Accuri C6 Flow Cytometer System
Originally published online as doi:10.1189/jlb.0707495 on October 30, 2007

Published online before print October 30, 2007
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(Journal of Leukocyte Biology. 2008;83:344-351.)
© 2008 by Society for Leukocyte Biology

Attenuated translocation of group IVa phospholipase A2 and up-regulated annexin-1 synthesis by glucocorticoid blocks β2-integrin adhesion in neutrophils

Angelo Y. Meliton*, Nilda M. Munoz*,1, Xiangdong Zhu* and Alan R. Leff*,{dagger}

* Departments of Medicine,
{dagger} Pediatrics, Pharmacology and Physiology and Committees on Molecular Medicine, Clinical Pharmacology and Pharmacogenomics, and Cell Physiology, The University of Chicago, Chicago, Illinois, USA

1 Correspondence: Department of Medicine, M6076, The University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA. E-mail: nmunoz{at}medicine.bsd.uchicago.edu


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ABSTRACT
 
We examined the effect of glucocorticoid stimulation in blocking β2-integrin adhesion of polymorphonuclear leukocytes (PMNs) isolated from human subjects. Surface expression of CD11b and ERK-1/2-mediated gIVaPLA2 phosphorylation, which are required for β2-integrin adhesion, were not affected by treatment with ≤10–6 M fluticasone propionate (FP) for PMNs activated by either 10–7 M LTB4 or 30 ng/ml TNF-{alpha} and caused no significant blockade of β2-integrin adhesion in vitro. Baseline expression of annexin-1 (ANXA1) synthesis was increased only after 10–6 M FP for PMNs; by contrast, comparable increase in ANXA1 expression was demonstrated in human eosinophils from the same subjects with 10–8 M FP. Viability of PMNs was verified by propidium iodide and by the persistence of β2-integrin adhesion in treated groups. Exogenous administration of ANXA1 mimetic peptide fragment blocked significantly and comparably the β2-integrin adhesion in PMNs activated by LTB4 and TNF-{alpha} and in eosinophils activated by IL-5. Translocation of gIVaPLA2 from the cytosol to the nucleus also was refractory for activated PMNs treated with ≥10–7 M FP; by contrast, complete blockade of nuclear translocation of cytosolic gIVaPLA2 was effected by 10–9 M FP in eosinophils. Our data indicate that the cell surface ANXA1 synthesis is capable of blocking β2-integrin adhesion in both PMNs and eosinophils. However, in contrast to eosinophils, FP does not cause either substantial ANXA1 synthesis or nuclear transport of cytosolic gIVaPLA2 in PMNs and thus does not block β2-integrin adhesion, a necessary step for granulocyte cell migration in vivo.

Key Words: fluticasone propionate • granulocytes • gIVaPLA2 • signal transduction • annexin-1 mimetic


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INTRODUCTION
 
Annexins are a well-conserved family of structurally related proteins that are implicated in the regulation of many physiological and pathophysiological functions, i.e., cell growth and differentiation [1 ], inflammation [2 , 3 ], and more recently, membrane trafficking [4 ]. The diverse biological properties of annexins depend on the N-terminal domain, which is unique in length and sequence and the potential sites for phosphorylation [5 ], glycosylation [6 ], and peptidase action [7 ].

Eosinophils and neutrophils express an inducible adhesion-promoting glycoprotein β2-integrin (CD11b/CD18), which facilitates adhesion of the circulating granulocytes to the endothelium [8 , 9 ]. Both eosinophils and neutrophils also express annexin-1 (ANXA1) [10 11 12 ], which plays a critical role in the anti-inflammatory effects of glucocorticoids [2 , 3 ]. We have reported previously that activation of eosinophils by glucocorticoid causes translocation of ANXA1 from the cytoplasm to the outer plasma membrane of eosinophils [13 ]; this event blocked β2-integrin-mediated adhesion [14 ]. Studies in vitro in polymorphonuclear leukocytes (PMNs) suggest that exogenous ANXA1 attenuates neutrophil migration at inflammatory sites [15 ]. However, corticosteroids also activate PMNs, and the role of glucocorticoids in preventing PMNs in migration into airways in chronic obstructive pulmonary disease (COPD) is controversial [16 ].

