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The Journal of Immunology, 2006, 176: 1878-1888.
Copyright © 2006 by The American Association of Immunologists

Lipoxin A4 Redistributes Myosin IIA and Cdc42 in Macrophages: Implications for Phagocytosis of Apoptotic Leukocytes1

Keira Reville*, John K. Crean*, Sharon Vivers{dagger}, Ian Dransfield{dagger} and Catherine Godson2,*

* School of Medicine and Medical Sciences, Conway Institute, University College Dublin, Belfield, and the Dublin Molecular Medicine Centre, Dublin, Ireland; and {dagger} The Rayne Laboratory, Medical Research Council Centre for Inflammation Research, University of Edinburgh Medical School, Edinburgh, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lipoxins (LXs) are endogenously produced anti-inflammatory agents that modulate leukocyte trafficking and stimulate nonphlogistic macrophage phagocytosis of apoptotic neutrophils, thereby promoting the resolution of inflammation. Previous data suggest a role for altered protein phosphorylation and cytoskeletal rearrangement in LX-stimulated phagocytosis but the exact mechanisms remain unclear. In this study we examine the effects of LXA4 on the protein phosphorylation pattern of THP-1 cells differentiated into a macrophage-like phenotype. THP-1 cells stimulated with LXA4 (1 nM) exhibit dephosphorylation of a 220-kDa protein. Using mass spectrometry, this protein was identified as MYH9, a nonmuscle myosin H chain II isoform A, which is involved in cytoskeleton rearrangement. THP-1 cells treated with LXA4 adopt a polarized morphology with activated Cdc42 localized toward the leading edge and MYH9 localized at the cell posterior. Polarized distribution of Cdc42 is associated with Akt/PKB-mediated Cdc42 activation. Interestingly, the annexin-derived peptide Ac2–26, a recently described agonist for the LXA4 receptor, also stimulates macrophage phagocytosis, MYH9 dephosphorylation, and MYH9 redistribution. In addition, we demonstrate that LXA4 stimulates the phosphorylation of key polarity organization molecules: Akt, protein kinase C{zeta}, and glycogen synthase kinase-3beta. Inhibition of LXA4-induced Akt and protein kinase C{zeta} activity with specific inhibitors prevented LXA4-stimulated phagocytosis of both apoptotic polymorphonuclear neutrophils and lymphocytes, highlighting a potential use for LXA4 in the treatment of autoimmune diseases. Furthermore, phosphorylation and subsequent inactivation of glycogen synthase kinase-3beta resulted in an increase in phagocytosis similar to that of LXA4. These data highlight an integrated mechanism whereby LXA4 regulates phagocytosis through facilitative actin cytoskeleton rearrangement and cell polarization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Inflammation is a dynamically regulated process, central to effective host defense. A critical aspect involves the resolution of an acute inflammatory state and such resolution is subverted in chronic inflammatory conditions. Intrinsic to the effective function and ultimate resolution of inflammation are phagocytic cells, predominantly the macrophage (1). It has been established that an important factor in the successful resolution of inflammation is the recognition, uptake, and degradation by phagocytes of apoptotic leukocytes (1, 2, 3). The acquisition of necrotic properties by apoptotic cells as a result of failed or inefficient clearance has proinflammatory consequences. Dying cells contain both Ags and adjuvants sufficient to initiate an autoimmune response (4) culminating in various systemic autoimmune diseases including systemic lupus erythematosus, autoimmune lymphoproliferative syndrome, and inflammatory rheumatoid arthritis. In addition to clearing cells before they undergo lysis, it has been suggested that ingestion of apoptotic cells results in the production of anti-inflammatory cytokines such as TGF-beta1 and the active suppression of proinflammatory mediators such as IL-8 (5, 6).

There is growing appreciation that resolution of inflammation may be regulated by endogenously produced mediators. Lipoxins (LXs)3 are endogenous anti-inflammatory mediators that have been described to act as braking signals in inflammation on the basis of their ability to inhibit polymorphonuclear neutrophil (PMN) function responses in vitro (chemotaxis, transmigration across endothelia and epithelia, superoxide anion production, and IL-1beta release) and to prevent leukocyte influx/recruitment (7). In contrast to their inhibitory effects on PMN function, LXs are also potent activators of monocytes, stimulating their chemotaxis and adherence (8). We have previously demonstrated that LXs are involved in the resolution of inflammation by promoting nonphlogistic phagocytosis of apoptotic PMN by macrophages in vitro (9) and in vivo (10). Additionally, pleiotropic agonists of the LXA4 receptor also stimulate phagocytosis of apoptotic PMN (10, 11).

The molecular mechanisms by which LXs stimulate phagocytosis of apoptotic cells are poorly understood, although it appears likely that this effect is mediated by posttranslational modifications of key signaling proteins (9). Previous data have implicated a role for protein phosphorylation as LX-stimulated phagocytosis is mimicked by the PKA inhibitor, Rp-cAMP, and inhibited by the cell permeant cAMP analog, 8-Br-cAMP, and the phosphatase inhibitor okadaic acid (10). cAMP is also intrinsically involved in the regulation of cytoskeletal rearrangement and function (12). More recently, it was reported that neutrophils stimulated with the chemoattractant fMLP exhibited intense phosphorylation of a 55-kDa protein that was blocked by ATLa (aspirin-triggered lipoxin) treatment (10–50 nM) (13). This 55-kDa protein was identified as leukocyte-specific protein 1, a downstream component of the p38-MAPK cascade in neutrophils.

Remodeling of the actin cytoskeleton is a prerequisite for all phagocytic processes (14). We have previously reported that LXA4 induces changes in ultrastructure and rearrangement of actin in monocytes and macrophages (15). These observations support the hypothesis that modulation of protein phosphorylation and of cytoskeletal reorganization play an important role in LXA4-mediated phagocytosis of apoptotic cells.

In this study we have investigated the effects of LXA4 on the macrophage phosphoproteome. We describe the identification of MYH9, a nonmuscle myosin H chain (NMMHC) class IIA protein in phosphoserine immunoprecipitates from vehicle and LXA4-treated macrophages. Furthermore, we report that LXA4-stimulated phagocytosis of apoptotic leukocytes is associated with decreased phosphorylation of MYH9, MYH9 redistribution, and cellular polarization coupled to Cdc42 activation and phosphorylation of the key signaling proteins Akt, protein kinase C (PKC){zeta}, and glycogen synthase kinase (GSK)-3beta.


