol 290: L703-L709, 2006. First published November 4, 2005; doi:10.1152/ajplung.00390.2005
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Involvement of phospholipase A2 in Pseudomonas aeruginosa-mediated PMN transepithelial migration

Bryan P. Hurley,1,2 Natecia L. Williams,1 and Beth A. McCormick1,2

1Mucosal Immunology Laboratories, Massachusetts General Hospital, Charlestown, Massachusetts; and 2Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts

Submitted 12 September 2005 ; accepted in final form 27 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Inflammation resulting from bacterial infection of the respiratory mucosal surface during pneumonia and cystic fibrosis contributes to pathology. A major consequence of the inflammatory response is recruitment of polymorphonuclear cells (PMNs) to the infected site. To reach the airway, PMNs must travel through several cellular and extracellular barriers, via the actions of multiple cytokines, chemokines, and adhesion molecules. Using a model of polarized lung epithelial cells (A549 or Calu-3) grown on Transwell filters and human PMNs, we have shown that Pseudomonas aeruginosa induces PMN migration across lung epithelial barriers. The process is mediated by epithelial production of the eicosanoid hepoxilin A3 (HXA3) in response to P. aeruginosa infection. HXA3 is a PMN chemoattractant metabolized from arachidonic acid (AA). Given that release of AA is believed to be the rate-limiting step in generating eicosanoids, we investigated whether P. aeruginosa infection of lung epithelial cells resulted in an increase in free AA. P. aeruginosa infection of A549 or Calu-3 monolayers resulted in a significant increase in [3H]AA released from prelabeled lung epithelial cells. This was partially inhibited by PLA2 inhibitors ONO-RS-082 and ACA as well as an inhibitor of diacylglycerol lipase. Both PLA2 inhibitors dramatically reduced P. aeruginosa-induced PMN transmigration, whereas the diacylglycerol lipase inhibitor had no effect. In addition, we observed that P. aeruginosa infection caused an increase in the phosphorylation of cytosolic PLA2 (cPLA2), suggesting a mechanism whereby P. aeruginosa activates cPLA2 generating free AA that may be converted to HXA3, which is required for mediating PMN transmigration.

lung inflammation; arachidonic acid; eicosanoid; polymorphonuclear cells; neutrophils

THE OPPORTUNISTIC PATHOGEN Pseudomonas aeruginosa is capable of causing inflammation in the lung resulting in severe lung tissue damage (19). This is most notable in cases of acute bacterial pneumonia in immune-compromised individuals and in chronic lung infections of individuals with cystic fibrosis (19, 21). One characteristic of each of these diseases involving P. aeruginosa infection is the accumulation of large numbers of polymorphonuclear cells (PMNs) or neutrophils in the airway lumen (6, 19, 21, 22). Although PMNs are generally thought to be responsible for eradicating pathogenic bacteria, PMN mechanisms for destruction of bacteria are nonspecific and, if excessive, can harm host tissue (29).

Several studies have suggested that the epithelial barrier at mucosal surfaces plays a key role in orchestrating the movement of PMNs from the bloodstream to the luminal space where the majority of P. aeruginosa are believed to reside (9, 14, 15, 19). To reach the airway lumen, PMNs are required to travel through several distinct tissue compartments within the alveolar wall (6, 22). PMNs must first escape the alveolar capillary, which involves the termination of their flow through the vessel followed by adherence to the lumen surface of the endothelium. The majority of PMNs migrate across the junctions that connect the endothelial cells. Once across the endothelial barrier, PMNs navigate the endothelial basement membrane through preexisting holes allowing PMNs to gain access to the interstitial space. PMNs then must travel though the extracellular matrix that encompasses the interstitial space, where fibroblasts are thought to provide further directional guidance. PMNs can then interact with the epithelial basement membrane and basolateral surface of the epithelium. Finally, PMNs migrate between the epithelial cells to the apical surface of the epithelial barrier where they gain access to the airway lumen (6, 22). This process involves the integrated actions of multiple cell types, various cytokines, adhesion molecules with specificity for particular ligands, and highly timed and compartmentalized secretion of various PMN-specific chemokines (6, 22).

