The FASEB Journal express article10.1096/fj.02-0941fje. Published online June 17, 2003.

A novel calcium-dependent proapoptotic effect of annexin 1 on human neutrophils Egle Solito,* Ahmad Kamal,† Francoise Russo-Marie,‡ Julia C. Buckingham,* Stefano Marullo,‡ and Mauro Perretti†

*Department of Neuroendocrinology, Imperial College London, Hammersmith Campus, Commonwealth Building, Du Cane Road London W12 ONN, London UK; †The William Harvey Research Institute, Bart’s and The London, Queen Mary SMD, Charterhouse Square, London EC1M 6BQ, UK; ‡Department of Cell Biology, Institut Cochin de Génétique Moléculaire, 75014 Paris, France

Corresponding author: Egle Solito, Department of Neuroendocrinology, Imperial College London, Hammersmith Campus, Commonwealth Building, Du Cane Road London W12 ONN, London UK. E.mail: e.solito@ic.ac.uk

ABSTRACT

The glucocorticoid-inducible protein annexin (ANXA) 1 is an anti-inflammatory mediator that down-regulates the host response. Endogenously, ANXA1 is released in large amounts from adherent polymorphonuclear neutrophils (PMN) and binds to their cell surface to inhibit their extravasation into inflamed tissues. The present study determined the effects of exogenous ANXA1 on several functions of human PMN in vitro. Addition of 0.1–1 µM human recombinant ANXA1 to the PMN provoked rapid and transient changes in intracellular Ca2+ concentrations that were blocked by the Ca2+ channel inhibitor SKF-96365. Although ANXA1 did not affect oxidant production and only minimally affected PMN chemotactic properties, the ANXA1-promoted Ca2+ influx was associated with two important functional effects: shedding of L-selectin and acceleration of PMN apoptosis. The latter effect was confirmed using three distinct technical procedures, namely, cell cycle, Hoechst staining, and ANXA5 binding assay. ANXA1-induced PMN apoptosis was insensitive to inhibitors of L-selectin shedding, whereas it appeared to be associated with dephosphorylation of the proapoptotic intracellular mediator BAD. In conclusion, exogenous ANXA1 displayed selective actions on human PMN. We propose that the new proapoptotic effect reported here may be part of the spectrum of ANXA1-mediated events involved in the resolution of acute inflammation.

Key words: inflammation • calcium channel • fMLP • chemotaxis • L-selectin

nnexins are a family of ubiquitous calcium and phospholipid binding proteins that are present in all eukaryotes examined to date except Saccharomyces cerevisiae. These proteins, of which 20 members are currently known, share a similar structure comprising

a core domain made of 70 amino acid repeats (usually 4 or 8 repeats) and a variable N-terminal domain. The core domain confers both calcium and phospholipid binding properties (1), whereas the N-terminal domain is believed to determine specific biological activities (1, 2).

A

Annexins are usually located intracellularly, either freely in the cytosol or associated with membranes or cytoskeleton in a calcium-dependent manner. However, annexins are also secreted and bound to the extracellular surface of the plasma membrane (3). Annexin 1 (ANXA1), a protein particularly abundant in monocytes (4) and polymorphonuclear leukocytes (PMN) (5), was originally identified as an endogenous mediator of the anti-inflammatory actions of glucocorticoids (6). Glucocorticoids stimulate expression and/or secretion of ANXA1 in vitro (7) and in vivo (8, 9). In experimental models of inflammation, administration of ANXA1 exerts potent inhibitory effects on the process of cell migration (10, 11). Accordingly, passive immunization against ANXA1 leads to a prolongation of the process of cell recruitment, indicating that the endogenous protein promotes resolution of inflammation (12).

The mechanisms proposed for the anti-inflammatory effects of ANXA1 are multiple. ANXA1 was first shown to inhibit the activity of phospholipase A2, the enzyme that initiates the biochemical cascade leading to eicosanoid’s synthesis (6, 13). A second mechanism by which ANXA1 interferes with the inflammatory process was identified in leukocytes and was correlated with their temporal and spatial activation. In resting PMN, most of the cytosolic ANXA1 is stored in gelatinase granules (14, 15). Once PMNs adhere to endothelial cells, an intense mobilization of ANXA1 on the cell surface occurs. The functional end point is a negative regulation of the PMN trans-endothelial passage (16). Two recent studies have provided new insight into the mechanism used by ANXA1 to block leukocyte extravasation. First, exogenous ANXA1 was reported to interact with the α-4 integrin at the surface of myelocytic cells, inhibiting its interaction with the endothelial integrin counter-receptor (17). Second, ANXA1 was shown to induce L-selectin shedding from the cell surface of human PMNs (18), preventing their transmigration through the endothelium.

An interesting twist in this line of research has been the proposition that ANXA1-derived N-terminal peptides, known to mimic some of the anti-inflammatory properties of the entire protein, altered PMN transmigration in vitro through mechanisms involving the receptor for formylated peptides (termed FPR) (19). In this study, putative FPR antagonists [butyloxycarbonyl-, or Boc for short, derivatives of formyl-Met-Leu-Phe (fMLP) (20) abrogated the effects of ANXA1-derived peptides on several assays of neutrophil activation (19). However, Strausbaugh and Rosen did not find an involvement of FPR in full-length ANXA1-induced L-selectin shedding from the cell surface of human PMNs (18). It is now clear that FPR is the prototype of a complex family of receptors known to bind several chemically unrelated molecules (21). The controversy that has evolved around the concept of ANXA1 acting at FPR is somehow justified by the fact that so-called Boc antagonists do not discriminate among the different members of the FPR family. More recently, we have provided evidence for an interaction between endogenous and exogenous full-length ANXA1 and ANXA1-derived peptides, with the lipoxin A4 receptor, a seven transmembrane G protein domain coupled receptor structurally related to FPR (22).