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

All cell culture materials were from Invitrogen Life Technologies and other reagents were from Sigma-Aldrich unless otherwise stated. LXA4 (5(S)-6(R)-15(S)-trihydroxy-7,9,13–trans–11–cis-eicosatetraenoic acid) was purchased from BIOMOL. The annexin 1 mimetic peptide Ac2–26 (Ac-AMVSEFLKQAWFIENEEQEYVQTVK) was prepared by the Advance Biotechnology Centre (Charing Cross and Westminster Medical School, London, U.K.) by using solid-phase stepwise synthesis. Anti-phosphoserine 16B4 Ab was purchased from Calbiochem. Anti-phospho-Akt (Ser473) Ab, anti-Akt Ab, anti-phospho-GSK-3beta (Ser9) Ab, and anti-phospho-PKC{zeta} (Thr410) Ab were obtained from Cell Signaling Technology. Anti-PKC{zeta} Ab was purchased from Upstate Biotechnology. Anti-Cdc42 Ab was obtained from Santa Cruz Biotechnology and anti-beta-actin Ab was purchased from Sigma-Aldrich. The specific Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, and the myristoylated PKC{zeta} pseudosubstrate inhibitor Myr-SIYRRGARRWRKL-OH were obtained from Calbiochem. The GSK-3beta inhibitor SB-415286 was purchased from Tocris Cookson and the myosin II inhibitor 2,3-butanedione monoxime (BDM) was from Sigma-Aldrich. An activity assay kit for Cdc42 and Super Signal West Pico and Dura Chemiluminescent substrates were purchased from Pierce.

Reagents were dissolved in DMSO or ethanol and further diluted in medium (final concentration, ≤0.1%). Equivalent concentrations of DMSO or ethanol were used as vehicle controls. The effect of pharmacological treatments on cell viability was monitored by flow cytometry using propidium iodide.

Cell isolation and culture

Human PMN were isolated from peripheral venous blood drawn from healthy volunteers, after informed written consent in accordance with institutional ethical guidelines (Mater Misericordiae University Hospital, Dublin, Ireland). PMN were separated by density gradient centrifugation on Ficoll-Paque (Amersham Biosciences) and dextran sedimentation (16). Following dextran sedimentation and hypotonic lysis of RBCs, PMN were incubated at 2 x 107 cells/ml with 10 µM CMFDA (5-chloromethylfluorescein diacetate; Molecular Probes) at 37°C for 15 min. Spontaneous apoptosis of CMFDA-labeled PMN was achieved by culturing 4 x 106 PMN/ml of RPMI 1640 medium supplemented with 10% autologous serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C for 24 h.

The human leukemic T cell lymphoblast cell line (Jurkat) was obtained from the European Cell Collection of Cell Cultures (ECACC). Jurkat cells were maintained in suspension in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, and 1% penicillin/streptomycin. Apoptosis was induced in CMFDA-labeled Jurkat T cells (at a concentration of 4 x 106 cells/ml) by exposure to UV irradiation at 254 nm for 2 min. Irradiated cells were then cultured overnight in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.

The human myelomonocytic cell line THP-1 (ECACC) was maintained in suspension in RPMI 1640 supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS. THP-1 cells were differentiated into a macrophage-like phenotype by treatment with 10 nM PMA for 48 h at 37°C (15).

Immunoprecipitation and Western blotting

Differentiated THP-1 cells (2 x 106 cells/ml), were serum-starved for 24 h and exposed to various agents as indicated. Lysates were harvested in RIPA lysis buffer (20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 5 mM ethylene diaminetetraacetic acid, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM NaF, 1 mM PMSF, 1 mM Na3VO4, 1 µM leupeptin, 0.3 µM aprotinin). The lysates were clarified by centrifugation at 14,000 rpm for 20 min and samples were normalized for total protein. For Western blot analysis, 60 µg of THP-1 protein extract was loaded onto each lane and separated by SDS-PAGE. Proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore), blocked with 5% (w/v) nonfat dried milk in TBST for 1 h at room temperature and probed overnight at 4°C with Abs raised against beta-actin (1/20,000), myosin IIA (1/1000), phosphoserine (1/500), total Akt (1/1000), phospho-Akt (1/500), phospho-GSK-3beta (1/1000), total PKC{zeta} (1/1000), and phospho-PKC{zeta} (1/1000). Membranes were incubated with HRP-conjugated secondary Abs (1/2000) for 1 h at room temperature and proteins were visualized by chemiluminescence.

For experiments as shown in Fig. 1, THP-1 cells were seeded at a density of 3 x 106 cells/ml onto 150-mm plates (six plates per condition), differentiated with 10 nM PMA for 48 h, serum-deprived, and stimulated with LXA4 (1 nM, 15 min) or vehicle control (0.1% ethanol, 15 min). Phosphoserine proteins were immunopurified from 4.7 mg of precleared lysates with 17 µg of mouse anti-phosphoserine 16B4 Ab and rocked overnight at 4°C. The 150 µl of protein G-agarose beads were added to the protein-Ab mixture and samples were rocked for a further 2 h at 4°C. Precipitated immunocomplexes were washed three times in fresh lysis buffer, boiled in sample buffer (250 mM Tris-HCl (pH 6.8), 10% (w/v) SDS, 0.5% (w/v) bromophenol blue, 25% (v/v) glycerol, and 6% (v/v) 2-ME), and resolved by SDS-PAGE. The gels were either stained with colloidal Coomassie blue (17) or transferred to PVDF membranes, and immunoblotted for phosphoserine.


