Microvasculature Receptor and Operative in the Inflamed 4 Formyl

Evidence for an Anti-Inflammatory Loop Centered on Polymorphonuclear Leukocyte Formyl Peptide Receptor 2/Lipoxin A4 Receptor and Operative in the Inflamed This information is current as Microvasculature of September 28, 2021. Vincenzo Brancaleone, Jesmond Dalli, Stefania Bena, Roderick J. Flower, Giuseppe Cirino and Mauro Perretti J Immunol 2011; 186:4905-4914; Prepublished online 11 March 2011; Downloaded from doi: 10.4049/jimmunol.1003145 http://www.jimmunol.org/content/186/8/4905 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/03/11/jimmunol.100314 Material 5.DC1 References This article cites 70 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/186/8/4905.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Evidence for an Anti-Inﬂammatory Loop Centered on Polymorphonuclear Leukocyte Formyl Peptide Receptor 2/Lipoxin A4 Receptor and Operative in the Inﬂamed Microvasculature

Vincenzo Brancaleone,*,† Jesmond Dalli,* Stefania Bena,* Roderick J. Flower,* Giuseppe Cirino,† and Mauro Perretti*

The importance of proresolving mediators in the overall context of the resolution of acute inﬂammation is well recognized, although little is known about whether these anti-inﬂammatory and proresolving molecules act in concert. In this article, we focused on

lipoxin A4 (LXA4) and annexin A1 (AnxA1) because these two very different mediators converge on a single receptor, formyl Downloaded from peptide receptor type 2 (FPR2/ALX). Addition of LXA4 to human polymorphonuclear leukocytes (PMNs) provoked a concentration- and time-dependent mobilization of AnxA1 onto the plasma membrane, as determined by Western blotting and ﬂow cytometry analyses. This property was shared by another FPR2/ALX agonist, antiﬂammin-2, and partly by fMLF or peptide Ac2-26 (an AnxA1 derivative that can activate all three members of the human FPR family). An FPR2/ALX antagonist blocked

AnxA1 mobilization activated by LXA4 and antiﬂammin-2. Analysis of PMN degranulation patterns and phospho-AnxA1 status