Studies in vitro indicate that ANXA1 blocks cytosolic gIVa phospholipase A2 (gIVaPLA2) activity, an essential enzyme in the regulation of β2-integrin-mediated adhesion in human eosinophils [14 ]. We have shown that at concentrations comparable to those achieved therapeutically by inhalation (10–7 M) [14 ], pretreatment of eosinophils with fluticasone propionate (FP) blocks the translocation of cytosolic gIVaPLA2 to the nuclear membrane and that blockade is reversed by ANXA1-blocking mAb [14 ]. More specifically, Ac2-26, a long fragment of ANXA1 synthetic peptide, consistently mimics the anti-inflammatory effect of FP on β2-integrin-mediated eosinophil adhesion [14 ].

In this study, we determined whether FP, a potent glucocorticoid used for inhalational treatment of allergic rhinitis and asthma, blocks 1) up-regulated surface CD11b expression, 2) β2-integrin-mediated adhesion, and 3) ERK-1/2-mediated gIVaPLA2 phosphorylation in activated PMNs. We also determined whether ANXA1 synthesis caused by FP down-regulates adhesion caused by LTB4 or TNF-{alpha} in PMNs. Our data suggest that antiadhesion effects of corticosteroids are substantially less in neutrophils than for eosinophils due to the relative refractoriness of FP in PMNs in causing 1) blockade of ERK-1/2 mediated gIVaPLA2 phosphorylation, 2) blockade of translocation of cytosolic gIVaPLA2 to the nuclear envelope, and 3) synthesis of endogenous ANXA1.


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MATERIALS AND METHODS
 
Antibodies and reagents
LTB4 and TNF-{alpha} were purchased from Biomol International (Plymouth, PA, USA) and Biosource (Camarillo, CA, USA), respectively. Polystyrene 96-well microplate wells were obtained from Costar (Cambridge, MA, USA). Phosphorylation-specific ERK-1/2 pAb (Promega, Madison, WI, USA) and nonspecific ERK-1/2 mAb was obtained from New England Biolabs (Beverly, MA, USA). Ser505 phosphorylation-specific cytosolic PLA2 (gIVaPLA2) mAb was obtained from Cell Signaling Technology (Beverly, MA, USA); gIVaPLA2 mAb was obtained from Santa Cruz Biotech (Santa Cruz, CA, USA), and Ac2-26 was obtained from Phoenix Pharmaceuticals (Belmont, CA, USA). Fluticasone propionate was provided by GlaxoSmithKline (Uxbridge, UK). BODIFY FL-conjugated goat anti-mouse secondary Ab was purchased from Molecular Probes (Eugene, OR, USA).

Isolation of human polymorphonuclear leukocytes and eosinophils
Human polymorphonuclear leukocytes (PMNs) and eosinophils were isolated from mildly atopic subjects according to a protocol approved by the University of Chicago Institutional Review Board. Informed consent was obtained from all volunteers in this study before participation. Volunteers with symptoms or subjects taking medication were excluded. The study included a total of 22 individuals age 20 to 45 years, 12 male and 10 female. All components of the project were in compliance with the University of Chicago and U.S. governmental guidelines for studies in which donors are not participating subjects.

PMNs were isolated by Ficoll-Paque sedimentation, as described previously [17 ]. Cells were resuspended in HBSS buffer + Ca++/0.2% BSA prior to counting.

The protocol for isolation of eosinophils has been recently described in detail [18 ]. The purity of eosinophils was determined by differential counts of H- and E-stained cytospin preparations. Purified PMNs and eosinophils stained by propidium iodide were >99% viable as analyzed by flow cytometric analysis. Cells were kept on ice until use.