Figure 1
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  		FIGURE 1. LXA4 induces dephosphorylation of a 220-kDa protein. A, Differentiated THP-1 cells were treated with vehicle or LXA4 (1 nM) before lysis in RIPA buffer. Phosphoserine proteins were immunoprecipitated (IP) from 1 mg of whole cell protein lysate using mAb 16B4. Immunocomplexes were denatured in sample buffer, separated by SDS-PAGE, transferred to PVDF membranes, and probed for phosphoserine (WB). Densitometry results are depicted graphically and represent the mean ± SEM (n = 3). Values given are expressed as fold induction relative to beta-actin compared with vehicle-treated cells (*, p < 0.005 relative to vehicle). B, Differentiated THP-1 cells were treated with vehicle or LXA4 (1 nM) before lysis in RIPA buffer. Phosphoserine proteins were immunoprecipitated from 1 mg of whole cell protein lysate using mAb 16B4. Immunocomplexes were denatured in sample buffer, separated by SDS-PAGE, and stained with colloidal Coomassie blue for subsequent analysis by mass spectrometry. The bands excised for MALDI-TOF mass spectrometry analysis are marked with an arrow. The location of molecular weight (MW) standards are as indicated.

 
For coimmunoprecipitation studies, THP-1 cells were differentiated, serum-starved, and stimulated with LXA4 (1 nM, 15 min) or vehicle control (0.1% ethanol, 15 min). Phosphoserine proteins were immunopurified from 1 mg of precleared lysate with 4 µg of mouse anti-phosphoserine 16B4 Ab and rocked overnight at 4°C. A total of 10 µl of protein G-agarose beads was added to the protein-Ab mixture and samples were rocked for a further 2 h at 4°C. Precipitated immunocomplexes were washed three times in fresh lysis buffer and boiled in sample buffer. Samples were subsequently resolved by SDS-PAGE, transferred onto PVDF membrane, and probed for myosin IIA or phosphoserine.

In-gel digestion

Gel bands were cut from colloidal Coomassie blue-stained one-dimensional SDS-PAGE gels, destained, in-gel digested with trypsin, and the peptides extracted using previously described methods (18).

Protein identification by peptide mass fingerprinting

Before peptide mass fingerprinting, the volumes of peptide containing solutions were adjusted to 5 µl by addition of 0.1% trifluoroacetic acid in 50% AcN. One microliter of each sample was deposited on a 1 x 96-well MALDI target plate and dried in a vacuum container. Equal volumes of matrix (10 mg/ml {alpha}-cyano-4-hydroxycinnamic acid in 50% AcN, 0.1% trifluoroacetic acid) were added to the previously loaded digest. Samples were dried using a vacuum container. Mass spectrometry measurements were conducted with a MALDI/TOF mass spectrometer Voyager super STR (Applied Biosystems equipped with a 337-nm nitrogen laser. The external calibration was performed on a tryptic digest of lysozyme C. The analyses were performed in the reflectron mode with an accelerating voltage of 20 kV, a delayed extraction parameter of 100–140 ns, and a low mass gate of 850 Da. Laser power was set slightly above threshold (10–15% higher than the threshold) for molecular ion production. Spectra were obtained by summation of 300 consecutive laser shots. Masses of the peaks were extracted from the spectra and used for protein identification using the MASCOT search engine (<www.matrixscience.com>) against SWISS-PROT and TrEMBL.

Cdc42 activity assay

Cdc42 activity was determined using an EZ-Detect Cdc42 activation kit. Briefly, THP-1 cells (2 x 106 cells/ml) were differentiated and stimulated as indicated. After treatment, cells were rinsed once with ice-cold PBS and scraped into 500 µl of lysis buffer. Samples were then vortexed and centrifuged at 16,000 x g for 15 min at 4°C and the supernatant transferred to a new tube. Active Cdc42 was then affinity purified with GST-Pak1-PBD (Pak1-p21-binding domain), separated by SDS-PAGE and measured by Western blotting using anti-Cdc42 Ab and compared with total Cdc42.

Immunocytochemistry

Differentiated THP-1 cells were grown at a concentration of 0.5 x 106 cells/ml on four-well Lab Tek permanox chamber slides (catalogue no. 177437; Nunc) and stimulated as indicated. Cells were washed with PBS and then fixed with 3.8% (w/v) paraformaldehyde in PBS for 20 min at room temperature. After rinsing in PBS, the cells were incubated with permeabilization buffer (0.1% (v/v) Triton X-100 in PBS) for 15 min at room temperature. Following incubation, fixed cells were washed twice with PBS. Cells were incubated in blocking buffer (3% (w/v) BSA in PBS) for 1 h at room temperature with gentle agitation. Slides were then incubated with primary Ab (1/100 dilution of a polyclonal rabbit anti-Cdc42 Ab or monoclonal mouse anti-myosin IIA Ab) in 1% (w/v) BSA/PBS overnight at 4°C. Slides were rinsed and incubated with secondary Ab (1/250 dilution of an anti-rabbit or anti-mouse FITC-conjugated Oregon green Ab) for 45 min at room temperature in dark conditions. Nuclei were counterstained with DAPI (4',6'-diamidino-2-phenylindole; 1 µg/ml, 30 s). Stained cells were visualized with a Leitz DM40 microscope and images captured using the Axiocam system and Axiovision 3.0.6 software (Carl Zeiss).

Phagocytosis of apoptotic leukocytes

THP-1 phagocytosis of apoptotic leukocytes was assessed using a flow cytometric assay as previously described (19). THP-1 cells (1 x 106 cells/ml) were differentiated to a macrophage-like phenotype by treatment with PMA (10 nM, 48 h) in 24-well plates (Costar). Differentiated THP-1 cells were exposed to experimental stimuli as indicated and incubated with CMFDA-labeled apoptotic cells (2 x 106 cells/ml) for 2 h at 37°C. After incubation of THP-1 cells with apoptotic cells, the medium was removed and wells were then incubated with 0.5% trypsin/0.2% EDTA for 5 min at 37°C to detach all THP-1 cells and remove noninternalized apoptotic cells. After 10 min incubation on ice, the proportion of THP-1 cells that exhibited increased fluorescence (corresponding to phagocytosis of fluorescent-labeled apoptotic cells) was determined by flow cytometry using an EPICS XL-MCL flow cytometer (Beckman Coulter) with System II software for EPICS XL-MCL v.3.0. Apoptotic target and THP-1 populations were identified by their distinct laser scatter properties. A minimum of 20,000 events within the THP-1 gate was acquired. The number of FL1-H-positive events in the THP-1 gate was divided by the total number of THP-1 cells to obtain the percentage of THP-1 cells that had internalized apoptotic cells.