suggested a model in which the two FPR2/ALX agonists mobilize the cytosolic (and not the granular) pool of AnxA1 through an http://www.jimmunol.org/ intermediate phosphorylation step. Intravital microscopy investigations of the inflamed mesenteric microvasculature of wild-type 2/2 and AnxA1 mice revealed that LXA4 provoked leukocyte detachment from the postcapillary venule endothelium in the former (>50% within 10 min; p , 0.05), but not the latter genotype (∼15%; NS). Furthermore, recruitment of Gr1+ cells into dorsal air- 2/2 pouches, inflamed with IL-1b, was significantly attenuated by LXA4 in wild-type, but not AnxA1 , mice. Collectively, these data prompt us to propose the existence of an endogenous network in anti-inflammation centered on PMN AnxA1 and activated by selective FPR2/ALX agonists. The Journal of Immunology, 2011, 186: 4905–4914. by guest on September 28, 2021 t is now appreciated that the process of acute inflammation Lipoxin A4 (LXA4) (2) and annexin A1 (AnxA1) are two ef- relies on the active involvement of a series of proresolving fectors of endogenous anti-inflammation (4) that are able to halt I mediators that ensure temporal and spatial containment of the leukocyte migration (5, 6) and promote macrophage phagocytosis host reaction (1). Therefore, a proinflammatory phase is followed by of infective agents as well as apoptotic leukocytes (7–9). In 2002, an anti-inflammatory and proresolving phase in line with “the we reported that this short-lived lipid, produced by transcellular beginning programs the end” concept recently put forward (2). synthesis and cooperation between lipoxygenases (10), and the However, whereas it is well accepted that proinflammatory media- glucocorticoid-regulated protein both converged on a specific re- tors (e.g., cytokines, chemokines, autacoids) often act in concert in ceptor target (11), the formyl peptide receptor type 2 [FPR2/ALX; a network-like fashion (3), the significance of such networks to currently used to identify the human receptor (12)]. inflammatory resolution has been poorly appreciated, and specific LXA4 and AnxA1 are very different not only chemically but mediators and pathways have often been investigated in isolation. also with respect to their biosynthesis (produced on demand or stored in cell sources) and metabolic fate; we questioned *William Harvey Research Institute, Barts and The London School of Medicine, whether a functional link existed between them that justified Queen Mary University of London, London EC1M 6BQ, United Kingdom; and a single receptor as the main transducer for their essentially † Dipartimento di Farmacologia Sperimentale, Universita´ degli studi di Napoli, similar proresolving properties. In addition, comparing the time 80129 Naples, Italy course of generation of these two mediators during acute in- Received for publication September 22, 2010. Accepted for publication February 13, 2011. flammation in mice and men reveals distinct windows for synthesis and secretion, with exudate LXA concentrations peaking This work was supported by the Wellcome Trust (Programme 086867/Z/08) and 4 forms part of the research themes contributing to the translational research portfolio at a much earlier time point than AnxA1 concentrations. In of Barts and the London Cardiovascular Biomedical Research Unit, which is sup- murine air-pouches, inflamed with the TLR2 agonist zymosan, ported and funded by the National Institutes of Health Research. LXA4 peaks at 4 h, whereas exudate AnxA1 expression is Address correspondence and reprint requests to Prof. Mauro Perretti, William Harvey highest at 24 h, coinciding with the peak of polymorphonuclear Research Institute, Barts and The London Medical School, Charterhouse Square, London EC1M 6BQ, United Kingdom. E-mail address: [email protected] leukocyte (PMN) influx (11). The same holds true in the zy- The online version of this article contains supplemental material. mosan peritonitis model (13, 14). Furthermore, 15-epi-lipoxin Abbreviations used in this article: AF2, antiflammin 2; AnxA1, annexin A1; CsH, induced by low-dose aspirin treatment inhibits leukocyte re- cyclosporin H; CTR, control; FPR2/ALX, formyl peptide receptor 2/lipoxin A4 recruitment in a human skin blister model of acute inflammation ceptor; LXA4, lipoxin A4; MFI, median fluorescence intensity; PMN, polymorpho- (15); in the same model, a delay in the onset of the inflammatory nuclear leukocyte; WRW , peptide WRWWWW. 4 response, seen in ∼40% of individuals, is associated with ab- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 normal production of 15-epi-lipoxin (16). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003145 4906 ANNEXIN A1 IS MOBILIZED BY FPR2/ALX AGONISTS