Flow cytometric analysis
PMNs were resuspended in HBSS buffer + Ca++/0.2% BSA containing 1 pg/ml GM-CSF and incubated with buffer control or 10–10 to 10–6 M FP for 24 h before activation with either 10–7 M LTB4 or 30 ng/ml TNF-{alpha} at 37°C for 15 min. This concentration (1 pg/ml GM-CSF) had no effect on either cell activation or adhesion in either control or experimental groups from the same subjects. For isolated eosinophils, IL-5 (10 pg/ml) was added to the culture media to maintain cell viability and similarly, had no effect on cell activation or adhesion (13). The incubation period was terminated by centrifugation at 400 g for 10 min, and the pellets were resuspended in PBS/1.0% BSA. Aliquots of 3 x 105 PMNs were incubated with 10 µl of the mAb directed against CD11b (clone Bear 1; Transduction Laboratory, Lexington, KY, USA) or isotype-matched control Ab at 4°C for 60 min. After 2 washes, the cells were incubated with an excess of fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse immunoglobulin for 30 min at 4°C. The cells were washed twice, resuspended in 300 µl of 1% paraformaldehyde, and kept at 4°C until analysis. Flow cytometry was performed on a FACScan (Becton Dickinson, Mountain View, CA, USA). Fluorescence intensity was determined on at least 10,000 cells from each sample.

In separate studies, the surface expression of ANXA1 was determined for eosinophils or PMNs treated with 10–8 M, 10–7 M, or 10–6 M FP and experimental procedures were performed as above using mAb directed against ANXA1 (Transduction Laboratory, Lexington, KY, USA).

Adhesion assay
A 96-microplate well was coated with 50 µl of soluble human BSA, a surrogate ligand for ICAM-1 [17 , 19 ], dissolved in 0.05 M NaHCO3 coating buffer (15 mM NaHCO3, 35 mM Na2CO3, pH 9.2), and incubated overnight at 4°C. Treated microplate wells were washed 2 times with HBSS buffer prior to use. The detailed protocol is described elsewhere [17 ].

Adhesion was assessed as residual myeloperoxidase (MPO) activity of adherent PMNs and as residual eosinophil peroxidase (EPO) activity of adherent eosinophils. Treated PMNs (4x104 cells) or eosinophils (1x104 cells) per 100 µl HBSS/0.1% gelatin were added to BSA-coated microplate wells and allowed to settle on ice for 10 min. Cells were pretreated with buffer control or with FP (24 h) before activation with 10 ng/ml IL-5 (eosinophils), 10–7 M LTB4 (PMNs), or 30 ng/ml TNF-{alpha} (PMNs) at 37°C for 15 min. A 24-h incubation was shown in preliminary time- and concentration-finding studies to be optimal. At the end of the activation period, microplate wells containing samples were gently washed 3 times with HBSS and thereafter, 100 µl of HBSS/0.1% gelatin was added to the reaction wells. A 100 µl of MPO substrate (1 mM H2O2, 1 mM O-dianizidine dihydrochloride, and 0.5% HTAB in Tris buffer, pH 5.5) or EPO substrate (1 mM H2O2, 1 mM O-phenylenediamine, and 0.1% TritonX-100 in Tris buffer, pH 8.0) was added to each well containing adherent cells prior to 30-min incubation at room temperature. The reaction mixture was terminated by addition of 50 µl of 4 M H2SO4. To generate a standard curve for each adhesion assay, serial dilutions of the original cell suspensions, were added to the empty wells, and the same procedure was conducted as above. Absorbance was measured at 405 nm (PMNs) or 490 nm (eosinophils) in a Thermomax microplate reader (Molecular Devices, Menlo Park, CA). All assays were performed in duplicate, and data were analyzed by Softmax (Molecular Devices, Sunnyvale, CA). The detection of MPO or EPO was linear between the concentrations of 1 x 103 to 1.5 x 104 cells/well as determined by a standard curve. No MPO or EPO activity was detected in the cell-free reaction supernatants following 30 min incubation, confirming that MPO or EPO was not present because of spontaneous granulocyte degranulation. Previous investigations have established the optimal concentration of IL-5, LTB4, and TNF-{alpha} used in this study [17 , 20 ]. The optimal time of activation of each agonist was then used for subsequent study (see Results).

In separate studies, eosinophils and PMNs were pretreated with 10 µM or 30 µM Ac2-26, a long fragment of ANXA1 mimetic peptide, for 20 min prior to cell activation and measurement of adhesion as performed above.

