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A novel calcium-dependent proapoptotic effect of annexin 1 on human neutrophils ¹

EGLE SOLITO^*,2, AHMAD KAMAL^#, FRANCOISE RUSSO-MARIE, JULIA C. BUCKINGHAM^*, STEFANO MARULLO and MAURO PERRETTI^#

^* Department of Neuroendocrinology, Imperial College London, Hammersmith Campus, London W12 ONN, London, UK; The William Harvey Research Institute, Bart’s and The London, Queen Mary SMD, London EC1M 6BQ, UK; and
Department of Cell Biology, Institut Cochin de Génétique Moléculaire, 75014 Paris, France

²Correspondence: Department of Neuroendocrinology, Imperial College London, Hammersmith Campus, Commonwealth Building, Du Cane Rd., London W12 ONN, London, UK. E-mail: e.solito{at}ic.ac.uk

SPECIFIC AIMS

The glucocorticoid-regulated protein annexin 1 (ANXA1) has been shown to inhibit the process of neutrophil (PMN) extravasation in several experimental settings. These anti-inflammatory effects are likely to be brought about by a direct interaction with specific receptors on the neutrophil cell surface. Except for receptor interaction, not many studies have investigated the effect of ANXA1 on neutrophil functions in vitro. 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, induction of a calcium-dependent early apoptotic signal. As apoptotic PMN are rapidly removed by phagocytosis, a novel model emerges where ANXA1 may limit PMN life span during inflammation.

PRINCIPAL FINDINGS

1. Analysis of ANXA1 effects on several parameters of PMN activation
Although it is clear that exogenous ANXA1 binds to human PMN, little is known about downstream events that this interaction can elicit. Here we show that once added to purified PMN, ANXA1 activated Ca²⁺ fluxes in a concentration-dependent manner. Signals were slower and dome-shaped compared with those elicited by the chemoattractant formyl-Met-Leu-Phe (fMLP). The structural related protein ANXA5 promoted weaker Ca²⁺ signals than ANXA1 on human PMN, whereas a chimera comprising the amino-terminal domain of ANXA1 and the core domain of ANXA5 promoted a strong Ca²⁺ signal even larger in amplitude than that elicited by native ANXA1. These data do not support the hypothesis that Ca²⁺ transport activity resides in the ANXA1 core domain, but rather suggest an effect mediated by the amino-terminal domain of the protein. Whereas fMLP and ANXA1-induced [Ca²⁺]_i responses were equally inhibited by the receptor antagonist Boc1, the calcium entry blocker SKF-96365 (25 µM) produced total inhibition of the response produced by ANXA1. Other signal events were promoted by fMLP, but not ANXA1, including PMN intracellular accumulation of inositol phosphates, superoxide generation, and cell chemotaxis. ANXA1-induced [Ca²⁺]_i fluxes were associated with shedding of L-selectin from the PMN cell surface. The functional link between the two effects was confirmed by the lack of effect of ANXA5 (whereas the chimera was active) and by the fact that L-selectin shedding was blocked by the receptor antagonist Boc1 and the calcium entry blocker SKF-96365.

2. Annexin 1-induced PMN activation is associated with accelerated cell apoptosis
PMN incubation with ANXA1 (6 h) resulted in a concentration-dependent manner (12–500 nM) acceleration of cell apoptosis. ANXA1-induced PMN apoptosis was observed using three distinct protocols, as shown in Fig. 1 : propidium iodide cell cycle analysis (Fig. 1A ), Hoechst^TM staining of nuclei (Fig. 1B ), and labeling of exposed phosphatidylserine with FITC-ANXA5 and flow cytometry (to monitor early apoptotic events; Fig. 1C ). Whereas ANXA5 did not increase PMN apoptosis comparatively to untreated cells (as determined by the cell cycle test), the chimera annexin 1-5 accelerated PMN apoptosis to a similar extent as ANXA1. This observation supports the hypothesis that the amino-terminal domain of ANXA1 is indeed involved in this novel biological action of the parent protein. The parallelism with ANXA1-induced PMN activation described above was partial, though, since ANXA1-induced cell apoptosis was equally sensitive to the calcium entry blocker SKF-96365 (25 µM) and to BAPTA (3 µM) (Fig. 2 ). Finally, ANXA1-induced apoptosis was preceded by a reduced degree of phosphorylated BAD, a phenomenon likely linked to reduced cell apoptosis. PMN incubation with ANXA1 (30 min at 500 nM) reduced the extent of serine phosphorylated BAD, as determined by immunoprecipitation and Western blot analysis, without changing total BAD levels. In line with the effects described above, SKF-96365 (25 µM) also prevented this action of ANXA1, pointing to a functional link between ANXA1-induced [Ca²⁺]_i responses and PMN apoptosis.

View larger version (43K): [in this window] [in a new window]   Figure 1. 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 ways. A) Representative FACS analysis of control and ANXA1-treated PMN obtained with PI DNA staining. B) Representative HoechstTM immunostaining of the previous donor. C) Dot plots showing ANXA5 (FL1 channel) and PI (FL2 channel) staining. Data shown in 3 panels have been repeated with at least 10 different donors.