AnxA1 is very abundant in human PMNs, representing between lysis buffer was used, and the cells were sheared through a 25-gauge 2 and 4% of total intracellular proteins (17). In resting PMNs, needle 10 times. Lysates were subsequently centrifuged for 2 min at 3003 3 a proportion (∼60%) of intracellular AnxA1 is localized in gela- g, and supernatants centrifuged again for 45 min at 800 g. The resulting supernatant (cytosolic fraction) was collected and the remaining pellet tinase and azurophilic granules (18, 19), with the remaining being resuspended in 20 mM Tris-HCl, 1% Triton X-100, pH 7.5 (100 ml), for 15 cytosolic in location. PMN adhesion to endothelial cell monolayers min (membrane fraction). Membrane-bound AnxA1 was recovered by provokes a rapid mobilization of AnxA1 onto the cell surface washing pelleted cells twice with 50 ml 1 mM EDTA. (20), where this agonist could encounter its receptor to activate Western blot analysis juxtacrine and/or autocrine signals (6), thus curtailing the extent of neutrophil trafficking (21). Nonetheless, the cytosolic nongra- Samples boiled in 63 Laemmli buffer were subjected to standard SDS- nular pool of the protein can also be mobilized, as, for instance, PAGE (12%) and electrophoretically blotted onto polyvinylidene difluor- ide membranes (PVDF; Millipore, Watford, U.K.). Membranes were in- in response to drug application (22–24), and the latter process cubated with the mouse mAbs anti-human AnxA1 [clone 1B; dilution, 27 entails a phosphorylation step at Ser (and possibly other resi- 1:1,000 (37)] and anti-human actin (1:10,000; Sigma-Aldrich) in TBST dues), which seems to be a prerequisite for secretion of the protein and 5% (w/v) nonfat dry milk overnight at 4˚C. Membranes were washed onto the cell surface (25). for 30 min with TBST, with the solution being changed at 10-min inter- Antiflammin 2 (AF2) is a nonapeptide corresponding to region vals; membranes were then incubated with secondary Ab (HRP-conjugated goat anti-mouse 1:5000; Dako, Cambridge, U.K.), for 2 h at room tem- 246–254 of AnxA1 (26). In experimental models of acute in- perature. Phospho-Ser27-AnxA1 was detected with rabbit Ser27 phospho- flammation, AF2 produces inhibitory properties that, at variance specific Ab (a kind gift from Dr. Egle Solito [Imperial College London, from initial observations (27), are not associated with inhibition of London, U.K.], 1:1000 in TBST, 1% BSA solution (23, 25). Proteins were phospholipase A but rather with an interference with PMN ac- then detected using the ECL Detection Kit and visualized on Hyperfilm Downloaded from 2 (Amersham Biosciences, Amersham, U.K.). tivation, chemotaxis, and adhesion to endothelial cells (28, 29). In the AnxA1 protein structure, residues 246–254 are exposed on the FACS analysis outer side of the protein (30) and are hence available for interac- Granule markers and FPR receptors. PMNs were treated with the different tion, in conjunction with the N-terminal region (31), with molec- FPR agonists, as described above, and incubated with mAbs against the ular targets. Congruent with this model, we found that AF2 binds different granule/vesicle markers CD35, CD66b, and CD63 (all FITC- conjugated mAbs, 1:10 final dilution; Serotec, Abingdon, U.K.). CD35 to FPR2/ALX, eliciting genuine postreceptor signaling responses http://www.jimmunol.org/ and inhibiting PMN interaction with endothelial monolayer un- recognizes secretory vesicles, CD66b is a marker for specific granules, and CD63 is a specific marker for azurophilic granules. These intracellular derflow (32). organelles are mobilized in a sequential manner by activated PMNs (38, 39). The present study was undertaken to determine whether LXA4 FPR1 and FPR2/ALX expression. FPR1 and FPR2/ALX cell surface ex- and AF2 might affect AnxA1 localization in human PMNs. The pression was measured by incubation with anti-FPR1 or anti-FPR2 mAb data obtained indicate the existence of an anti-inflammatory net- (1:10 dilution in both cases; from R&D Systems, Abingdon, U.K., and work centered on FPR2/ALX that operates to regulate PMN ac- Genovac, Brussels, Belgium, respectively); a final staining with a rabbit anti-mouse IgG (1:200 dilution, clone STAR9B; Serotec) was then con- tivation and trafficking in the microcirculation. ducted. With flow cytometry, $10,000 events were analyzed using a FACSCa- libur flow cytometer (Becton Dickinson, San Jose, CA) with CellQuest by guest on September 28, 2021 Materials and Methods