To investigate the final step in this complex process, namely the migration of PMNs across the epithelial barrier into the air space, we have developed an in vitro model using lung epithelial cells grown on permeable Transwell filters (15). Using this model, we have discovered that P. aeruginosa infection of the apical surface of lung epithelial monolayers results in the directed migration of PMNs from the basolateral side to the infected apical side (15). This process is mediated by epithelial production and release of the PMN chemoattractant hepoxilin A3 (HXA3). HXA3 is an eicosanoid that is produced by lung epithelial cells in response to infection by P. aeruginosa in a protein kinase C-dependent manner (15).

Eicosanoids represent a class of bioactive lipid molecules that are derived from arachidonic acid (AA) and have been reported to be involved in numerous inflammatory processes (7). In addition to our report implicating the eicosanoid HXA3 as a mediator of inflammation, several other eicosanoids have been shown to be involved in inflammation, including prostaglandins and leukotrienes, and these molecules have quite distinct roles despite their structural similarities (7). Increased production of a specific eicosanoid, such as HXA3, might occur by a shift in activity of the numerous enzymes such as the lipoxygenases and cyclooxygenases within a cell that metabolize AA, transforming it into the various eicosanoids. Alternatively, increased production of certain eicosanoids may result from an increase in the activity of enzymes responsible for the release of AA, the eicosanoid precursor, from membrane phospholipids. It has been suggested that the rate-limiting step for production of eicosanoids by various cell types is the availability of the precursor molecule AA (7, 17). Two mechanisms to generate free AA for conversion to eicosanoids by lipoxygenases and cyclooxygenases are by the actions of phospholipase A2 (PLA2) or by the enzyme diacylglycerol (DAG) lipase. PLA2 is capable of breaking down phospholipid membranes into free AA (5, 17, 18). There are numerous reports demonstrating the induction of enhanced PLA2 activity in response to various inflammatory stimuli, suggesting that PLA2 is a major player in the inflammatory response (17, 23, 30, 31). DAG lipase, on the other hand, acts in sequence with monoacylglycerol lipase resulting in the liberation of AA from DAG rather than directly from membrane phospholipids (5, 18). The objective of this report is to determine whether P. aeruginosa infection results in an increase in the release of AA, and, if so, whether PLA2 and/or DAG lipase play a role in the process of P. aeruginosa-induced PMN transepithelial migration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Growth and maintenance of epithelial cells. The A549 cell line was originally derived through explant culture of lung carcinomatous tissue from a 58-yr-old Caucasian male. These cells display properties of type II alveolar epithelial cells and also form barriers when grown on permeable filters, although they do not develop transepithelial electrical resistance (TEER) (3, 9, 15). A549 cells are maintained in Ham's F-12K medium with 2 mM L-glutamine, 1.5 g/l NaHCO3, 10% fetal bovine serum, and 100 units of penicillin/streptomycin. Calu-3 lung adenocarcinoma cells display properties of the bronchial epithelium and are able to form polarized barriers on Transwell filters with TEER values of 400–1,500 {Omega}·cm2 (28). Polarized monolayers of A549 and Calu-3 are grown and maintained on the underside of 0.33-cm2 collagen-coated Transwell filters to study PMN migration in the physiological basolateral-to-apical direction as previously described (15, 20).

Bacterial strains. P. aeruginosa (PAO1) and the Escherichia coli K12 strain (MC1000) were grown in Luria-Bertani overnight at 37°C under aerobic conditions to a stationary growth phase. For infection of epithelial cells, overnight cultures were washed once in HBSS and resuspended at a concentration of 6 x 107 bacteria/ml of HBSS.

PMN isolation. PMNs are isolated from whole blood anticoagulated with acid citrate/dextrose obtained from healthy human volunteers. The protocol for PMN isolation was approved by the Massachusetts General Hospital institutional review board, and informed written consent was obtained from each volunteer. The buffy coat was obtained by centrifugation. Plasma and mononuclear cells were removed by aspiration, and the majority of the red blood cells (RBCs) were removed using a 2% gelatin sedimentation technique. Residual RBCs were removed by lysis in cold NH4Cl lysis buffer. This technique allows for rapid isolation of functionally active PMNs (>98%) at 90% purity (15, 20).

PMN transmigration assay. The PMN transmigration assay using inverted cell culture monolayers of polarized cells has been described (15, 20). Briefly, the apical surface of lung epithelial cells was exposed for 1 h to 25 µl of various doses of bacteria with the standard concentration for the majority of the experiments being 6 x 107 bacteria/ml. For a positive control, 10–8 µM formyl-methionyl-leucyl-phenylalanine (fMLP) was added to the bottom (apical) chamber of uninfected monolayers. PMNs (1 x 106) were then added to the top (basolateral) chamber, and the plate was placed at 37°C for 2 h. PMNs were quantified by the myeloperoxidase assay. Data are displayed as means (SD) of at least three independent monolayers/condition.