Western blot analysis
Isolated PMNs were pretreated with 10–10 M to 10–6 M FP for 24 h and then activated for 15 min with either 10–7 M LTB4, 30 ng/ml TNF-{alpha}, or buffer control at 37°C. The cell pellet was lysed in 70 µl of lysis buffer (20 mM Tris-HCl, 30 mM Na4P2O7, 50 mM NaF, 40 mM NaCl, 5 mM EDTA, 1% Nonidet-40, one protease inhibitor tablet), and the disrupted cells were centrifuged at 500 g for 1 min to remove nuclear and cellular debris. A total of 65 µl of supernatant was mixed with 14 µl of 6x loading buffer and boiled for 5 min. Samples were loaded to SDS-PAGE using 10% acrylamide gels under reducing conditions, and electrotransfer of proteins to a nitrocellulose membrane was achieved using a semi-dry system. The membrane was blocked with 1% BSA in TBS-T buffer for 60 min before the addition of 2 µg/ml anti-phosphorylated ERK-1/2 Ab (Promega) or 2 µg/ml of anti-phosphorylated Ser505 gIVaPLA2 Ab (Cell Signaling Technology). After washing, the membranes were incubated with 1:3000 dilutions of goat anti-rabbit Ig conjugated with HRP and analyzed by an enhanced chemiluminescence system (ECL; Amersham, Arlington Heights, IL, USA).

The identified phosphorylated bands were scanned by GS-710 Calibrated Imaging Densitometer (Bio-Rad, Hercules, CA, USA), and data were analyzed and expressed as the increase in optical density from baseline.

Confocal microscopy: distribution of cytosolic gIVaPLA2
Because gIVaPLA2 is an essential enzyme for induction of β2-integrin adhesion [20 ], we further examined whether FP blocks the translocation of cytosolic gIVaPLA2 to the nuclei of activated eosinophils or PMNs. Treated cells were harvested and were stained with mAb directed against gIVaPLA2, as described previously [14 , 21 ]. The slides containing treated cells were incubated with 20 µg/ml BODIFY FL-conjugated goat anti-mouse secondary Ab for 60 min at room temperature, and translocation of cytoplasmic gIVaPLA2 within the nuclei was analyzed by confocal microscopy.

Statistical analysis
All data are expressed as the mean ± SEM. Differences between groups were assessed by paired t-test. Where more than 2 groups were compared, differences among groups were assessed by one-way ANOVA. Where differences were found, comparisons among groups were made by Fisher’s least-protected difference test. Statistical significance was claimed where P < 0.05.


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RESULTS
 
Effect of FP on cell viability
We first tested the long-term effect of FP on viability of eosinophils and PMNs used in this study. After 24 h incubation with 10–10 M to 10–6 M FP, treated cells were stained with propidium iodide and demonstrated >92% viability, as determined by FACScan. Unstimulated cells (buffer control) served as a negative control. Fig. 1 is a representative dot-plot analysis from treated cells isolated from 3 isolations using 3 different donors. In all subsequent studies, cells were preincubated with increasing concentrations of FP for 24 h prior to experimental procedures. Studies using trypan blue exclusion dye also produced comparable results (data not shown).


Figure 1
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  		Figure 1. Viability of cells: Eosinophils (EOS) and polymorphonuclear leukocytes (PMNs) were incubated with either 10–8 M fluticasone propionate (FP) or 10–6 M FP for 24 h prior to propidium iodide staining. Representative dot-plots for EOSs (top) and PMNs (bottom) in the presence or absence of FP. Viability of cells was determined by FACS analysis. The control indicates no treatment with FP.

Effect of FP on surface expression of CD11b
We next examined the inhibitory effect of 10–10 M to 10–6 M FP on surface CD11b expression activated with buffer control, 10–7 M LTB4, or 30 ng/ml TNF-{alpha} on PMNs. The constitutive CD11b expression of PMNs was 46.8 ± 14.9 specific fluorescence intensity (SFI; mean fluorescence intensity minus isotype-matched control, no treatment) for unstimulated cells stained with mAb directed against CD11b (Fig. 2 ). Activation with LTB4 up-regulated the surface CD11b expression to 80.6 ± 22.6 SFI (Fig. 2A ; P<0.02 vs. unstimulated cells) and 70.4 ± 23.5 SFI for cell activated with 30 ng/ml TNF-{alpha} (Fig. 2B ; P<0.05 vs. unstimulated cells). FP (10–10 M to 10–6 M) did not affect the surface CD11b expression caused by either LTB4 or TNF-{alpha} activation.