For inhibitor studies, differentiated THP-1 cells were washed with RPMI 1640 before treatment with a specific Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (10 µM, 30 min) (20), the GSK-3beta inhibitor, SB-415286 (30 µM, 30 min) (21), a PKC{zeta} pseudosubstrate inhibitor (10 µM, 60 min) (22), the myosin II inhibitor, BDM (10 µM, 30 min) (23), or appropriate vehicles before stimulation with LXA4 (1 nM, 15 min, 37°C) or vehicle control (0.1% ethanol, 15 min, 37°C). The treated cells were washed with RPMI 1640 before incubation with CMFDA-labeled apoptotic cells as described earlier.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LXA4 alters the protein phosphorylation pattern of THP-1 cells

To investigate the effect of LXA4 on the macrophage phosphoproteome, differentiated THP-1 cells were stimulated with vehicle (0.1% (v/v) ethanol) or LXA4 (1 nM) for 15 min at 37°C and serine-phosphorylated proteins were prefractionated by immunoprecipitation. This fraction was subsequently separated by SDS-PAGE on a 10% (v/v) gel. This method allowed us to obtain high concentrations of relatively low abundant proteins, membrane proteins, and proteins greater than 100 kDa (24). Following electrophoresis, gels were either transferred to PVDF membranes and immunoblotted with an anti-phosphoserine Ab to detect changes in protein phosphorylation (Fig. 1A) or stained with colloidal Coomassie blue for subsequent analysis by mass spectrometry (Fig. 1B). Both the colloidal Coomassie blue-stained gel and the phosphoserine immunoblot demonstrate a significant dephosphorylation of a protein ~220 kDa (p220) in size following stimulation with LXA4.

Identification of p220 as MYH9

Colloidal Coomassie blue-stained polyacrylamide gel slices corresponding to the p220 serine-phosphorylated protein were excised (Fig. 1B; marked by black arrows) and subjected to in-gel digestion by trypsin. After digestion, a small portion of the supernatant was removed and analyzed by high-accuracy peptide mass fingerprinting using MALDI. The peptide masses obtained by MALDI analysis were used to search protein databases. Forty-eight tryptic peptides were identified by peptide mapping that matched sequences in human myosin, heavy polypeptide 9, nonmuscle type A, NMMHC IIA (MYH9), which amounted to a total protein coverage of 29% (Fig. 2A). A MALDI mass spectrum for the tryptic digest of p220 is presented in Fig. 2B. Both Western blotting and immunoprecipitation experiments were undertaken to confirm that p220 was MYH9 (Fig. 3). To determine whether treatment with LXA4 dephosphorylates serine residues within MYH9, we prefractionated the phosphoserine proteins by immunoprecipitation before immunoblotting for MYH9 with a specific Ab. As shown in Fig. 3A, MYH9 undergoes significant serine dephosphorylation in response to stimulation with LXA4 (1 nM). Total cellular levels of MYH9 remain unchanged after treatment with LXA4 (Fig. 3A). In contrast, treatment of freshly isolated PMN with LXA4 did not promote serine dephosphorylation of MYH9 (Fig. 3B). A peptide mimetic of annexin 1, Ac2–26 also promotes significant serine dephosphorylation of MYH9 (Fig. 3C).


Figure 2
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  		FIGURE 2. Identification of p220/MYH9 by mass spectrometry. A, Peptides were analyzed by peptide mass fingerprinting and matched to sequences in human MYH9. The sequences of peptides are shown along with the total protein coverage of MYH9 (boldface). B, MALDI-TOF mass spectrometry of peptides obtained by in-gel trypsin digestion of phosphorylated protein is shown as spectrum of MYH9 220-kDa protein.

 

Figure 3
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  		FIGURE 3. Confirmation that p220 is MYH9 by Western blotting (WB) and immunoprecipitation (IP) experiments. A, Differentiated THP-1 cells treated with vehicle or LXA4 (1 nM). B, Freshly isolated PMN treated with vehicle or LXA4. C, Differentiated THP-1 cells treated with vehicle or Ac2–26 (100 nM) lysed in RIPA buffer. Phosphoserine proteins were immunoprecipitated from 1 mg of whole cell protein lysate using mAb 16B4. Immunocomplexes were denatured in sample buffer, separated by SDS-PAGE, transferred to PVDF membranes, and probed for MYH9 using a mAb against NMMHC IIA. Total cellular MYH9 was determined by immunoblotting for MYH9. Expression of beta-actin was examined as a loading control.

 
LXA4 treatment induces cellular polarization and MYH9 rearrangement in differentiated THP-1 cells

Myosin IIA has been implicated as having a role in establishing cell polarity (25). Given this observation, we investigated whether treatment with LXA4 and subsequent dephosphorylation of MYH9 altered myosin distribution within THP-1 cells. Differentiated THP-1 cells were exposed to LXA4 (1 nM) or vehicle for 15 min at 37°C and stained for MYH9. Control THP-1 cells demonstrated a spherical shape and homogenous MYH9 staining (Fig. 4A). Stimulation with LXA4 induced a shape change with cells adopting a polarized structure and MYH9 appearing to localize at the cell posterior (Fig. 4A). Microscopic analysis revealed that 30% of LXA4-treated cells demonstrated changes in cell shape and MYH9 distribution. Stimulation of differentiated THP-1 cells with Ac2–26 induced a shape change and MYH9 distribution similar to that of LXA4 (Fig. 4B). Microscopic investigation showed that 22% of Ac2–26-treated cells adopted a polarized shape with altered MYH9 distribution.


Figure 4
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  		FIGURE 4. Effect of LXA4 on MYH9 localization and function in phagocytosis of apoptotic leukocytes. Differentiated THP-1 cells were exposed to vehicle or LXA4 (1 nM) (A) or vehicle or Ac2–26 (100 nM) (B). Cells were fixed with paraformaldehyde and stained with a mAb against NMMHC IIA. Nuclei were counterstained with DAPI. Arrows indicate MYH9 localization. C, Differentiated THP-1 cells were treated with BDM (10 µM, 30 min) at 37°C before stimulation with vehicle or LXA4 (1 nM). THP-1 cells were then incubated with apoptotic PMN or apoptotic Jurkat T cells for 2 h and phagocytosis was assayed by flow cytometry. Data represent mean ± SEM (n = 3) and are expressed as fold induction over basal (*, p < 0.005 vs vehicle; #, p < 0.005 vs LXA4; and ##, p < 0.001 vs LXA4).