AA release assay. This assay has been previously described (25). A549 and Calu-3 cells were grown in 24-well plates and used 5–7 days after seeding. Cells were washed three times with PBS(–), treated with media containing 0.2 µC/ml [3H]AA, and incubated for 18–24 h to allow [3H]AA incorporation into membrane phospholipids. After 18–24 h, cells were washed three times to remove unincorporated [3H]AA and treated with 0.5 ml of bacteria (6 x 107 bacteria/ml). Supernatants (100 µl) were collected at various time points, filtered, and measured by scintillation counting. At the completion of 5 h, cells were solubilized with 500 µl/well of 1% SDS and 1% Triton X-100 and then sampled (250 µl) for measurement by scintillation counting.

Drug treatment. For experiments involving drug treatments, epithelial cells were pretreated for 2 h with either of the PLA2 inhibitors ONO-RS-082 and N-(p-amylcinnamoyl) anthranilic acid (ACA) or with the DAG lipase inhibitor RHC-80267 (Biomol) (5, 26). After pretreatment, the drug was removed by washing before lung epithelial cells were infected with PAO1. The inhibitors presented in this study did not affect cell viability in the presence or absence of bacterial infection as assessed by both the lactose dehydrogenase (LDH) assay and the barrier integrity assay. None of the drugs had any major effect on the amount of bacteria adhering to the A549 monolayers.

Cell viability assay. To determine whether the bacterial infection resulted in toxicity to the A549 monolayers grown on either Transwells or 24-well plates, release of the enzyme LDH into the supernatant of monolayers under infected conditions was compared with uninfected monolayers. As a positive control for cell death, Triton X-100 was added to monolayers. Monolayers were washed and equilibrated in HBSS for ~30 min. Monolayers were then treated for 1 h with HBSS alone or with bacteria. After 1 h, cells were washed three times and placed in HBSS or Triton X-100 for 2–5 h. The amount of LDH released into the supernatant was quantified using the LDH assay (Sigma, St. Louis, MO). In some experiments, monolayers were pretreated for 2 h with PLA2 inhibitors before assay was performed.

Detection of cytosolic PLA2 activation. A cell fractionation protocol was used to obtain membrane-soluble proteins, as cytosolic PLA2 (cPLA2) becomes phosphorylated and accumulates in the membrane upon activation (12). A549 monolayers seeded on six-well plates or 4.5-cm2 permeable filters were treated with HBSS alone, 1 µM PMA, or 6 x 107 bacteria/ml for 45 min. Cells were scraped off the permeable filters in buffer containing 150 mM Tris, pH 8.0, 15 mM EDTA, 6 mM EGTA, 200 mM PMSF, 4 mM Na3VO4, 40 mM NaF, and 1 Complete Mini Protease Inhibitor Cocktail tablet/10 ml buffer. Scraped cells were subjected to sonication followed by centrifugation at 55,000 rpm for 1 h. Cell pellets were resuspended in a lysis buffer containing 0.1% Triton X-100, 0.2% SDS, 50 mM Tris, pH 8.0, 5 mM EDTA, 2 mM EGTA, 200 mM PMSF, 4 mM Na3VO4, 40 mM NaF, and 1 Complete Mini Protease Inhibitor Cocktail tablet/10 ml buffer. Lysates were again subjected to sonication followed by centrifugation at 30,000 rpm for 10 min. The supernatant was collected and concentrated using Centricon filters (30,000 mol wt cut off), and lysates were normalized for protein concentration and electrophoresed on an 8–16% gradient polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) followed by transfer to nitrocellulose. Blots were probed with anti-cPLA2 antibody or anti-phospho cPLA2 antibody (Cell Signaling) followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody and detection with ECL reagent.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PMN transmigration across A549 alveolar epithelial cell monolayers in response to infection with P. aeruginosa was mediated by the induced epithelial cell production and secretion of HXA3 from the apical surface (15). One potential mechanism underlying the increase in epithelial cellular production of HXA3 was through increased availability of the substrate necessary for HXA3 synthesis (17). AA, a precursor shared by many eicosanoids, can be converted to 12-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HpETE) by the actions of 12-lipoxygenase (32). 12-HpETE can then be converted to HXA3 by hepoxilin synthase. Thus it is conceivable that as more AA is liberated from membrane phospholipids, more substrate is potentially available for 12-lipoxygenases.