Irrespective of the receptors involved, not many studies have investigated the effect of ANXA1 on neutrophil functions in vitro. A peptide drawn from the ANXA1 N terminus was shown to inhibit neutrophil activation produced with interleukin-8 or platelet-activating factor (23), a feature also demonstrated for the full-length protein (24). More recently, the ability of ANXA1-derived peptides (19) and full-length ANXA1 (22) to cause transient changes in intracellular calcium has been reported. The present study investigated the effects of human recombinant

ANXA1 on several markers of in vitro PMN activation, with particular emphasis on a novel biological activity for this protein, that is, induction of a calcium-dependent early apoptotic signal. As apoptotic PMN are rapidly removed by phagocytosis (25), a novel model emerges where ANXA1 may limit PMN life span during inflammation.

MATERIALS AND METHODS

Materials

fMLP, bovine serum albumine (BSA), sodium tetraborate, ammonium formate, formic acid, boric acid, lithium chloride, fura 2/acetoxymethylester (Fura 2/AM), 1-[β-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-imidazole, HCl (SKF-96365, hydrocloride), 1,2-bis(o-aminophenoxy)ethan-N,N,N'N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA), and Hoechst H33342 were purchased from Sigma (St. Louis, MO). Boc1 (butyloxycarbonyl-MDLP) was from ICN-flow (Emerald Biostructure, Bainbridge Island, WA). Dextran was from Pharmacia Biotech AB (Uppsala, Sweden) and Lymphocytes Separation Medium was from Eurobio (Les Ullis, France). Dowex AG1-X8 ion-exchange resin (200–400 mesh, formate form) was from Bio-Rad (Hercules, CA), and myo(2-[3H])inositol (740 Gbq/mmol) was from ICN Pharmaceuticals (Costa Mesa, CA). Hepes and Hank’s balanced salt solution (HBSS) were from GIBCO (Life Science Cergy, Pontoise, France); the scintillation cocktail Hisafe3 was from Pharmacia LKB (Bois d’Arcy, France). RO31-9790 and RO-28-2653 were a generous gift from Dr. H.R. Krell (Roche Diagnostic GmbH Pharma Research, Penzberg Nonnenwald, Germany). Human recombinant ANXA1, ANXA5, and chimeric ANXA1-5 (containing the first 44 aa of ANXA1 attached to the 320-aa core of annexin 5) were produced in Escherichia coli and purified by FPLC as already reported (11). Endotoxin contamination was <20 pg/ml as measured by the Lymulus amebocyte chromogenic assay.

Human PMN isolation

PMNs were isolated from whole blood of healthy donors by a two-step procedure consisting of dextran T500 (Pharmacia) sedimentation (45 min at room temperature) followed by centrifugation at 1500 rpm for 20 min on Lymphoprep gradient (density 1.077 g/ml, Flobio, France). Cells were used immediately after isolation. An enriched population containing >95% PMN (red blood cell contamination was between 3 and 6% of the total cells) was routinely prepared (26).

Functional studies on isolated PMNs

Changes in the cytosolic-free calcium concentration were measured in PMN loaded with 1 mM Fura 2/AM at 37°C for 1 h (27). Fura 2 fluorescence assay was performed with aliquots of 5 × 106 PMN in 2 ml HBSS, using a fluorimeter (Jobin Yvon 3D, Lonjumeau, France) equipped with a thermally controlled cuvette holder and a magnetic stirrer. Excitation and emission wavelength for Fura 2 fluorescence assays were 340 and 510 nm, respectively. Cytosolic calcium concentrations were calculated as previously described (28). Tracings were reproduced and scanned using an Agfa Snap CAM with version F-3.0 color software (Apple Macintosh). Tracings are shown for one population of cells and are representative of at least three experiments.

PMN (5×107/ml in HBSS supplemented with 0.025% BSA) (26) were labeled with 100 µM of myo-(2-[3H])inositol/ml and incubated for 4 h at 37°C with gentle shaking. At the end of the incubation period, cells were washed three times and resuspended in phosphate-buffered saline (PBS) containing 20 mM Hepes, 0.025% BSA, and 10 mM LiCl (for inhibiting the enzymes of InsP-dephosphorylation). After washing, cells were pooled again, counted, and resuspended in 20 mM H-HBSS, supplemented with 0.01% BSA and 10 mM LiCl. Cells (107/600 µl) were aliquoted in Eppendorf and incubated for 10 min at 37°C in a water bath. The analysis of the inositol was performed as described previously (29). After centrifugation, the hydrosoluble perchloric supernatants were diluted and neutralized. The [3H]inositol-phospate was separated by anion-exchange chromatography on Dowex AG1-X8 column (30). Radioactivity was determined after adding 16 ml of scintillation cocktail to a 2-ml sample volume aliquot of each fraction.