Figure 2
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  		Figure 2. Blockade of up-regulated surface expression of CD11b induced by LTB4 and TNF-{alpha}. Polymorphonuclear leukocytes (PMNs) were incubated with increasing concentrations of FP ranging from 10–10 M to 10–6 M for 24 h before activation with 10–7 M LTB4 (A) or 30 ng/ml TNF-{alpha} (B) for 15 min. The presence of CD11b of the surface of cells was assessed by FACS analysis. The control (baseline CD11b) indicates no treatment. The results are expressed as specific fluorescence intensity (SFI; mean fluorescence intensity minus isotype-matched control) and the standard error for 5 independent experiments. *, P < 0.02 vs. activated cells.

Effect of FP on β2-integrin-mediated adhesion
We examined whether the increased surface expression of CD11b elicited by LTB4 or TNF-{alpha} above baseline constitutive levels caused increased adhesion of PMNs to BSA-coated microplate wells. We have demonstrated previously that BSA is a full surrogate for ICAM-1 [17 , 19 ]. LTB4 (Fig. 3A ) and TNF-{alpha} (Fig. 3B) increased β2-integrin adhesion to 23.8 ± 2.54% and 23.78 ± 2.38%, respectively, vs. ~5% adhesion at baseline (unstimulated cells; P<0.05); 10–6 M FP caused only minimal blockade of adhesion caused by LTB4 (P<0.05) had no significant effect at any concentration on adhesion elicited by TNF-{alpha}.


Figure 3
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  		Figure 3. Blockade of stimulated β2-integrin adhesion by FP. Polymorphonuclear leukocytes (PMNs) were incubated with increasing concentrations of fluticasone propionate (10–10 M–10–6 M FP) prior to activation with 10–7 M LTB4 (A) or 30 ng/ml TNF-{alpha} (B) for 15 min. Unbound cells were washed with buffer, and % cellular adhesion was determined by measuring the residual myeloperoxidase activity of adherent cells using microplate reader. The composite data were expressed as the mean value and SE for 5 different experiments. *, P < 0.05 vs. LTB4-activated cells

Effect of FP on ERK-1/2-mediated gIVaPLA2 phosphorylation
We next investigated whether LTB4- or TNF-{alpha}-induced adhesion of β2-integrin involved activation of ERK-1/2 and subsequent gIVaPLA2 phosphorylation in PMNs. We have shown previously that phosphorylated ERK-1/2 is required for activation of gIVaPLA2 and induction of β2-integrin adhesion in human eosinophils [20 , 22 ]. Immunoblotting analysis demonstrated that 10–7 M LTB4 (Fig. 4A , top) or 30 ng/ml TNF-{alpha} (Fig. 4A , below) predominantly caused phosphorylation of ERK-2. FP did not block ERK-2 phosphorylation caused by LTB4 or TNF-{alpha} even at high concentration (10–6 M FP).


Figure 4
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  		Figure 4. Activation of ERK-1/2-mediated gIVaPLA2 phosphorylation. Phosphorylation of ERK-1/2 (A) and gIVaPLA2 (B) caused by LTB4 or TNF-{alpha} was examined in polymorphonuclear leukocytes (PMNs) in the presence or absence of increasing concentrations of FP. The phosphorylated ERK-1/2 or gIVaPLA2 was developed with anti-phosphorylated ERK-1/2 Ab or anti-phosphorylated Ser505 gIVaPLA2 Ab, respectively, and analyzed by an enhanced chemiluminescence system. (C) Densitometric analysis: Effect of fluticasone propionate (FP) on LTB4- and TNF-{alpha}-induced gIVaPLA2 phosphorylation (n=3 experiments per group). Data represented the fold increase in optical density from baseline and were expressed as optical density/mm2.

In four separate isolations from four different donors, gIVaPLA2 phosphorylation was identified by Ser505 gIVaPLA2 phosphorylated mAb [21 , 23 ]. Cytosolic gIVaPLA2 phosphorylation was evident after activation with LTB4 (Fig. 4B , top) or TNF-{alpha} (Fig. 4B , below). However, FP had no inhibitory effect on activated gIVaPLA2 phosphorylation even at the greatest concentration used as assessed by densitometric analysis (Fig. 4C) . The total gIVaPLA2 protein bands identified by pAb against gIVaPLA2 were used as a marker to demonstrate equal loading of samples (Fig. 4B)