 
Myosin assembly is required for LXA4-stimulated phagocytosis of apoptotic leukocytes

Myosin IIA activity has previously been demonstrated to play a role in particle internalization during both Fc{gamma}R and complement receptor type 3-mediated phagocytosis (26). To determine whether myosin filament assembly is involved in LXA4-induced phagocytosis of apoptotic cells, we made use of the chemical inhibitor BDM, which blocks the ATPase activity of myosin II (27). Differentiated THP-1 cells were treated with 10 µM BDM for 30 min before stimulation with vehicle or 1 nM LXA4 for 15 min. THP-1 cells were then incubated with apoptotic CMFDA-labeled PMN or lymphocytes for 2 h at 37°C. As previously reported (9), LXA4 stimulated an increase in phagocytosis of apoptotic PMN (Fig. 4C). Pretreatment of differentiated THP-1 cells with BDM reduced LXA4-stimulated phagocytosis of apoptotic PMN (Fig. 4C). Similarly, LXA4 stimulated an increase phagocytosis of apoptotic lymphocytes and pretreatment of differentiated THP-1 cells with BDM reduced LXA4-stimulated phagocytosis of apoptotic lymphocytes (Fig. 4C). Pretreatment of differentiated THP-1 cells with BDM did not affect cell viability (data not shown).

LXA4 induces Akt-mediated activation of Cdc42, accompanied by localization of Cdc42 expression at the leading edge of THP-1 cells

Having previously observed that LXA4 stimulates RhoA- and Rac-dependent cytoskeleton reorganization (15) and given data from other groups implicating Rho GTPases as important modulators of actin cytoskeleton function during phagocytosis (28, 29), we subsequently investigated the involvement of Cdc42 in LXA4-induced actin rearrangement and polarization. Cdc42 has previously been shown to be active toward the front of polarized cells (30). We consequently examined Cdc42 distribution within LXA4-treated THP-1 cells. Differentiated THP-1 cells were exposed to LXA4 (1 nM) or vehicle for 15 min and stained for Cdc42. Control THP-1 cells exhibited uniform Cdc42 staining throughout the cytoplasm (Fig. 5A). Stimulation with LXA4 induced a polarized phenotype with Cdc42 accumulating at the leading edge of the cells (Fig. 5A). Microscopic analysis revealed that 27% of LXA4-treated cells demonstrated changes in cell shape and Cdc42 distribution.


Figure 5
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  		FIGURE 5. Effect of LXA4 on Cdc42 localization and activation. A, Differentiated THP-1 cells were exposed to vehicle or LXA4 (1 nM). Cells were fixed with paraformaldehyde and stained with a polyclonal Ab against Cdc42. Nuclei were counterstained with DAPI. Arrows indicate Cdc42 localization. B, Differentiated THP-1 cells were treated with a specific Akt inhibitor (10 µM, 30 min) at 37°C before stimulation with vehicle or LXA4 (1 nM). Activity of the GTP-bound Cdc42 in THP-1 lysates was assessed by a pulldown assay using glutathione-Sepharose beads coupled to a GST-Pak1-PBD fusion protein. Bound and total Cdc42 proteins were detected by Western blotting with a polyclonal Cdc42 Ab.

 
To probe the role of Cdc42 in LXA4-induced actin cytoskeleton rearrangement and polarization, cells were treated with LXA4 and lysates assayed for active Cdc42 using a GST-Pak-PBD pulldown assay. LXA4 induced an increase in levels of active Cdc42 (Fig. 5B). The PI3K pathway, which is required for cell polarization, activates Rac and Cdc42 GTPases at the leading edge of migrating cells (31). To determine whether LXA4-stimulated activation of Cdc42 is mediated by Akt, we exposed differentiated THP-1 cells to a specific Akt inhibitor (10 µM, 30 min at 37°C) before stimulation with LXA4 (1 nM, 15 min). Pretreatment of differentiated THP-1 cells with the specific Akt inhibitor blocked LXA4-stimulated activation of Cdc42 (Fig. 5B).

This observation led us to further investigate the role of Akt in LXA4 signaling. Treatment of differentiated THP-1 cells with LXA4 (1 nM, 15 min) stimulated the rapid phosphorylation (within 2 min) of Akt (Fig. 6A). Furthermore, LXA4-triggered Akt phosphorylation is concentration-dependent (EC50 ~1 x 10–9 M; range 1 x 10–12–1 x 10–7 M) (Fig. 6B). To investigate the involvement of Akt phosphorylation in LXA4-stimulated phagocytosis, THP-1 cells were preincubated with a specific Akt inhibitor 30 min before treatment with LXA4 and incubation with either apoptotic CMFDA-labeled PMN or lymphocytes for 2 h at 37°C. LXA4-induced phagocytosis of both apoptotic PMN and apoptotic lymphocytes (Fig. 6C) was blocked by pretreatment with the inhibitor. Pretreatment of differentiated THP-1 cells with the Akt inhibitor did not affect cell viability (data not shown).