We, therefore, sought to determine whether infection of A549 cells with PAO1 resulted in an increase in release of free AA from epithelial cells. To accomplish this, we utilized the previously described AA release assay (25). The A549 cells were preincubated with [3H]AA to allow incorporation into membrane phospholipids. Tritiated cells were then washed to remove unincorporated [3H]AA and infected with either the lung pathogenic P. aeruginosa strain PAO1 or the nonpathogenic E. coli K12 strain MC1000. As controls, cells were incubated in HBSS alone or in the presence of PMA, a global signaling pathway activator previously shown to induce the release of AA from the membranes of certain cell types (12). As shown in Table 1, PAO1 results in a significant increase in the percentage of AA released into the cell supernatant as soon as 1 h when compared with untreated cells. The magnitude of difference between PAO1-infected and untreated A549 cells increases over time and reaches a difference of >10-fold after 5 h of infection. Infection with the nonpathogenic E. coli MC1000 resulted in only minimal increases in AA release over the first 3 h, with a small but significant increase at 5 h. PMA did not cause any significant increase of AA release before 5 h of treatment. The substantial release of [3H]AA from PAO1-infected A549 cells was not a consequence of a loss in A549 cell viability. A549 cells infected with the same concentration of PAO1 did not release any additional LDH over the course of 5 h when compared with uninfected A549 cells, indicating that the lung epithelial cells remained viable under the experimental conditions (data not shown).


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  		Table 1. Time course of AA released from A549 alveolar epithelial cells in response to P. aeruginosa infection

 
Because we observed an increase in the amount of AA released from A549 cells in response to PAO1 infection, we next sought to determine the potential mechanism by which this phenomenon occurs. Two enzymes responsible for generating AA have been described, PLA2, which liberates AA from cellular and organelle membranes, and DAG lipase, which liberates AA from DAG (5, 17, 18). Because of this attribute, these enzymes are potentially important to the production of various eicosanoids (7, 17, 18). To address whether the PAO1-induced AA release from A549 cells involved the actions of PLA2, we employed inhibitors that are specific for their ability to block PLA2 activity (ONO-RS-082 and ACA) (18, 26). For assessment of the contribution of DAG lipase, we utilized the DAG lipase-specific inhibitor RHC-80267 (18). A549 cells prelabeled with [3H]AA were treated for 2 h with ONO-RS-082, ACA, or RHC-80267 followed by washing and infection with PAO1 for 1 h. After washing away unbound PAO1, we incubated A549 cells for 2 h in HBSS, at which point the supernatant was collected and measured using a scintillation counter. The particular experimental design chosen above to measure AA release in the presence and absence of inhibitors aims to mimic the optimal conditions previously established for PAO1-induced PMN transepithelial transmigration. As shown in Fig. 1, both PLA2 inhibitors and the DAG lipase inhibitor significantly reduce the amount of [3H]AA released from PAO1-infected A549 cells. These inhibitors also significantly interfere with induction of AA release in A549 cells by known activators of AA release such as the calcium ionophore A-23187 (data not shown). None of the inhibitors completely shut down the PAO1-induced response at the doses of inhibitors employed, thus the data imply that PAO1-induced release of AA may involve both PLA2 and DAG lipase. None of the drugs used in these experiments caused any modulation in LDH release, suggesting that these inhibitors were not toxic to the A549 cells at these doses.


Figure 1
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  		Fig. 1. Pseudomonas aeruginosa-induced [3H]arachidonic acid (AA) release is partially, but significantly, inhibited by PLA2 and diacylglycerol (DAG) lipase inhibitors. A549 cells were pretreated for 2 h with the PLA2 inhibitor ONO-RS-082 (10 µM), the PLA2 inhibitor 12-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (ACA, 25 µM), or the DAG lipase inhibitor RHC-80267 (100 µM) followed by infection with PAO1 for 1 h and collection of supernatant after an additional 2 h. The amount of AA released in the absence of infection (2.1 ± 0.6%) was subtracted from the amount released in the presence of infection (8.2 ± 0.4%; 4-fold increase, P < 0.05). This value (6.1%) was set at 100%, and each value for PAO1-induced AA release in the presence of inhibitor was displayed as a percentage of this value. Each condition was performed in triplicate; *P < 0.05 when a given data set was compared with the (no inhibitor) control. Data presented are representative of experiments performed at least 3 times using cells of a different passage.