Superoxide anion generation was measured by reduction of ferricytochrome C (horse heart type III) as previously described (31). PMN (106 cells/ml) and 75 µl ferricytochrome C (5 mg/ml) were incubated with or without recombinant ANXA1 for 10 min at 37°C. Incubation was stopped by placing the tubes in an ice-water bath after which they were centrifuged at 1200 rpm for 10 min. The absorbance of the supernatants was read at 550 nm in a spectrophotometer (DU 40 Beckmann, Gagny, France). The results were expressed in nmol of released O2

–/min/106 cells.

A modified version of the original Boyden chamber technique was used for determining PMN chemotaxis (32). In brief, 0.1 ml of PMN suspension (106 cells/ml) in HBSS supplemented with 1% bovine serum albumin (BSA) was added to the upper compartment of the Boyden chamber and 0.2 ml of chemotactic substances to the lower compartment. A cellulose filter (Millipore Rond-Point des Sangliers, Guyancourt, France) of pore diameter 3 µm was placed between the two compartments. Following 90 min at 37°C chemotaxis in response to fMLP with or without ANXA1 addition, filters were removed and stained with hematoxylin (Sigma, St Louis, MO). Cell migration was determined by measuring the distance of the leading front within the filter (33), using a light microscope and an oil immersion 100× objective. Five high-power fields were analyzed for each filter by an observer unaware of the different treatments.

A whole blood protocol was used to minimize the risk of PMN activation. Venous blood was collected in tubes containing anticoagulant from human volunteers. Blood aliquots (200 µl) were treated with different stimuli at 37°C for 15 min. At the end of incubation, blood aliquots were incubated for 10 min on ice, before erythrocyte lysis, which was produced by addition of an equal volume of Immuno-Lyse solution (Coulter, Miami FL). A monoclonal antibody against human L-selectin (CD62L; 20 µg/ml of clone MCA1076G, Serotec, Oxford, UK) was added for 1 h at 4°C, followed by a 30-min incubation with a FITC-labeled goat anti-mouse IgG (Serotec 1:200). After three washes, cells were analyzed by flow cytometry. Each leukocyte population was identified by its forward and side scatter profile and by using specific cell markers (CD14 for monocytes and CD16 for neutrophils). Fluorescence in the FL1 (green) channel was quantified as a measure of L-selectin expression vs. a background control obtained with cells stained with FITC-mouse IgG alone.

Studies of PMN apoptosis

Apoptosis was determined by staining DNA with propidium iodide (PI), using a procedure recently described in detail (34). In brief, resting or treated cells were washed twice with cold

PBS and fixed in cold 80% ethanol for 30 min. After they were washed in PBS, cells were incubated in a PI buffer [PBS pH 7.4, 0.1 mM ethylenediaminetetraacetatic acid (EDTA), 0.1% Triton X-100] containing 5% RNase T1, 1 mg/ml RNase A, and 100 µg/ml PI (final concentrations). Distribution of cell cycle phases with different DNA content was determined by flow cytometry. A pass filter of 585 nm was used to collect PI fluorescence, acquiring 10,000 events for each sample.

In some experiments, apoptosis was also measured by Hoechst nuclear staining (35). After appropriate treatments, cells were resuspended in 50 µl at a concentration of 1 × 106 cells/ml, and 5 µl of a solution of Hoechst H33342 (10 µg/ml) were added. Cells were then further incubated for 20 min at 37°C and subsequently fixed with 10 µl of cold formaldehyde (40% w:v). Cells were then analyzed by fluorescence microscopy with an Olympus BH-2-RFCA microscope. Apoptosis was assessed on the basis of chromatin structure and condensation. For each condition, at least 200 cells were counted.

Finally, to differentiate between early and late apoptosis, we quantified FITC-annexin 5 (FITC-ANXA5) binding as recently described (34). In brief, 106/ml PMN were washed in cold PBS and resuspended in 1× binding buffer in the presence of FITC-ANXA5 (5 µl; BD-Bioscience, Oxford, UK) + 10 µl of PI (50 µg/ml) and incubated at room temperature for 15 min in the dark. Analysis by flow cytometry was performed within 1 h using a FACScan II analyzer (Becton Dickinson, Cowley, Oxford, UK), with 15 mW argon ion laser (488 nm) and Consort 32 computer running Lysis II software. FITC-ANXA5 staining was detected in the FL1 (green) channel, whereas PI staining was monitored in the FL2 (red) channel. The following controls were used to set up compensation and quadrants: unstained cells and cells stained with FITC-ANXA5 or with PI alone. Other samples were also incubated with FITC-ANXA5 in buffer without calcium to control for nonspecific binding.

Analysis of BAD phosphorylation

PMN (107) were incubated at 37°C for 30 min with human recombinant ANXA1 (0.5 µM) in the presence or absence of SKF 96365 (25 µΜ). Cells were then lysed in 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100 containing aprotinin (0.15 U/ml), 20 mM leupeptin, and 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM NaF, and 1 mM NaVO4. Immunoprecipitation experiments were performed on cell lysates. In brief, 200 µl were incubated with rabbit polyclonal anti-BAD [directed against a synthethetic peptide (KLH conjugated) corresponding to Bad residues 112–130 (Cell Signaling, New England Biololabs, Beverly, MA); this antibody does not discriminate between phosphorylated and nonphosphorylated BAD protein], with gentle rocking overnight at 4°C. Protein A agarose beads were than added, and samples were further incubated for 3 h at 4°C. Samples were washed in lysis buffer 5 times, and pellets were resuspended in 3× SDS sample buffer and then applied on 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were first blocked with 3% milk powder for 1 h, followed by the incubation with the above anti-BAD polyclonal antibody (1:1000) or anti-serine (Clone 4A9; BIOMOL, Plymouth Meeting, PA) monoclonal antibody (0.5 µg/ml) overnight. The secondary antibody was an anti-rabbit or anti-mouse IgG-horseradish peroxidase conjugate (IgG-HRPO, Sigma) and was used at a final dilution of 1:2500.