Figure 6
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  		FIGURE 6. Involvement of Akt, PKC{zeta}, and GSK-3beta in LXA4–stimulated phagocytosis of apoptotic leukocytes. A, Differentiated THP-1 cells were treated with vehicle or LXA4 (1 nM) for the indicated time period at 37°C before lysis in RIPA buffer. Phosphorylation of Akt was monitored by Western blotting with a polyclonal phospho-Akt Ab, and expression of total Akt was also examined by Western blotting with a polyclonal total Akt Ab. B, Differentiated THP-1 cells were treated with vehicle or the indicated concentration of LXA4 for 15 min at 37°C. Phosphorylation of Akt was monitored by Western blotting with a polyclonal phospho-Akt Ab and expression of total Akt was also examined by Western blotting with a polyclonal total Akt Ab. C, Differentiated THP-1 cells were treated with a specific Akt inhibitor (AI, 10 µM for 30 min) at 37°C before stimulation with vehicle or LXA4 (1 nM). THP-1 cells were then incubated with apoptotic PMN or apoptotic Jurkat T cells for 2 h and phagocytosis was assayed by flow cytometry. Data represent mean ± SEM (n = 3) and are expressed as fold induction over basal relative to vehicle-treated cells (*, p < 0.0005 vs vehicle; #, p < 0.005 vs LXA4; **, p < 0.001 vs vehicle; ##, p < 0.001 vs LXA4). D, Differentiated THP-1 cells were treated with vehicle or LXA4 (1 nM) for (Figure legend continues) the indicated time period at 37°C before lysis in RIPA buffer. Phosphorylation of PKC{zeta} was monitored by Western blotting with a polyclonal phospho-PKC{zeta} Ab and expression of total PKC{zeta} was examined as a loading control. E, Differentiated THP-1 cells were treated with vehicle or the indicated concentration of LXA4 for 15 min at 37°C. Phosphorylation of PKC{zeta} was monitored by Western blotting with a polyclonal phospho-PKC{zeta} Ab and expression of total PKC{zeta} was also examined by Western blotting with a polyclonal total PKC{zeta} Ab. F, Differentiated THP-1 cells were treated with a pseudosubstrate inhibitor (PKC{zeta}I; 10 µM, 60 min) at 37°C before stimulation with vehicle or LXA4 (1 nM). THP-1 cells were then incubated with apoptotic PMN or apoptotic Jurkat T cells for 2 h and phagocytosis was assayed by flow cytometry. Data represent mean ± SEM (n = 3) and are expressed as fold induction over basal relative to vehicle-treated cells (*, p < 0.05 vs vehicle; #, p < 0.05 vs LXA4; **, p < 0.005 vs vehicle; ##, p < 0.005 vs LXA4). G, Differentiated THP-1 cells were treated with vehicle or LXA4 (1 nM) for the indicated time period at 37°C before lysis in RIPA buffer. Phosphorylation of GSK-3beta was monitored by Western blotting with a polyclonal phospho-GSK-3beta Ab and expression of beta-actin was examined as a loading control. H, Differentiated THP-1 cells were treated with a GSK-3beta inhibitor SB-415286 (SBI; 30 µM, 30 min) at 37°C before stimulation with vehicle or LXA4 (1 nM). THP-1 cells were then incubated with apoptotic PMN or apoptotic Jurkat T cells for 2 h and phagocytosis was assayed by flow cytometry. Data represent mean ± SEM (n = 3) and are expressed as fold induction over basal relative to vehicle-treated cells (*, p < 0.0005 vs vehicle; #, p < 0.005 vs vehicle; **, p < 0.001 vs vehicle.

 
LXA4 induces the phosphorylation of PKC{zeta} and GSK-3beta, regulating cell polarization and phagocytosis of apoptotic leukocytes

To investigate the possibility that LXA4 may stimulate phagocytosis in part through cell polarization, we examined the phosphorylation status of components of the cell polarizing regulatory pathway, namely PKC{zeta} and GSK-3beta. Addition of LXA4 to differentiated THP-1 cells caused the rapid and transient phosphorylation of PKC{zeta} (Fig. 6D). Furthermore, LXA4-triggered PKC{zeta} phosphorylation is concentration-dependent (EC50 ~1 x 10–9 M; range 1 x 10–12–1 x 10–7 M) (Fig. 6E). We then investigated whether the PKC{zeta} pathway is involved in LXA4-stimulated phagocytosis of apoptotic leukocytes. THP-1 cells were preincubated with the specific myristoylated PKC{zeta} pseudosubstrate peptide inhibitor (10 µM) for 1 h before treatment with LXA4 for 15 min and incubation with either apoptotic CMFDA-labeled PMN or lymphocytes for 2 h at 37°C. LXA4-induced phagocytosis of apoptotic PMN and apoptotic lymphocytes (Fig. 6F) was blocked by the addition of the inhibitor. Pretreatment of differentiated THP-1 cells with the pseudosubstrate inhibitor did not affect cell viability (data not shown).

Given the observations that LXA4 treatment induces THP-1 cell polarization and the rapid phosphorylation of Akt, we subsequently investigated another component of the polarization complex and known Akt substrate GSK-3beta. Phosphorylated (activated) Akt phosphorylates (inactivates) GSK-3beta. In this inactive state, phosphorylated GSK-3beta promotes polarization (32). We demonstrate that GSK-3beta is rapidly phosphorylated and consequently inactivated in response to treatment with LXA4 (Fig. 6G). The effect of phosphorylated GSK-3beta on phagocytosis was investigated using the GSK-3beta inhibitor SB-415286. SB-415286 phosphorylates, and therefore inactivates GSK-3beta, resulting in an increase in phagocytosis of apoptotic PMN and apoptotic lymphocytes (Fig. 6H) mimicking that of LXA4. Pretreatment of differentiated THP-1 cells with SB-415286 did not affect cell viability (data not shown).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we used a single-step affinity-based purification (phosphoserine immunoprecipitation) followed by one-dimensional electrophoresis and mass spectrometry to identify a protein involved in LXA4 signaling. In this study, we report that LXA4 (1 nM, 15 min) promotes dephosphorylation of MYH9 and alters phosphorylation/activation of components of a signaling cascade mediating cell polarization and phagocytosis. Therefore, MYH9 dephosphorylation may serve as a regulatory mechanism. The concentration of LXA4 that was effective against MYH9 was consistent with that shown to stimulate macrophage phagocytosis of apoptotic PMN (9, 10). Thus, we have identified a site of action for LXA4 in macrophages that may explain some of the physiological effects of this mediator.

MYH9 is a class IIA NMMHC (1961 amino acid polypeptide) with a molecular mass of 220 kDa. In humans, there are three known class II NMMHC (IIA, IIB, and IIC), encoded by distinct genes (MYH9, MYH10, and MYH14) (33). Most cell tissues contain both NMMHC IIA and NMMHC IIB (34), although some cell types are selectively enriched in either NMMHC IIA (platelets, lymphocytes, neutrophil granulocytes, and brush border cells) (35) or NMMHC IIB (neuronal tissue) (36).