 
PAO1 infection of lung epithelial cells results in a significant increase in AA liberation. The release of AA into A549 cell supernatant appears to be mediated, in part, by the actions of PLA2 and DAG lipase. Because increased AA release may be a significant event in the PMN transepithelial migration process, as AA is the precursor for HXA3, we sought to determine whether inhibitors of PLA2 (ONO-RS-082 and ACA) and/or DAG lipase (RHC-80267) interfere with PAO1-induced PMN transmigration. To accomplish this, we pretreated A549 cells grown on Transwells for 2 h with inhibitors, infected them for 1 h, and added PMNs to the basolateral surface and allowed them to migrate for 2 h. Both inhibitors of PLA2 resulted in complete inhibition of PMN transmigration (Fig. 2). Figure 2A demonstrates that ONO-RS-082 at 10 and 20 µM significantly blocks PAO1-induced PMN transmigration. For ACA, 50 and 100 µM treatment was required to block PAO1-induced migration (Fig. 2B). In contrast, doses of the DAG lipase inhibitor RHC-80267 as high as 100 µM resulted in no significant effect on PMN transmigration (Fig. 2C). None of the drugs altered the ability of PMNs to migrate across A549 epithelial monolayers in response to an alternative PMN chemoattractant (fMLP) added to the apical chamber of uninfected monolayers at the same time PMNs were added to the basolateral surface. This result suggests that the blocking effect of the PLA2 inhibitors is not due to a generalized inhibition in PMN movement across the epithelium. None of these drugs interfered with A549 barrier properties at any of the doses used (data not shown).


Figure 2
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  		Fig. 2. P. aeruginosa-induced neutrophil (PMN) transepithelial transmigration is inhibited by PLA2 inhibitors. Various doses of the PLA2 inhibitors ONO-RS-082 (A) and ACA (B) or various doses of the DAG lipase inhibitor RHC-80267 (C) were used to pretreat A549 cells for 2 h followed by a 1-h infection with PAO1 and quantification of migrated PMNs after an additional 2 h. Each condition was performed in triplicate; *P < 0.05 when the data set for a given condition was compared with the amount of PMNs migrating in response to PAO1 in the absence of drug treatment. Data presented are representative of experiments performed at least 3 times using cells of a different passage. fMLP, formyl-methionyl-leucyl-phenylalanine.

 
Given that PLA2 thus appears to play a crucial role in PAO1-induced PMN transepithelial transmigration, we next proceeded to determine whether there is any evidence of P. aeruginosa-induced activation of PLA2. One of the major cellular PLA2 enzymes implicated in mediating AA release for eicosanoid production is the cPLA2 (17). This enzyme is expressed in numerous lung epithelial cell lines and can be induced by inflammatory cytokines such as TNF-{alpha}, IL-1{alpha}, and IFN-{gamma} (30, 31). Activation of cPLA2 involves phosphorylation at various serine residues and translocation from the cytosol to the membrane where it can interact with phospholipids to cause the liberation of AA (17).

To investigate whether cPLA2 is activated, we assessed the amount of phosphorylated cPLA2 present in A549 cell membrane lysates. Briefly, A549 monolayers were infected with PAO1 at a concentration of 6 x 107 bacteria/ml for 45 min and subjected to a lysis protocol to isolate and concentrate cell membrane proteins. Membrane-associated protein fractions were normalized for the amount of protein they contain and were electrophoresed on SDS-PAGE gels and subjected to Western blotting and probed with either cPLA2 or phosphorylated cPLA2 antibodies. Infection of A549 cells with PAO1 resulted in a 13.6-fold increase in the amount of phosphorylated cPLA2 compared with untreated HBSS buffer control (Fig. 3). Although infection with the nonpathogenic E. coli strain MC1000 also resulted in an increase in phosphorylated cPLA2 (6.7-fold), the total amount was less substantial compared with PAO1 infection. Treatment with the cell activator PMA, a strong activator of many cellular processes, resulted in an increase of cPLA2 phosphorylation as expected. The amount of total cPLA2 was unchanged in each of the cell fractions regardless of the cell treatment condition (Fig. 3). Together, these data indicate that PAO1 infection leads to phosphorylation of cPLA2, and this event may contribute to the AA release described above.