Effect of FP on cell surface ANXA1
We next examined the effect of 10–10 M, 10–8 M, and 10–6 M FP on cell surface ANXA1 expression in both eosinophils and PMNs from the same donors. Cells were stained with mAb directed against ANXA1, and measurement of membrane-bound ANXA1 expression was analyzed by flow cytometric analysis. A representative histogram of surface ANXA1 expression caused by FP in both eosinophils and PMNs is shown in Fig. 5 . In eosinophils, ANXA1 expression was up-regulated with 10–8 M FP and was greatest at 10–6 M FP. By contrast, 10–8 M FP did not up-regulate ANXA1 expression in PMNs; modest up-regulation of ANXA1 comparable to that observed at 10–10 M FP in eosinophils was achieved only with 10–6 M FP in PMNs.


Figure 5
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  		Figure 5. Up-regulation of surface ANXA1 expression. Eosinophils (EOS) and polymorphonuclear leukocytes (PMN) from the same donors were incubated with 10–10 M to 10–6 M FP for 24 h prior to determination of surface ANXA1 expression using FACS analysis. Inset: Composite data were calculated as % increase from baseline and expressed as mean ± SEM from 4 experiments using 4 different donors. *, P < 0.05 vs. activated cells; **, P < 0.02 vs. activated cells.

Composite data for these studies are shown in Fig. 5 (inset). In eosinophils (Fig. 5 , EOS), ANXA1 expression was 174.9 ± 37.6% of baseline level after 10–8 M FP (P<0.05); by contrast (Fig. 5 , PMNs), minimal increase in ANXA1 expression was observed for PMNs receiving 10–8 M FP (109.5±18.7% of baseline level; P=NS). At the highest concentration tested (10–6 M FP), ANXA1 expression further increased to 225.7 ± 38.7% for eosinophils (P<0.02 vs. baseline level) and only 125.9 ± 13.6% for PMNs (P<0.05 vs. baseline level).

Effect of ANXA1 mimetic peptide on PMN and eosinophil adhesion
We have reported previously that ANXA1 N-terminus peptide, Ac2-26, mimics the effect of endogenous ANXA1 synthesis caused by FP in blocking eosinophil adhesion [14 ]. Accordingly, we examined the comparative effect of Ac2-26 in adhesion of eosinophils and PMNs to BSA-coated microplate wells in response to activation by IL-5 (eosinophils), LTB4 (PMNs), or TNF-{alpha} (PMNs), respectively. Baseline adhesion was 4.26 ± 0.52% for resting eosinophils (unstimulated cells) and increased to 26.83 ± 4.1% after IL-5 activation (Fig. 6 , EOS; P<0.02). Pretreatment of PMNs for 20 min with Ac2-26 inhibited β2-integrin adhesion of eosinophils caused by 10 ng/ml IL-5. At 10 µM Ac2-26, adhesion caused by 10 ng/ml IL-5 decreased to 15.64 ± 1.97% (P<0.05) and further to 12.53 ± 1.79% for cells treated with 30 µM Ac2-26 (P<0.05 vs. IL-5-activated cells)


Figure 6
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  		Figure 6. Blockade of adhesion by ANXA1 mimetic peptide. Eosinophils (EOS) and neutrophils (PMN) were exposed to 10 µM or 30 µM Ac2-26 prior to activation with either 10 ng/ml IL-5 (EOS), 10–7 M LTB4 (PMN), or 30 ng/ml TNF-{alpha} (PMNs) and adhesion assay. Unbound cells were washed with buffer, and adhesion was measured as residual eosinophil peroxidase (EOS) or myeloperoxidase (PMN) activity of adherent cells using a microplate reader. Data are expressed as mean ± SEM from 4 experiments with different donors. *, P < 0.05 vs. activated cells.

Blockade of adhesion also was observed to a lesser, but significant, degree for PMNs treated with Ac2-26 prior to activation with 10–7 M LTB4 or 30 ng/ml TNF-{alpha} (Fig. 6 , PMN). Baseline adhesion was 5.0 ± 0.85% in unstimulated PMN. Activation with LTB4 increased β2-integrin adhesion of PMN to 16.07 ± 2.21%; activation by TNF-{alpha} caused an 18.67 ± 2.15% increase in β2-integrin adhesion (P<0.02 vs. unstimulated cells, both groups). At 10 µM, the ANXA1 fragment, Ac2-26, caused ~36% blockade of adhesion caused by LTB4 and for cells activated with TNF-{alpha} (P<0.05 vs. activated cells in both groups); 30 µM Ac2-26 did not promote further blockade of adhesion.