Statistical analysis

Experiments were performed in duplicate and triplicate and repeated at least three times to obtain a mean ±SE. Differences between two experimental groups were analyzed by the Student’s t test, whereas comparisons among three or more groups were made by ANOVA followed, if significant, by Bonferroni post hoc test. P< 0.05 was considered significant.

RESULTS

ANXA1 produces Ca2+ fluxes in human PMN

Previous studies showed that exogenously added ANXA1 binds to the extracellular surface of human neutrophils (4). We used a preparation of a recombinant purified protein (ANXA1), produced in E. coli, shown to possess antimigratory activity in vivo (11), to study signaling and functional effects associated with ANXA1 binding to human PMN.

ANXA1 added to human PMN produced transient changes in intracellular Ca2+ concentrations ([Ca2+]i) (Fig. 1A, 1B). The traces appeared slightly slower in their kinetics and dome-shaped compared with those elicited by fMLP (10 nM; Fig. 1A). The effect of ANXA1 was concentration-dependent, detectable at 200 nM and maximal at 1 µM (Fig. 1B). The homologous protein ANXA5 produced a much weaker Ca2+ signal than ANXA1, whereas a chimeric that contained the N-terminal domain of ANXA1 and the core domain of ANXA5 promoted a strong Ca2+ signal, which was even larger in amplitude than that elicited by ANXA1 (Fig. 1C). These data do not support the hypothesis that the putative Ca2+ transport activity of the annexin core domain (36, 37) is involved in the Ca2+ signal observed with human PMN. Conversely, they suggest a specific role for the unique ANXA1 N terminus. Coincubation with the calcium-entry blocker SKF-96365 produced a concentration-dependent inhibition of ANXA1-induced [Ca2+]i fluxes (Fig. 1D, 1E). Although this compound produced total inhibition of the response elicited by ANXA1, it affected only the second phase of the response elicited by fMLP (Fig. 1D). In fact, PMN activation by fMLP has long been characterized by an initial increase in [Ca2+]i levels by opening intracellular stores, followed by a slow inward phase sustained by entry of the cation from the extracellular medium through voltage-dependent calcium channels present in the plasma membrane (38, 39).

Due to the recently proposed link between ANXA1 and FPR we compared, at least in part, the calcium signals by fMLP with the one produced by ANXA1. Figure 2 summarizes this set of data. Both fMLP- and ANXA1-induced [Ca2+]i responses were inhibited by the FPR antagonist Boc1 (Fig. 2A, 2B). Cross-desensitization analysis showed that fMLP desensitized ANXA1-induced [Ca2+]i response, whereas the opposite did not occur (Fig. 2C). This is identical to what we have recently reported for the ANXA1-derived N terminus peptide (22).

Therefore, the Ca2+ responses produced by fMLP and ANXA1 in human PMN are quite different despite being equally susceptible to the effect of the FPR antagonist Boc1. Based on these initial analyses, we selected a concentration of 500 nM ANXA1 for most of the subsequent experiments.

Other PMN responses

The diversity of effects between fMLP and ANXA1 was also evident with respect to classical markers of PMN activation. In contrast to fMLP, ANXA1 failed to activate InsP formation (Fig. 3A) and superoxide release (Fig. 3B). In these assays, addition of ANXA1 alone to cells did not produce any significant effect. Slightly different was the result obtained in the chemotaxis assay. ANXA1 did not inhibit the marked chemotactic effect of fMLP (Fig. 3C); however, at 250 nM, it produced by itself a modest yet significant chemotactic response.

Rapid cleavage of L-selectin from the cell surface is a well-established marker of PMN activation. Using a whole blood protocol, we found that human recombinant ANXA1, but not ANXA5, produced L-selectin shedding (Fig. 4A illustrates a representative experiment, with the histograms displaying the fluorescence measured in the FL1 channel). In analogy to that observed in terms of [Ca2+]I, the chimeric protein ANXA1/5 retained full activity. Similar data were obtained for the monocytes (data not shown). Cumulative data of this series of experiments is shown in Figure 4B.

Next we performed some mechanistic experiments using L-selectin expression as a read-out of PMN activation. First, we linked ANXA1-induced L-selectin shedding to the changes seen in [Ca2+]i (see Fig. 1 and 2). Addition of the calcium-entry blocker SKF-96365 abrogated ANXA1-induced L-selectin shedding (Fig. 5A), whereas this compound was essentially inactive on the fMLP response. Second, we used the FPR antagonist Boc1 (20 µM) that reverted the shedding produced by either fMLP or ANXA1 (Fig. 5B). With either stimulus, L-selectin shedding was genuinely provoked by the specific metalloprotease recently identified and characterized (40, 41). In fact, the selective inhibitor RO31-9790 used at a concentration of 50 µM, which retains selectivity (18), but not the unrelated compound RO28-2653, a gelatinase inhibitor, prevented fMLP- and ANXA1-induced L-selectin shedding (Fig. 5C).