Class II myosins have been proposed to have a crucial role in providing the motor activity for diverse functions such as cytokinesis, phagocytosis, and muscle contraction and polarity. Class II myosins exist as a hexameric complex composed of two myosin H chain subunits and two pairs of L chain subunits. The H chain subunit contains the ATPase activity, providing energy that is the driving force for the contractile processes previously mentioned. To participate in contractility, nonmuscle myosin II must successfully assemble into functional bipolar filaments. First, individual H chains must dimerize by folding into an {alpha}-helical coiled-coil structure. The second assembly step involves lateral associations of H chain dimers to form functional bipolar filaments. In principle, defects in either step could result in the inability to form functional nonmuscle myosin IIA filaments (37).

Lower eukaryotes such as Dictyostelium and Acanthamoeba possess a single myosin II gene, and thus are suitable model organisms for genetic studies on the general cellular functions of myosin II (38, 39). Dictyostelium nonmuscle myosin II has been studied in detail and has a molecular structure very similar to that of mammalian myosin II. In lower eukaryotes, myosin II filament assembly, contraction, and disassembly are highly regulated by phosphorylation and are important in the establishment of cell polarity, the retraction of the posterior cell body and the regulation of pseudopod extension (40, 41, 42). Although the mechanisms of regulation of mammalian MYH9 phosphorylation are poorly understood, it has recently been proposed that Mts1, a member of the S100 family of Ca2+-binding proteins, is involved in regulating filament assembly by binding to residues 1909–1924 of nonmuscle myosin IIA (43). It is possible that serine phosphorylation of MYH9 may regulate filament assembly by modulating the formation of the Mts1-MYH9 complex.

Treatment of THP-1 cells with LXA4 resulted in MYH9 localization at the cell posterior (Fig. 4A). Such data are consistent with the localization of myosin II at the rear and sides of neutrophils (44). Similarly, MYH9 is enriched at the uropod in T cells, particularly at the head-tail junction (45).

Phagocytosis is a highly regulated process, which requires remodeling of the macrophage cytoskeleton for successful engulfment of the apoptotic target (14). The signals generated by the phagocyte-apoptotic cell interaction target the cytoskeleton (46), inducing changes that lead to engulfment, followed by a process of internalization (14). To our knowledge, this study is the first demonstration of a specific role for MYH9 in the regulation of phagocytosis of apoptotic cells by LXA4. Previously, a proteomics approach was adopted to identify the proteins associated with latex bead phagosomes (47). A number of actin binding proteins were identified, including NMMHC IIA. However, the phosphorylation status of MYH9 was not investigated in this study (47). These findings correlate with our hypothesis that MYH9 is critically involved in phagocytosis of apoptotic cells.

Myosins have previously been implicated in Fc{gamma}R- and complement receptor-mediated phagocytosis, localizing on phagosomes (26, 48). Complement receptor-mediated phagocytosis specifically uses MYH9 to assist in the assembly of actin cups around particles (26). Our data demonstrate that, through specific inhibition of class II myosin, macrophages require MYH9 motor activity to successfully engage in LXA4-stimulated phagocytosis of apoptotic cells (Fig. 4B). We propose that LXA4-mediated dephosphorylation and localization of MYH9 induces a polarized phenotype, consequently priming the macrophage to rapidly respond to and ultimately phagocytose apoptotic cells.

It is noteworthy that pretreatment with a peptide mimetic of annexin 1, Ac2–26, promotes serine dephosphorylation of MYH9 and redistribution of MYH9 within polarized cells. Recent data have demonstrated pretreatment with Ac2–26 promotes the nonphlogistic phagocytosis of apoptotic PMN acting through the pleiotropic agonists of the LXA4 receptor (11).

Cell polarity is important for phagocytosis. Establishment and maintenance of cell polarity requires the activation of separate molecular processes at the cell anterior and posterior (49). Primed mammalian and Dictyostelium cells have strongly biased axial polarity, with F-actin assembled primarily in the leading edge and myosin II in the rear cell body (50). The Rho GTPases, Rho, Rac, and Cdc42 have been shown to regulate actin assembly and rearrangement in response to external stimuli, hence providing the driving force for polarization, cell migration, and phagocytosis of particles (51). A role for the Rho GTPase Cdc42 in controlling cell polarization in diverse cell types including fibroblasts, macrophages, astrocytes, and mesangial cells is well established (52, 53, 54). Cdc42 has also been shown to regulate actin reorganization during phagocytosis by promoting pseudopod extension and phagosome closure (29, 55). Treatment of THP-1 cells with LXA4 resulted in Cdc42 localization at the cell anterior (Fig. 5A) and increased levels of active Cdc42 (Fig. 5B). These observations are consistent with previous studies demonstrating the localization of Cdc42 to the leading edge of polarized motile cells (53) and that Cdc42 localization to the leading edge is usually associated with activation (30).

There is growing evidence that a key step in mediating polarity is the activation of PI3K and the downstream effector Akt (56). Our results indicate that LXA4-induced activation of Cdc42 is mediated by the serine/threonine kinase Akt, which is a downstream target of PI3K (Fig. 5B). Activation of the PI3K pathway is also important in macrophage phagocytosis of apoptotic cells (29). We have previously observed that pretreatment of THP-1 cells with the PI3K inhibitor LY294002 inhibited LXB4-stimulated phagocytosis of apoptotic PMN (10). However, in LXA4-treated renal mesangial cells, inhibition of platelet-derived growth factor receptor phosphorylation is coupled to an attenuation of downstream PI3K activity and subsequent Akt phosphorylation. These differing observations may reflect the heterogeneity of PI3K isoform expression in cells of myeloid and nonmyeloid lineage (57) or the multiplicity of receptors. FPRL2, a receptor for peptide agonists, is expressed on monocytes/macrophages but not on PMN (58). LX and its analogs act via the agonists of the LXA4 receptor to inhibit PMN chemotaxis. In contrast, LXs have been reported to activate monocyte chemotaxis, and this effect is shared by agonists of the LXA4 receptor peptide ligands. LXs have been demonstrated to induce changes in the ultrastructure and F-actin reorganization in human monocytes and macrophages, but not in PMN (15). This contrasting data obtained from peptide and LX ligands in monocytes and PMN suggests the involvement of the FPRL2 receptor. These observations raise the intriguing possibility that LXA4 may exert its effects through the FPRL2 receptor in macrophages.