software (Becton Dickinson). Results are reported as median fluorescence Human PMN isolation intensity (MFI) units. Peripheral blood was collected from healthy volunteers by i.v. withdrawal in In vivo analyses in AnxA1-deficient mice 3.2% sodium citrate solution (1:10). PMNs were isolated from blood by density centrifugation on Histopaque 1119/1077 (Sigma-Aldrich, Poole, AnxA1-deficient (AnxA12/2) (40) and wild-type C57BL/6J (AnxA1+/+) U.K.) gradient according to the manufacturer’s instructions and suspended mice were obtained from B&K Limited (Hull, U.K.). Male age-matched 5- in PBS containing 0.5% BSA. Contaminating erythrocytes were removed to 8-wk-old mice were used for all experiments, approved and performed by lysis with cold MilliQ water. All healthy volunteers gave oral and written under the guidelines of the Ethical Committee for the Use of Animals, consent, and cell separation was covered by ethical approval 05/Q0603/34 Barts and The London School of Medicine and Home Office regulations (East London and The City Research Ethics Committee 1). (Scientific Procedures Act, 1986). PMN recruitment of postcapillary venules: the detachment effect. Intravital PMN incubation with FPR agonists microscopy was performed as previously reported (41, 42). Briefly, mice were anesthetized, and the left jugular vein was cannulated with poly- To monitor AnxA1 mobilization, peptide Ac2-26 (Ac-AMVSEFLKQAW- FIENEEQEYVQTVK; Tocris, Bristol, U.K.), AF2 (HDMNKVLDL; Tocris, ethylene tubing (PE-10; internal diameter, 0.28 mm). A cautery incision was made along the abdominal region, and the superior mesenteric artery Bristol, U.K.), LXA4 (EMD Chemicals, Darmstadt, Germany), or fMLF (Sigma-Aldrich, Dorset, U.K.) was added to freshly prepared PMNs (4 3 106 was exposed and clamped with a microaneurysm clip to induce ischemia in the mesentery for 30 min, followed by a 45-min reperfusion phase. Toward cells/well) for 10–60 min; incubations were then stopped by rapid centri- ∼ fugation at 4˚C and freezing. In some experiments, PMNs were stimulated the end of the reperfusion period (i.e., after 35 min), the mesenteric vascular bed was exteriorized, placed on a viewing Plexiglas stage, and with selected agonists in the presence of the FPR1 antagonist cyclosporin 2 m mounted on a Zeiss Axioskop FS with a water-immersion objective lens H (CsH, 10 min; Biomol, Hamburg, Germany), which was used at 10 M, 3 as recently reported (33, 34). In other cases, the FPR2/ALX antagonist (magnification 40; Carl Zeiss, Welwyn Garden City, U.K.) and an eye- piece (magnification 310; Carl Zeiss). Tissue preparations were trans- WRWWWW (35) (WRW4;10mM, 210 min; EMD Chemicals) was used. Altogether, these experiments aimed at investigating the involvement of illuminated with a 12-V, 100-W halogen light source. A Hitachi charge- FPR1 and FPR2/ALX in PMN AnxA1 mobilization after application of the coupled device color camera (model KPC571; Hitachi, Tokyo, Japan) four agonists used in the current study. Finally, to measure rapid formation acquired images that were displayed on a Sony Trinitron color video of phospho-Ser27-AnxA1, PMNs were incubated with selected agonists for monitor (model PVM 1440QM; Sony, Tokyo, Japan) and recorded on 10 min. In all cases, cell-free supernatants were collected, with cells being a Sony super-VHS videocassette recorder (model SVO-9500 MDP) for processed for membrane and cytosolic fractions. AnxA1 localization and subsequent offline analysis. A video time-date generator (FOR.A video expression were monitored by Western blotting and FACS analysis (see timer, model VTG-33; JVC, Tokyo, Japan) projected the time, date, and below). stopwatch function onto the monitor. Mesenteries were superfused with thermostated (37˚C) bicarbonate-buffered solution (grams per liter: NaCl, Isolation of PMN membrane and cytosolic fractions 7.71; KCl, 0.25; MgSO4, 0.14; NaHCO3, 1.51; and CaCl2, 0.22; pH 7.4, gassed with 5% CO2/95% N2) at a rate of 2 ml/min. When a suitable After treatment, cells (4 3 106) were resuspended in lysis buffer (20 mM postcapillary venule was selected (diameter, 20–40 mm; with equivalent Tris-HCl, pH 7.5, with a protease and phosphatase inhibitor mixture; numbers of 5–8 adherent leukocytes per 100-mm vessel length), PBS (100 Sigma-Aldrich) and processed as previously shown (36). Briefly, 100 ml ml), LXA4, or AF2 was administered (1-mg dose) via the jugular vein, and The Journal of Immunology 4907 Downloaded from http://www.jimmunol.org/