ANXA1 induced human PMN apoptosis

Because changes in [Ca2+]i sometimes have been linked to induction of cell apoptosis, we next investigated whether the anti-inflammatory mediator ANXA1 would accelerate PMN apoptosis.

Human PMN incubation with ANXA1 (6 h) resulted in a concentration-dependent acceleration of PMN apoptosis as determined by FACS analysis, using the cell cycle/PI procedure. This incubation time was chosen in preliminary experiments and was found to be optimal, because longer incubations (e.g., overnight) resulted in a marked degree of spontaneous apoptosis (>50–60%) and were therefore more difficult to modulate. Figure 6A shows a representative FACS profile for PI staining in control and ANXA1-stimulated PMN. This novel property of ANXA1 was confirmed using another classical procedure to detect programmed cell death. Figure 6B shows representative pictures of nuclear fragmentation as visualized with the Hoechst staining. Finally, double staining with FITC-ANXA5 and PI also detected ANXA1-induced early (FITC-ANXA5+, PI– cells) and late (FITC-ANXA5+, PI+) apoptosis (Fig. 6C). Out of three experiments performed with different donors, PMN incubation with 500 nM ANXA1 produced a 6 h degree of apoptosis that ranged between 15% and 50% (always significantly higher than the one measured in untreated PMN, ranging from 3–5% to 25%, respectively).

Previous studies showed an increase in caspase-3 activity during constitutive apoptosis of aging PMN and apoptosis induced by anti-Fas antibodies (42). In contrast, apoptosis induced by the neutrophil activator PMA as well as tumor necrosis factor (TNF)-α is caspase-3 independent, indicating that this cell type possesses at least two different modes for entering into programmed cell death (42, 43). The same may apply to ANXA1, because caspase-3 activity after ANXA1 incubation (at 500 nM for 6 h) was not different from that measured in control cells (data not shown). Moreover, PMA- and ANXA1-mediated apoptosis were similar in their extent and only partially additive (Fig. 7A), suggesting the possible activation of a common pathway. In these experiments, the effect of fMLP was also tested, finding induction of apoptosis (Fig. 7A). However, more accurate analysis by the FITC-ANXA5 and PI double staining indicated that the fMLP effect was delayed compared with ANXA1 (data not shown). PMN apoptosis was not mimicked by ANXA5, whereas the chimeric protein ANXA1/5 produced again an effect that was qualitatively and quantitatively similar to that of ANXA1 (Fig. 7B).

Consistently, ANXA1-induced apoptosis was abrogated by the blocking channel SKF 96365 in a concentration-dependent manner (Fig. 8A). This blocker was ineffective on fMLP-induced PMN apoptosis (data not shown). Furthermore, the calcium-chelating agent BAPTA attenuated the effect of the protein, whereas the L-selectin sheddase inhibitor RO31-9790 was not active in this experimental context (Fig. 8B).

Finally, PMN incubation with ANXA1 produced dephosphorylation of Bad, a classical proapoptotic signal molecule (44). Figure 9A shows Bad immunoreactivity in PMN protein extracts immunoprecipitated with a polyclonal antibody against Bad and blotted against a serine antibody (Fig. 9A, top panel) or a polyclonal antibody against total Bad (Fig. 9A, bottom panel). Out of three experiments, an approximate 40% reduction in phospho-BAD was calculated following PMN incubation with ANXA1 (Fig. 9B). Again, these effects were susceptible to inhibition by the SKF-96365 compound (Fig. 9A, 9B).

DISCUSSION

The main result of the present study is the novel notion that ANXA1-induced activation of human PMN is associated with an accelerated degree of cell apoptosis. Addition of ANXA1 to these cells produced selective changes in specific markers of cell activation (e.g., [Ca2+]i and L-selectin shedding), and these appeared to be genuinely linked to the process of PMN apoptosis, which is spontaneously switched on following in vitro cell incubations. Finally, all the effects observed here are likely to be due to the specific N terminus region of the ANXA1 protein, as evinced by comparing the actions of the native protein to those of human recombinant ANXA5 and the chimeric protein ANXA1-5.

For many years, inhibition of phospholipase A2 was believed to be the principal mechanism for the anti-inflammatory properties of ANXA1 (6, 13). This mechanism of action was subsequently challenged by reports showing that ANXA1 and ANXA1-derived peptides inhibited PMN recruitment in animal models in which inhibitors of arachidonic acid catabolism were inactive (10, 45). Subsequent studies demonstrated that these pharmacological antimigratory actions also had pathophysiological relevance, because the endogenous protein displayed similar antimigratory effects (16, 46). The model proposed for the PMN was that upon cell adhesion on an endothelium, the protein would be exported from the cytosol onto the cell surface and at this

level it would operate through an unknown mechanism to down-regulate the extent of PMN emigration across the endothelium (47). In an in vivo context, exogenous and endogenous ANXA1 provoked detachment of adherent PMN (11, 46).

More recently, two membrane targets for ANXA1 have been identified: One is the fMLP receptor or FPR (19), and the other is the α4β1 integrin (17). It is possible that both molecular targets could be responsible for the antimigratory actions of ANXA1 discussed previously, although it is more likely that the interaction with α4β1 integrin is more relevant to cells of the mono-myelocytic lineage (17, 48). In addition, more recent data indicate that FPR might not be the sole receptor for ANXA1 and its peptides (22). The present study was planned to investigate ANXA1 effects that would be produced downstream from the interaction with its cell surface target(s). In this manner, we would fill a gap in the field of ANXA1 biology and PMN functions.