Figure 3
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  		Fig. 3. P. aeruginosa-induced activation of phosphorylated (P) cytosolic PLA2 (cPLA2). A549 monolayers were treated for 45 min with buffer alone (HBSS), 1 µM PMA, P. aeruginosa PAO1 (1 ml/well at 6 x 107 bacteria/ml), or Escherichia coli K12 MC1000 (1 ml/well at 6 x 107 bacteria/ml). Membrane fractions were collected, normalized for protein concentration, subjected to Western blotting, and probed with anti-phospho-cPLA2 or anti-cPLA2. Densitometry was accomplished using Adobe Photoshop, and the values listed above the bands for each treatment are represented as fold increase over values for HBSS bands for anti-phospho-cPLA2 after compensating for any slight differences observed in density of anti-cPLA2 bands. Image represents an experiment performed at least 3 times with similar results.

 
Our studies in this report as well as our previous studies were performed using the A549 cell line as an appropriate model for the lung epithelium (3, 9, 15). We next set out to determine whether P. aeruginosa could induce PMN transmigration across other lung epithelial models. To accomplish this, we chose an alternative human lung epithelial cell line that has been widely used as a model for the lung epithelial barrier (28). Calu-3 is a bronchial-like epithelial cell line that forms tight barriers on Transwells and is capable of not only restricting the flux of protein molecules (i.e., horseradish peroxidase) as we observed with A549 barriers, but also forming measurable electrical resistance (400–1,500 {Omega}·cm2) after growing 7–10 days on Transwell filters.

We performed the PMN transmigration assay across PAO1-infected Calu-3 monolayers using a wide range of infection concentrations (1.25 x 104 – 2 x 108 PAO1/monolayer). We observed a strikingly similar pattern as previously observed in P. aeruginosa-induced PMN migration across A549 monolayers (15). At the highest concentrations of PAO1 infection, very little migration occurred, whereas significant migration occurred at the mid-level concentrations of PAO1 infection, reaching a maximal response at 3.1 x 106 PAO1/monolayer (Fig. 4). As PAO1 is further diluted from the mid-level concentrations, the PMN transmigration response diminished in a dose-dependent fashion. As reported with A549 cells (15), the loss in PMN transmigration response in Calu-3 monolayers infected with high concentrations of bacteria was not due to destruction of the Calu-3 monolayers, as determined using the LDH assay.


Figure 4
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  		Fig. 4. Bacteria-induced PMN transmigration across lung epithelial cells. Calu-3 monolayers were infected with a range of PAO1 concentrations from 1.25 x 104 to 2 x 108 bacteria/monolayer. HBSS represents uninfected monolayers. Each PAO1 concentration was performed ~6 times, and data presented are as a compilation of multiple experiments using cells of different passages. *PMN migration response that is significantly reduced (P < 0.05) compared with the maximal response, which occurs at 3.1 x 106 PAO1/monolayer. CFU, colony-forming unit.

 
Next we investigated whether PAO1 infection of Calu-3 cells resulted in any increases in [3H]AA release into the cell supernatant. PAO1 induced significant increases in AA release within 1 h of infection, and this difference increased over time (Table 2). Although the overall magnitude of AA release in PAO1-infected Calu-3 cells was less than that which occurred in PAO1-infected A549 cells (Table 1 vs. Table 2), the overall pattern of response to various stimuli was similar in both cell lines. The global cell pathway activator PMA caused a significant increase in AA release at 1 h, whereas the nonpathogenic E. coli K12 MC1000 strain failed to induce any significant AA release over the 5-h experiment (Table 2). In addition to causing an increase in AA release in Calu-3 cells, PAO1 infection also caused a 2.6-fold increase in the amount of phosphorylated cPLA2 (Fig. 5). This is less substantial than the increase in phosphorylated cPLA2 that occurred in PAO1-infected A549 cells under similar conditions (Fig. 3).