View larger version (14K): [in this window] [in a new window]   Figure 2. 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 RO28-2653 (50 µM). PMN apoptosis determined with the PI cell cycle technique; ANOVA test *P < 0.005 from ANXA1-treated PMN. Data are mean ± SE of experiments.

CONCLUSIONS AND SIGNIFICANCE

The novel observation of this study is that the anti-inflammatory protein ANXA1 activates a Ca²⁺ influx that either directly or indirectly accelerates PMN apoptosis. Exudate PMN have an threefold higher lifespan than blood PMN, which undergo programmed cell death in a matter of hours. As extravasated PMN may contribute to the tissue injury seen in chronic pathologies with an inflammatory etiology, we reasoned that part of the anti-inflammatory profile of ANXA1 could be linked to PMN apoptosis.

In human PMN, ANXA1 is mostly intracellular and is externalized by activated cells. Recent data proposed that once on the cell surface, ANXA1 can interact with the human receptor for lipoxin A₄, also termed ALXR or FPRL1 (for its analogy to the receptor for fMLP, or FPR). Direct interaction between ANXA1 and derived peptides with ALXR/FPRL1, but not FPR, has been reported. Nonetheless, the antagonist Boc1, which does not discriminate between FPR and ALXR/FPRL1, is known to block the rise in [Ca²⁺]_i recently reported solely for short ANXA1-derived peptides. The present study sought to analyze the effects of the full-length protein on downstream events that might be produced after interaction with PMN receptor or receptors. Cell incubation with ANXA1 produced selective cell activation with changes in [Ca²⁺]_i and L-selectin levels, whereas no effect was seen on inositol phosphate formation and superoxide generation. The specificity of these cellular responses was confirmed by the lack of effect of ANXA5, with the efficacy of the chimera ANXA1-5, as well as by the blockade produced by Boc1. As the rise in [Ca²⁺]_i has been reported to activate apoptotic pathways in other cell types, including thymocytes and eosinophils, we tested the effect of ANXA1 on PMN apoptosis. PMN incubation with ANXA1 promoted a high degree of apoptosis when tested at 6 h, as demonstrated using three distinct technical procedures. Similar to the more rapid responses (e.g., [Ca²⁺]_i and L-selectin shedding), ANXA1-induced PMN apoptosis was prevented by cell incubation with calcium entry blockers SKF-96365 and BAPTA. Finally, the chimera ANXA1-5, but not human recombinant ANXA5, mimicked the effect of ANXA1. In the final part of the study we focused on BAD, a member of the Bcl-2 family of proteins known to regulate cell apoptosis, whose phosphorylation status is controlled by calcium-dependent pathways. For instance, phosphorylated BAD heterodimerizes with anti-apoptotic proteins such as Bcl-2 and Bcl-x_L, promoting cell death. For these reasons, we checked whether ANXA1-induced PMN activation and apoptosis could modify BAD phosphorylation. Short PMN incubation with ANXA1 almost doubled the percentage of Bad that was not phosphorylated; this effect was again susceptible to inhibition by SKF-96365. Figure 3 shows our current working hypothesis. Upon PMN adhesion, ANXA1 moves to the cell surface compartment, where it acts to cause cell detachment, reducing the extent of PMN extravasation. This mechanism of action may well be responsible for the role that ANXA1 plays in the phase of resolution of inflammation. We report that besides promoting controlled PMN activation, ANXA1 accelerates the induction of PMN apoptosis. This may affect PMN biology and resolution of inflammation in two ways. First, ANXA1-detached PMN will enter into apoptosis and clear from the bloodstream in a much faster and more efficient manner (Fig. 3 , option 1). Second, ANXA1 may promote apoptosis of PMN already at the site of inflammation (Fig. 3 , option 2). We now know that, once extravasated, PMN switch on ANXA1 synthesis and that higher intracellular ANXA1 levels have been associated with a degree of susceptibility to apoptosis. In conclusion, the data presented and discussed here indicate that the anti-inflammatory mediator ANXA1 may have an important effect on the phase of resolution of inflammation: 1) ANXA1 can be a stop signal for cells that are beginning the process of extravasation; 2) ANXA1 can be a go signal for disruption and possibly removal of the cells that have already reached the inflammatory site.

View larger version (33K): [in this window] [in a new window]   Figure 3. Hypothesis for ANXA1-induced PMN apoptosis in the context of an inflammatory scenario. There are at least two options by which ANXA1-induced PMN apoptosis can affect the PMN. 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 the bloodstream in a much faster and more efficient manner (option 1). Second, ANXA1 may promote apoptosis of PMN that are already at the site of inflammation, expressed as a dotted oval area in option 2. In either case, ANXA1-induced PMN apoptosis will promote resolution of inflammation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0941fje; doi: 10.1096/fj.02-0941fje

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