Historically, several members of the annexin family (ANXA1, 2, 5–7, and 12) have been reported to possess Ca2+ channel activities (49, 50). These activities were investigated only in in vitro settings and often with reconstituted systems and were possibly mediated by the large core common to these proteins. The core retains the highest homology among members of the annexin superfamily, and it is thought to be responsible for annexin ability to bind phospholipids in the presence of Ca2+ (1). In fact, the comparison of Ca2+ transport activities of several annexins was conducted using large unilamellar vesicles in which purified annexins were inserted. In these experiments, ANXA5 was reported to be a much better transporter than ANXA1 (threefold difference at any tested concentration) (51). In a more physiological context, ANXA5 was also shown to mediate peroxide-induced Ca2+ influx in B lymphocytes (52).

A recent study by Walther et al. (19) used ANXA1-derived peptides to mobilize Ca2+ in human PMN, using concentrations in the 10–20 µM range. The present study began by testing the effect of submicromolar concentrations of full-length ANXA1 added to human PMN in a test tube. ANXA1 produced transient changes in [Ca2+]i; this effect was concentration-dependent and required an intact ANXA1 N terminus, as demonstrated by the results produced with human recombinant ANXA5 and the chimeric protein ANXA1-5. ANXA1-induced [Ca2+]i flux was linked to a modest chemotactic effect and, more clearly, to shedding of L-selectin from the cell surface. The ANXA1 concentrations used in these in vitro experiments are lower than the maximal extracellular concentration reported so far for the protein (2 µM) (53). In addition, resting PMN have high intracellular levels of ANXA1 (∼3–4% of total cytosolic protein) (5), and the large majority of this (∼50–70%) is exported on the plasma membrane upon cell adhesion (16). Thus, it is likely that high local concentrations of the protein can be reached on the PMN cell surface within the microenvironment of an adherent PMN.

In view of the recent data linking ANXA1 to receptors of the FPR family, in most of the experimental settings we used fMLP as an internal control. Data obtained in the experiments of [Ca2+]i flux and second-messenger generation indicated a disparity of effects between the two PMN activators. Note that at variance from Walther et al. (19), Strausbaugh and Rosen (18) failed to demonstrate that ANXA1-induced L-selectin shedding was brought about by an interaction with FPR. This is in contrast to the data obtained in the present study, in which Boc1 blocked fMLP and ANXA1-induced L-selectin shedding. The reason for this discrepancy may lie in the enantiomer used, because only the D-Leu containing Boc derivatives act as antagonists at this receptor (54) as well as the lipoxin A4 receptor (22). ANXA1-induced [Ca2+]i flux was

blocked by the FPR antagonist Boc1 and the calcium-entry blocker SKF-96365. The same applied to ANXA1-induced L-selectin shedding, indicating a mechanistic link to receptor activation and transient changes in intracellular calcium.

PMN are terminally differentiated cells with a short life span once released from the bone marrow in the blood stream. In a matter of hours, they undergo programmed cell death and are then rapidly removed from circulation by the reticulo-endoplasmic system. PMN apoptosis may be either accelerated or delayed by multiple specific agents (reviewed in ref 55), and delayed PMN apoptosis is associated with several inflammatory diseases characterized by sustained neutrophil activation (56). Interestingly, a functional association between UV-induced PMN apoptosis and activation of the metalloprotease that mediates shedding of L-selectin has been proposed (57). This is possibly associated, at least in part, to the fact that engagement of PMN adhesion molecules had already been shown to modulate programmed cell death (58). For instance, cross-linking of CD11a or CD11b, but not CD18, with specific mAbs produced a delay in spontaneous PMN apoptosis (59). In contrast, cross-linking of L-selectin with its sulfatide ligands accelerated the degree of the apoptotic process (59). For these reasons, we tested the effect of exogenous ANXA1 on PMN apoptosis, reporting, for the first time, a promoting effect of the protein.

ANXA1-induced acceleration of PMN apoptosis occurred in a concentration-dependent manner, and it was demonstrated using three distinct detection systems. The concentrations required were in line with those necessary to activate the cell in terms of [Ca2+]i flux and L-selectin shedding. Conversely, inhibitors of [Ca2+]i flux prevented ANXA1-induced apoptosis. Many cell types have been shown to enter into apoptosis following a rise in intracellular Ca2+, for example, thymocytes and eosinophils (55). However, sustained elevation of [Ca2+]i with calcium ionophores has also been associated with a retardation in neutrophil apoptosis (60). It is plausible that different kinetics, amplitudes, or compartmentalization of the Ca2+ fluxes may generate opposite effects on PMN apoptosis. Alternatively, the proapoptotic effect of ANXA1 seen here may not depend on the intracellular Ca2+ rise per se but on another yet unidentified ANXA1-promoted signal, which is Ca2+-dependent.

This proapoptotic effect of ANXA1 resembles more that produced by TNF-β rather than fMLP. fMLP induces the process of apoptosis in a superoxide release-dependent pathway, whereas TNF-β-induced apoptosis appears to be unrelated to activation of the respiratory burst oxidase (61). ANXA1-induced PMN apoptosis was linked to Bad dephosphorylation. It is thought that following an increase in cytosolic Ca2+, the Ca2+-activated protein phosphatase calcineurin dephoshorylates Bad, which allows Bad to associate with mitochondria, heterodimerize with Bcl-XL, and promote apoptosis (62, 63). The endpoint would be accelerated apoptosis. Our data are in accord with this finding, and further studies will clarify this pathway in more detail.