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  		Table 2. Time course of AA released from Calu-3 bronchial epithelial cells in response to P. aeruginosa infection

 

Figure 5
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  		Fig. 5. P. aeruginosa-induced activation of phosphorylated cPLA2. Calu-3 monolayers were treated for 45 min with buffer alone (HBSS), 1 µM PMA, P. aeruginosa PAO1 (1 ml/well at 6 x 107 bacteria/ml), or E. coli K12 MC1000 (1 ml/well at 6 x 107 bacteria/ml). Membrane fractions were collected, normalized for protein concentration, subjected to Western blotting, and probed with anti-phospho-cPLA2 or anti-cPLA2. Densitometry was accomplished using Adobe Photoshop, and the values listed above the bands for each treatment are represented as fold increase over values for HBSS bands for anti-phospho-cPLA2 after compensating for any slight differences observed in density of anti-cPLA2 bands. Image represents an experiment performed at least 3 times with similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The inflammatory response mounted against bacterial pathogens infecting the mucosal surface of the lung is highly complex and multifaceted. One of the destructive consequences of an overaggressive inflammatory response is the accumulation of activated PMNs in the airway lumen that can damage lung tissue (6, 19, 21, 22). Over the past several years, numerous studies have presented evidence that the epithelial cells lining the luminal cavity and separating the luminal contents from the underlying tissue are key players in orchestrating innate immune responses (2, 4, 8). Through interactions with Toll-like receptors, bacteria can stimulate epithelial cells to produce cytokines and chemokines such as the PMN chemoattractant IL-8, which likely plays a major role in recruiting PMNs from the bloodstream to the epithelium (2, 16). Given the complexity of the route that PMNs must travel to reach the airway lumen, i.e., through the endothelium, the basement membrane, the interstitial space, the epithelial basement membrane, and the epithelial cell layer, it is likely that multiple PMN chemoattractants participate at discrete steps in this recruitment process (6, 22). Indeed, we have previously shown using an in vitro model of the lung epithelium that bacteria-induced PMN migration across the epithelial barrier is not dependent on IL-8 production; instead, the production and apical secretion of the eicosanoid HXA3 are necessary for directing PMNs across the infected epithelium (15).

In this report, using the same in vitro lung epithelial model, we have further defined the mechanism preceding HXA3 production and PMN transepithelial migration in response to infection with P. aeruginosa. Infection of A549 cells with P. aeruginosa resulted in a substantial increase in release of AA, suggesting that a potential mechanism for production of HXA3 from A549-infected cells is through the increase in availability of the HXA3 precursor. The P. aeruginosa-induced increase in AA release observed in our system was partially prevented by pretreatment of A549 cells with specific inhibitors of PLA2 and DAG lipase. Thus P. aeruginosa-induced enhancement of AA release from A549 cells, at least in part, involves the actions of both PLA2 and DAG lipase. Interestingly, at doses affecting P. aeruginosa-induced AA release, the PLA2 inhibitors completely inhibited the PMN transepithelial migration response to P. aeruginosa, whereas the DAG lipase inhibitor did not have any effect on PMN transmigration. These data suggest that PLA2 activity is crucial for mediating PMN transmigration, most likely through its ability to generate the precursor AA for the 12-lipoxygenase, which catalyzes the conversion of AA to HXA3. AA generated by the actions of DAG lipase may not be made available to 12-lipoxygenase since inhibition of DAG lipase does not interfere with P. aeruginosa-induced PMN transepithelial migration.

In line with our evidence that PLA2 activity was increased in A549 cells in response to P. aeruginosa infection, we detected a significant increase in the amount of phosphorylated cPLA2 enzyme. Phosphorylation of cPLA2 significantly enhances its activity and represents a mechanism by which the cell regulates this enzyme's function. The activation of cPLA2 has been implicated as a key step in the production of multiple eicosanoids in various cell types, including lung epithelial cells (17, 30, 31). For example, bronchial epithelial cells produce PGE2 in response to infection with influenza virus (23). The phosphorylation of cPLA2 induced by influenza virus is strongly implicated as a prerequisite event to the increased production and release of PGE2 from infected bronchial epithelial cells (23). These data lead us to the hypothesis that PAO1 infection of A549 cells causes the phosphorylation of cPLA2, which leads to an increase in the liberation of AA from cellular membranes. This event potentially creates a pool of free AA that can then be converted by 12-lipoxygenase to HXA3. The HXA3 produced is then released from the apical membrane of A549 cells and establishes a gradient that directs PMNs across the infected lung epithelial monolayers.