The observation that a specific Akt inhibitor blocked LXA4-induced activation of Cdc42 (Fig. 5B) led us to further investigate the involvement of Akt as an intracellular modulator of LXA4 signaling. We proceeded to demonstrate that treatment of THP-1 cells with LXA4 results in the phosphorylation of Akt (Fig. 6A). This activated Akt appears to modulate LXA4-induced phagocytosis of apoptotic cells because exposure of THP-1 cells to a specific Akt inhibitor before LXA4 stimulation was shown to block LXA4-induced phagocytosis (Fig. 6B). We propose that LXA4 contributes to the activation of Akt and subsequently Cdc42, thereby promoting enhanced uptake of apoptotic cells.

Previously, we observed that pretreatment of THP-1 cells with the PKC inhibitor GF109203X inhibited LX-stimulated phagocytosis of apoptotic PMN (9), suggesting the involvement of PKC in LXA4 signaling. Members of the PKC family (novel PKC{delta} and/or PKC{epsilon}) have previously been implicated in Fc{gamma}R-mediated phagocytosis (59). It is interesting to note that PKC{zeta} has previously been demonstrated to associate with myosin IIA in fibroblasts (60). In this study we show that treatment of THP-1 cells with LXA4 results in phosphorylation of PKC{zeta} (Fig. 6C). This activated PKC{zeta} appears to be involved in driving LXA4-induced phagocytosis of apoptotic cells because exposure of THP-1 cells to the myristoylated peptide inhibitor Myr-SIYRRGARRWRKL-OH before LXA4 stimulation was found to abrogate LXA4-induced phagocytosis (Fig. 6D). We propose that PKC{zeta} plays a role in regulating LXA4-stimulated cytoskeletal rearrangements given previous observations that the Cdc42 target protein, PKC{zeta}, is essential for defining cell polarity in migrating astrocytes (53) and mesangial cells (54).

Intriguingly, inhibitors directed against MYH9, Akt, and PKC{zeta} failed to completely block phagocytosis of both apoptotic PMN and apoptotic Jurkat T cells, thus suggesting that activation of a pathway involving these signaling components is required for LXA4-stimulated phagocytosis of apoptotic leukocytes. Previous data have also indicated that LXA4-mediated augmentation of phagocytosis is both PI3K- and PKC-dependent (10). It is of interest to note that inhibitors directed against MYH9, Akt, and PKC{zeta} did not affect phagocytosis of apoptotic cells by macrophages untreated with LXA4. These data may suggest that these signaling proteins are not involved in normal clearance of apoptotic cells. However, in our experiments the level of phagocytosis observed basally is low (5%) so that it may not be possible to observe a further decrease in basal phagocytosis in response to inhibitors. Lack of detectable effect does not preclude a possible inhibition. We propose that LXA4 specifically dephosphorylates MYH9 and activates Akt and PKC{zeta}, causing the phagocyte to adopt a polarized phenotype, engaging the phagocyte for a rapid response to the presence of apoptotic cells and their subsequent engulfment.

Active Cdc42 further promotes polarization by recruiting and activating a cytoplasmic mPar6-PKC{zeta} complex, which in turn interacts with and regulates GSK-3beta activity (32). GSK-3beta has also been implicated in the regulation of polarity governing connective tissue growth factor-mediated mesangial cell migration (54). Activated Akt specifically phosphorylates GSK-3beta (an Akt substrate) on Ser9 thus inhibiting its catalytic activity (61). Following treatment with LXA4, GSK-3beta undergoes rapid phosphorylation at Ser9, and subsequent inactivation (Fig. 6E). Treatment with the GSK-3beta inhibitor SB-415286 induces phosphorylation/inactivation of GSK-3beta, resulting in an increase in phagocytosis mimicking that of LXA4, thus highlighting the importance of GSK-3beta in the establishment of LXA4-induced polarity and phagocytosis (Fig. 6F).

Importantly, we demonstrate for the first time the ability of LXA4 to stimulate macrophage phagocytosis of apoptotic lymphocytes. This observation is of significant interest in light of recent evidence that increased lymphocyte apoptosis and defects in macrophage removal of apoptotic cells have been proposed to contribute to the development of the autoimmune disease, systemic lupus erythematosus (62, 63, 64). Consequently, LXA4-induced modulation of macrophage phagocytosis may be considered an attractive target for therapeutic intervention. A recent investigation into host defense against Mycobacterium tuberculosis further highlighted the involvement of LXA4 in maintaining a balance between inflammation and resolution (65). Administration of the stable lipoxin analog ATLa2 to M. tuberculosis-infected 5-lipoxygenase knockout mice restored both pulmonary mycobacterial loads to levels comparable to those observed in infected wild-type animals, thereby negatively regulating protective Th1 responses.

In conclusion, this study has yielded the intriguing finding that MYH9 may be a regulatory site for LXA4-induced effects on the macrophage cytoskeleton. THP-1 cells treated with LXA4 adopt a polarized morphology with MYH9 and Cdc42 demonstrating a spatial distribution within the cell. In addition, our observations that LXA4 stimulates the phosphorylation of key signaling molecules (Akt, PKC{zeta}, and GSK-3beta) concurrent with increased levels of activated Cdc42 demonstrates that THP-1 cell polarization is a key, early event in LXA4-induced phagocytosis (Fig. 7). Collectively, these data provide an insight into the complex signal-transduction network controlling clearance of apoptotic cells and highlight the significant anti-inflammatory potential of LX in promoting the resolution of inflammation.


Figure 7
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  		FIGURE 7. Proposed signaling events governing LXA4-stimulated macrophage polarization and phagocytosis.

 

   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by the Health Research Board, Ireland, Enterprise Ireland, the European Union and the Wellcome Trust, and funded under the Programme for Research in Third Level Institutions by the Higher Education Authority. Back

2 Address correspondence and reprint requests to Prof. Catherine Godson, School of Medicine and Medical Sciences, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. Back

3 Abbreviations used in this paper: LX, lipoxin; PMN, polymorphonuclear neutrophil; PKC, protein kinase C; GSK, glycogen synthase kinase; BDM, 2,3-butanedione monoxime; PVDF, polyvinylidene difluoride; NMMHC, nonmuscle myosin H chain. Back

Received for publication June 28, 2005. Accepted for publication November 4, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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