The following scheme summarizes the findings reported here in the context of PMN migration in inflammation. Figure 10 shows the current working hypothesis. Several studies have shown that upon PMN adhesion ANXA1 moves to the cell surface compartment, and here it acts, probably through FPR or related receptors, to cause cell detachment, thereby reducing the extent of PMN extravazation. This has been demonstrated for both exogenous (11) and endogenous (46) ANXA1. This mechanism of action may well be responsible for the role that ANXA1 plays in the phase of resolution of inflammation. Accordingly, passive immunization of animals against

ANXA1 prolongs the extent of PMN recruitment into a specific tissue site (12). Here, we report that besides promoting controlled PMN activation (likely to be necessary to induce the cell detachment phenomenon as discussed previously), ANXA1 accelerates the induction of PMN apoptosis. This may impact on PMN biology and resolution of inflammation in two manners. First, ANXA1-detached PMN will enter into apoptosis and clear from the blood stream in a much faster and efficient manner. It is likely that previously adherent PMN may somehow be primed and at least in part activated, hence these cells could be of some danger; their quick removal by the nonphlogistic process of apoptosis (Fig. 10, option 1) is therefore more compatible with host survival. Second, ANXA1 may promote apoptosis of PMN that are already at the site of inflammation (Fig. 10, option 2). We now know that once extravazated, PMN switch on ANXA1 synthesis (15), and that higher intracellular ANXA1 levels have been associated with a degree of susceptibility to apoptosis (34). In addition, and more relevant to the data here, produced with exogenously applied ANXA1, extracellular ANXA1 is present at the site of inflammation, and its expression is associated with PMN influx (15, 64–66). More recent kinetics studies indicate the presence of higher ANXA1 levels in inflammatory exudates in concomitance with the phase of resolution, that is, the lowering of PMN numbers after the peak of cell influx (22).

Glucocorticoids are known to retard neutrophil apoptosis (67, 68). As ANXA1 is a glucocorticoid-regulated protein, it is difficult to reconciliate these findings with the data presented here. It is tempting to propose that glucocorticoid regulation of PMN ANXA1 may be more relevant in the context of cell adhesion and detachment (46), whereas exudates expression of the protein could be mostly due to the cell exudation process, and less susceptible to glucocorticoid regulation. For instance, extracellular ANXA1 can have different effects from the intracellular protein. Studies with cells expressing different ANXA1 levels, and their potential susceptibility to glucocorticoid-mediated regulation of apoptosis, will help clarify this apparent contrasting point.

ACKNOWLEDGMENTS

We thank Dr. M. Tissot for the calcium analysis and Mrs. S. Wheller for technical support. This work was sponsored by the CNRS and the Wellcome Program Grant 051887/Z/97/A. M.P. is a senior research fellow of the Arthritis Research Campaign UK (grant P0583). We thank Dr. H.R. Krell of Roche Diagnostic GmbH Pharma Research Penzberg Nonnenwald (Germany) for the generous gift of compounds RO31-9790 and RO28-2653.

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Received December 12, 2002; accepted May 2, 2003.

Fig. 1

Figure 1. ANXA1 activates transient [Ca2+]i fluxes in human PMN. A) PMN were loaded with Fura-2 and stimulated with the indicated compounds, and the resulting [Ca2+]i-dependent fluorescence changes were recorded. Shown is a comparison of the Ca2+ signals promoted by 10 nM fMLP and 500 nM ANXA1. B) Concentration-related response for exogenous ANXA1 on [Ca2+]i. C) Comparison of the Ca2+ signals promoted by ANXA1, annexin 5 (ANXA5), and chimera ANXA1-5 (all tested at 500 nM). D) Signals recorded in the presence of 50 µM SKF-96365 (SKF). E) Concentration-related response for the Ca2+ influx blocker SKF-96365 on the ANXA1 response. A, D) Recordings are from single donor samples and are representative of results obtained from 3 to 10 different donors. B, E) *P<0.05 vs. unstimulated cells (n=3 experiments).

Fig. 2

Figure 2. ANXA1-induced [Ca2+]i in PMN is blocked by Boc1 and SKF-96365. A, B) PMN were loaded with Fura-2 and [Ca2+]i signals promoted by fMLP (100 nM) or ANXA1 (500 nM). Recordings in the absence or presence of Boc1 (20 µM) are shown. C) Cross-desensitization between fMLP (100 nM) and ANXA1 (500 nM). In all cases, experiments correspond to recordings on single donor samples and are representative of results obtained from ≥3 different donors.