Interestingly, a P. aeruginosa-secreted exotoxin known as ExoU has recently been identified as a PLA2 with homology to the catalytic domain of human cPLA2 (27). This exotoxin has been established as a virulence factor displaying cytotoxicity toward airway epithelial cells whose potency is dependent on functional PLA2 activity (27). It is currently unknown whether this molecule, which is injected directly into cells by a type III secretion system, can modulate the generation of eicosanoids; however, it is believed that the PLA2 activity exhibited by ExoU is an important contributor to disease pathogenesis (27). Many pathogenic P. aeruginosa strains, including PAO1 used in this study, lack the gene encoding ExoU. Here we show that an ExoU negative P. aeruginosa strain is capable of increasing the activity of epithelial cell-derived cPLA2, and this event may be important to the disease process.

Although this P. aeruginosa-mediated increase in phosphorylated cPLA2 could explain the P. aeruginosa-induced increase in AA release, it must be pointed out that there are other PLA2 enzymes that may also be contributing to AA release, and we cannot rule out their potential importance in mediating PMN transmigration. In addition to cPLA2 there are two other groups of PLA2 enzymes termed secretory PLA2 (sPLA2) and calcium-independent PLA2 (iPLA2) (1, 24). The sPLA2 subfamily consists of small (14–19 kDa) enzymes that are secreted by cells and act on the luminal surface of cell membranes to liberate AA, which can then be processed further by lipoxygenases or cyclooxygenases (24). The iPLA2 group consists of enzymes that are similar in size to the cPLA2 family, ~85 kDa, and, like cPLA2 enzymes, reside in the cytosol and act in the membrane, but unlike cPLA2, their activation is independent of calcium (1). Future studies will explore the relative contribution of each of the three PLA2 groups in terms of mediating AA release, HXA3 production, and PMN transepithelial migration to determine which activity is critical to these processes. Furthermore, it is also conceivable that PAO1 could possess a functional PLA2 enzyme that might contribute to generation of free AA in airway epithelial cells. Several bacterial pathogens possess functional PLA2(s) including Yersinia species (YplA and pldA), E. coli (PldA), Klebsiella pneumoniae (PldA), and Legionella pneumoniae (PlaB) (4, 10, 13). Determination of the particular PLA2 responsible for orchestrating PMN transepithelial migration may lead to targeted therapies designed to dampen inflammation in the lung.

Because our in vitro model investigates mechanisms of bacteria-induced PMN transepithelial transmigration in the absence of other cellular compartments that PMNs must travel through to reach the lumen, such as blood vessels and interstitial space, clearly it will be important to test whether this inflammatory pathway (involving the actions of PLA2 and the production of HXA3) is active in the epithelium under in vivo conditions. Thus, as an important future extension of these current studies, it will be important not only to identify the specific PLA2 gene(s) mediating this process, but also to determine how prevalent this pathway is using alternative in vitro lung epithelial models (11). To this end, we have extended our finding beyond one isolated lung epithelial cell line. In this report, we demonstrate that P. aeruginosa induces PMNs to migrate across the bronchial epithelial cell line Calu-3 in a pattern remarkably similar to the A549 cells. In addition, P. aeruginosa activates cPLA2 and causes an increase in the amount of AA released. Although the existence and relative importance of this newly described inflammatory pathway need to be evaluated in more complex models, its potential relevance is reinforced by our observation of its existence in other lung epithelial cell in vitro models. Thus this observation justifies further analysis of this inflammatory pathway.

In summary, we have shown that PLA2 activity is required for PAO1-induced PMN transepithelial transmigration. Upon infection, lung epithelial cells phosphorylate cPLA2 and release significantly more AA from membrane stores. It is our hypothesis that increased PLA2 activity, which mediates AA release, is required for production of the eicosanoid HXA3, which is responsible for orchestrating PMN movement across lung epithelial monolayers.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grant DK-56754 (B. A. McCormick) and by a Pilot and Feasibility Award from the Cystic Fibrosis Foundation (B. A. McCormick). Salary support for B. P. Hurley was provided by the Ruth L. Kirschstein National Research Service Award Individual Fellowship from the National Institute of Allergy and Infectious Diseases.


   ACKNOWLEDGMENTS
 
We thank Mike Pazos for expert technical assistance. We also thank Dr. W. Allan Walker for continued support. We are grateful to Karen L. Mumy for critical review of this manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. P. Hurley, Mucosal Immunology Laboratories, Massachusetts General Hospital, CNY 114 (114-3503), Charlestown, MA 02129 (e-mail: Bphurley{at}partners.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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