Blockade of cytosolic gIVaPLA2 localization by FP
Cytosolic gIVaPLA2 is an essential enzyme in induction of β2-integrin adhesion [22 ]. This study was designed to visualize the actual localization of activated cytosolic gIVaPLA2 within the nuclei in the presence or absence of increasing concentrations of FP (Fig. 7 ). Immunofluorescence microscopy studies were carried out using mAb directed against gIVaPLA2 [14 , 21 ]. In resting (unstimulated) eosinophils and PMNs, antigenic adsorption of gIVaPLA2 mAb revealed a homogenous distribution of gIVaPLA2 in the cytoplasm (Fig. 7B and 7H) . However, the level of cytosolic gIVaPLA2 decreased after activation with IL-5 and localized in the nuclei of eosinophils (Fig. 7C , EOS). Pretreatment of eosinophils with 10–9 M FP blocked fully the cytosolic gIVaPLA2 translocation to the nucleus (Fig. 7D , EOS). In contrast to eosinophils, substantial (but still not complete) blockade of cytosolic gIVaPLA2 translocation to the nuclei was demonstrated only with 10–7 M to 10–6 M FP in PMNs activated with LTB4 or TNF-{alpha} (Fig. 7 , K, L, O, P).


Figure 7
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  		Figure 7. Localization of cytosolic gIVaPLA2. EOS (A–F) and polymorphonuclear leukocytes (PMN; G–P) were preincubated with increasing concentrations of FP ranging from 10–9 M to 10–6 M prior to activation with 10 ng/ml IL-5, 10–7 M LTB4 or 30 ng/ml TNF-{alpha}. Cytoslides were prepared, and treated cells were stained with FITC-conjugated gIVaPLA2 Ab, and translocation of cytosolic gIVaPLA2 to the nuclei was analyzed by confocal microscopy (x100 original magnification) as described in the Materials and Methods section. Control cells (A and G) indicate no gIVaPLA2 mAb staining; unstimulated cells (B and H) indicate baseline gIVaPLA2 stained with gIVaPLA2 mAb. Results are representative of cytostained cells from 4 experiments using 4 different donors.


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DISCUSSION
 
The objective of this investigation was to determine whether corticosteroid treatment with FP was efficacious in blocking cell adhesion of human PMNs to its counterligand in vitro. We found that even at high concentrations, FP was substantially less effective in blocking adhesion of β2-integrin in PMNs than for eosinophils isolated from the same donors. Subsequent studies demonstrated some potential mechanisms in cell signaling accounting for these differences.

There are at least 4 essential steps in β2-integrin adhesion of granulocytes: 1) up-regulation of surface CD11b/CD18 expression [20 , 24 , 25 ]; 2) increase conformational change (affinity) of CD11b/CD18 [26 ]; 3) intracellular translocation of cytosolic gIVaPLA2 to the perinuclear membrane [14 , 21 ]; and 4) focal clustering (avidity) of β2-integrin [24 , 26 ].

Prior studies have demonstrated that intracellular CD11b is comparably up-regulated in activated eosinophils and PMNs [20 , 22 , 24 , 25 ] by cell-specific cytokines, chemokines, or receptor-specific lipid mediators [20 , 22 , 24 , 25 ]. In these studies, comparable stimulated expression of β2-integrin caused a comparable degree of adhesion in both eosinophils and PMNs (Fig. 3) . In prior investigations, we have demonstrated two unique mechanisms by which glucocorticoids block β2-integrin adhesion in eosinophils [14 , 21 ]: 1) selective blockade of intracellular translocation of cytosolic gIVaPLA2 to the nucleus [14 , 21 ]; and 2) subsequent translocation of newly synthesized ANXA1 to the outer plasma membrane of eosinophils [14 ].