Fig. 3

Figure 3. Comparison of PMN responses elicited by fMLP and ANXA1. A) PMN were labeled with [3H]-myo-inositol and incubated at 37°C with 100 nM fMLP or 500 nM ANXA1 for various times (30 s and 1 min for fMLP; 30 s and 1, 5, and 10 min for ANXA1). In some experiments, PMN were preincubated with ANXA1 for 10 min before addition of fMLP. Inositol phosphate analysis was performed as indicated in Materials and Methods. Results, corresponding to the maximal observed stimulation (30 s for fMLP, 5 min for ANXA1), are the mean ±SE of triplicates in one experiment and are representative of three independent experiments; *P<0.05 vs. control inositol species generation, as calculated on original data. B) Superoxide anion production by PMN incubated for 10 min at 37°C with 100 nM fMLP and/or the indicated concentrations of ANXA1, as measured by the reduction of ferricytochrome C. Histograms are the mean of triplicates and are representative of three independent experiments. C) PMN chemotaxis promoted by fMLP and ANXA1. Cells were added to the upper compartment of Boyden chambers with 10 nM fMLP alone or with the indicated concentrations of ANXA1 being added to the lower compartment. Chambers were incubated for 90 min at 37°C. Cell migration was determined by measuring the distance of the leading front (see Materials and Methods), and specific migration was calculated by subtracting the distance of the leading front measured in control chambers in the absence of chemoattractant. Data are the mean ±SE of three independent experiments performed in triplicate. ANOVA test, *P<0.05 vs. unstimulated cells.

Fig. 4

Figure 4. ANXA1 induces L-selectin shedding from human PMN cell surface. A) Single donor FACS profile comparing ANXA1, ANXA5, and the chimera ANXA1-5 (0.7 µM in all cases), with the response produced by 10 nM fMLP on L-selectin PMN surface expression. B) Cumulative data performed with three different donors in triplicate. ANOVA test, *P<0.05 vs. relative basal L-selectin expression (B group).

Fig. 5

Figure 5. Effect of SKF-96365, Boc1, and RO31-9790 on ANXA1-induced L-selectin shedding. Whole blood aliquots were pretreated with SKF-96365 (25 µM; A), Boc1 (20 µM; B), RO31-9790 or RO28-2653 (50 µM; C), or vehicle for 3 min followed by fMLP (10 nM) or ANXA1 (0.7 µM) for 30 min at 37°C. PMN surface L-selectin levels were measured by flow cytometry. Data represent the means ± SE of three experiments (three different donors) performed in triplicate. ANOVA test, #P< 0.05 vs. corresponding vehicle group, *P<0.05 vs. relative basal L-selectin expression (85±15 MFI units, C).

Fig. 6

Figure 6. ANXA1 induces apoptosis in human neutrophils. Human PMN were incubated with or without 500 nM ANXA1 for 6 h at 37°C. The degree of cell apoptosis was measured in three distinct manners. A) Representative FACS analysis of control and ANXA1-treated PMN obtained with PI DNA staining. B) Representative Hoechst immunostaining of the previous donor. C) Dot plots showing ANXA5 (FL1 channel) and PI (FL2 channel) staining. Data shown in the three panels have been repeated at least with 8–10 different donors.

Fig. 7

Figure 7. Comparative effect on neutrophils apoptosis induced by ANXA1, ANXA5, and chimeric ANXA1-5. A) Effect of 1 nM PMA, 10 nM fMLP, or 500 nM ANXA1 on PMN apoptosis followed by 6 h incubation as determined with the PI cell cycle technique. B) As in A, but after cell incubation with 500 nM of each recombinant annexin. ANOVA test, *P<0.05 from untreated PMN (basal). #P<0.05 from ANXA1-treated PMN. Data are mean ±SE of five experiments.

Fig. 8

Figure 8. Calcium blockers inhibit ANXA1-induced apoptosis in PMN. Human PMN were incubated with or without ANXA1 and the reported inhibitors/antagonists for 6 h at 37°C. A) Inhibition of ANXA1 concentration-response curve for PMN apoptosis by the Ca2+ influx blocker SKF-96365 (SKF; 25 µM). B) ANXA1-promoted apoptosis was reverted by the Ca2+ calcium chelator BAPTA (3 µM) but not by the L-selectin shedding inhibitors RO31-9790 (RO; 50 µM). PMN apoptosis was determined with ANXA5/PI staining; ANOVA test, *P<0.005 from ANXA1-treated PMN. Data are mean ±SE of three experiments.

Fig. 9

Figure 9. ANXA1 induces BAD dephosphorylation. Detection of BAD and phospho-BAD in human neutrophils following a 30-min treatment with human recombinant ANXA1 (500 nM) in the presence or absence of SKF-96365 (25 µM). A) Upper panel: phospho-BAD immunoreactivity as detected with an antibody to phospho-serine; lower panel: total BAD immunoreactivity. Western blot profiles are from a single donor. C) Histogram (% of phosphorylated BAD) was obtained as mean ±SE from three donors. ANOVA test, P<0.05 vs. untreated PMN.

Fig. 10

Figure 10. Hypothesis for ANXA1-induced PMN apoptosis in the context of an inflammatory scenario. There are at least two options by which ANXA1 can affect PMN during inflammation. First, ANXA1 can cause detachment of adherent PMN, likely through a process of controlled activation. It is possible that the “detached” portion of PMN will enter into apoptosis and clear from the blood stream in a much faster and efficient manner. As adherent cells are also activated, this mechanism is compatible with host survival. Second, ANXA1 may promote apoptosis of PMN that are already at the site of inflammation, expressed as a dotted oval area. Many studies have shown ANXA1 expression in inflammatory exudates; therefore, it is possible that the proapoptotic effect of the protein may be relevant to shortening the life expectancy of extravasated cells (which otherwise is much longer in respect to PMN circulating in the blood). In either case, ANXA1-induced PMN apoptosis will promote resolution of inflammation.