We previously have shown that IL-5-induced adhesion was blocked fully by 10–7 M FP [14 ], whereas, significant blockade of LTB4-or TNF-{alpha}-induced adhesion was not affected by FP in PMNs extracted from the same donors (Fig. 3) . While 10–8 M FP significantly blocked eosinophil β2-integrin adhesion caused by eotaxin-1 [14 ], 10–6 M FP was required to attenuate even minimally the LTB4-induced adhesion in PMNs (Fig. 3A) . Low concentrations of FP blocked completely the intracellular translocation of cytosolic gIVaPLA2 to the nucleus caused by IL-5 in eosinophils but were substantially less effective (~2 log) in blocking the transport of cytosolic gIVaPLA2 to the nuclear membrane of PMNs in response to LTB4 and TNF-{alpha} (Fig. 7 ).

As for eosinophils [14 , 21 ], treatment of PMNs with FP, even at high concentrations, had no effect on phosphorylation of ERK-1/2 (Fig. 4A) or subsequent phosphorylation of the 85-kDa cytosolic gIVaPLA2 (Fig. 4B) , which is a requisite step in β2-integrin adhesion [22 ]. Previous investigations have demonstrated that ANXA1 blocks eosinophil and PMN migration to the site of inflammation [27 , 28 ]. We have shown that up-regulation of ANXA1 expression caused by FP blocks eosinophil synthesis of cysteinyl LTC4 [13 ], affinity of β2-integrin to ICAM-1 [14 , 21 ], and translocation of cytosolic gIVaPLA2 to the perinuclear membrane [14 , 21 ]. Subsequent data obtained through fluorescence-activated cell sorting confirmed that FP is notably more potent and efficacious in augmenting the membrane-bound ANXA1 expression in eosinophils than in PMNs (Fig. 5) . However, the mechanisms by which FP down-regulates β2-integrin adhesion through increase in ANXA1 synthesis and blockade of gIVaPLA2 activity in both eosinophils and PMNs remain to be elucidated. In the present study, refractoriness to translocation to the cell membrane of ANXA1 appears to account, in part, for decreased blockade of PMN adhesion (vs. eosinophils) after corticosteroid treatment. However, even with comparable concentrations of exogenous ANXA1 fragment, blockade of PMN adhesion is still less substantial than for eosinophils from the same donors treated with the same concentrations (Fig. 5) . Thus, PMNs may be intrinsically less susceptible by other unidentified mechanisms to ANXA1 regulation in the blockade of β2-integrin adhesion.

It is important to note some limitations of our findings. These studies were performed under static conditions in vitro. To achieve the necessary stimulus isolation, we used isolated PMNs and eosinophils and evaluated solely the effect of FP only for β2-integrin mediated adhesion in both cells. While stimulus and cell-specific isolation are essential to assess signal transduction mechanisms in a specific cell type, these data do not necessarily reflect the more complex environmental conditions that exist in vivo. Nonetheless, the demonstration of relative refractoriness of PMNs vs. eosinophils to both ANXA1 synthesis and gIVaPLA2 activity may suggest a potential mechanism for the substantially greater efficacy of inhaled corticosteroids in treating eosinophils-mediated diseases of the lung compared to the inflammatory process mediated predominantly by PMNs.

We conclude that in activated eosinophils, FP is more potent and efficacious in 1) blocking β2-integrin adhesion; 2) up-regulating ANXA1 expression; and 3) inhibiting the transport of cytosolic gIVaPLA2 to the nucleus compared with PMNs. Our findings thus suggest some potential mechanisms for the relative lesser efficacy of corticosteroids in blocking some forms of neutrophilic inflammation in the lung than for inflammatory diseases involving eosinophils.


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ACKNOWLEDGEMENTS
 
This work was supported by GlaxoSmithKline (GSK, USA) research grant (to A. R. L.), GlaxoSmithKline (GSK; UK) Center of Excellence in Asthma (to A. R. L.), National Heart Lung and Blood Institute grants HL-46368 (to A. R. L.) and HL-85779 (to A. R. L.). We thank Anissa Lambertino, Evan Boetticher, and David Binder for their technical assistance.

Received July 26, 2007; revised September 12, 2007; accepted October 4, 2007.


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A. Y. Meliton, N. M. Munoz, C. M. Osan, L. N. Meliton, and A. R. Leff
Leukotriene D4 activates {beta}2-integrin adhesion in human polymorphonuclear leukocytes
Eur. Respir. J., February 1, 2010; 35(2): 402 - 409.
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