FIGURE 1. AnxA1 disposition in human PMNs upon agonist stimulation. A, Representative blots showing AnxA1 localization in unstimulated and

LXA4-treated human PMNs; AnxA1, mainly present in cytosol (Cyt), is mobilized toward the membrane surface (Mem) upon stimulation with agonists (LXA4). Data are from three distinct PMN preparations. B, Human PMNs were treated with vehicle (control [CTR]), AF2 (10 mM), peptide Ac2-26 (10 mM), LXA4 (100 nM), or fMLF (1 mM) for 10–60 min; then expression of AnxA1 in cytosolic and membrane fractions was determined by Western blotting. Blots are shown as line graphs reporting expression versus time. Data, mean 6 SEM, are calculated as percentage of vehicle-treated cytosolic AnxA1 expression taken as 100. *p , 0.05, **p , 0.01, ***p , 0.001 versus CTR; n = 3. fMLP, formyl-methionyl-leucyl-phenylalanine. by guest on September 28, 2021 the fate of the adherent leukocytes was monitored for up to 10 min. Offline 0.5% carboxymethyl-cellulose, as described (43). Four hours later, air- analysis monitored i) the number of adherent cells that rejoined the pouches were lavaged and exudate fluids stained with Turk’s solution bloodstream and ii) the number of newly adherent leukocytes, as described for total cell counting in light microscopy. The extent of PMN influx was previously (41, 42). determined by FACS analysis using a FITC-conjugated mAb for Ly-6G/ PMN trafficking into inflamed air-pouches. Air-pouches were formed on the Gr1 (clone RB6-8C5; 10 mg/ml final concentration; eBioscience, San dorsal area of C57Bla6 mice by injection of 2.5 ml air on days 0 and 3. On Diego, CA) or isotype controls (eBR2a; eBioscience). FACS analysis was day 6, 10 ng mouse IL-1b (Peprotech, London, U.K.) was injected in 0.5 ml conducted as above, determining the percentage of Gr1-positive (Gr1+)

FIGURE 2. AnxA1 mobilization in human PMN: concentration-response studies. Treatment of human PMNs either with AF2

(1–10 mM) (A) or LXA4 (10–100 nM (B) for 30 min provoked a decrease in cytosolic AnxA1 levels and a parallel increase in AnxA1 membrane surface expression. Blots are representative of three different experiments. Data, mean 6 SEM, are calculated as percentage of vehicle-treated cytosolic AnxA1 expression taken as 100. *p , 0.05, **p , 0.01, ***p , 0.001 versus vehicle, CTR. 4908 ANNEXIN A1 IS MOBILIZED BY FPR2/ALX AGONISTS

FIGURE 3. WRW4 prevents LXA4- and AF2- elicited mobilization of AnxA1 in human PMNs.

Human PMNs were pretreated with WRW4 (10 mM, 10 min), prior to addition of vehicle (CTR),

Ac2-26 (10 mM), LXA4 (100 nM), or AF2 (10 mM) for an additional 30 min. Subcellular fractions were then processed and analyzed by Western blotting. A, Representative blots from three different experiments in the presence (+) or absence (2)ofWRW4. B–D, Densitometric analysis for AnxA1 expression in cytosol (B), membrane (C), and supernatant (D). Data, mean 6 SEM, are calculated as percentage of vehicle- treated cytosolic AnxA1 expression taken as 100. *p , 0.05, **p , 0.01, ***p , 0.001 x xx versus CTR (i.e., no WRW4), p , 0.05, p , xxx , 0.01, p 0.001 versus vehicle (i.e., in pres- Downloaded from ence of WRW4 only).

cells and calculating the total number of PMNs per air-pouch, taking into experiments showing a ratio of ∼80 6 12% intracellular AnxA1 http://www.jimmunol.org/ account total cell number. versus 20 6 12% membrane AnxA1 (10 experiments, performed Data analysis in triplicate). This finding is congruent with the predominant intracellular localization of the protein in resting PMNs (20). 6 Western blot data are reported as mean SEM, expressed as percentage of However, cell activation with soluble stimuli would mobilize the vehicle-treated cytosolic AnxA1 expression from at least three experiments performed in triplicate and plotted as an X(time)/Y(expression) protein onto the cell surface in an EDTA-labile pool, where partial graph. The n number of mice has been reported for in vivo experiments. cleavage would occur—hence the appearance of the doublet (36). Differences were evaluated using the Mann–Whitney U test for non- Incubation of PMNs alone over a 60-min time course provoked parametric data (in vitro analyses) or by ANOVA followed by Dunnett , only mild changes in AnxA1 expression in the absence of recep- posttest (in vivo analyses). In all cases, a p value 0.05 was taken as the m by guest on September 28, 2021 threshold for rejecting the null hypothesis. tor agonists. Cell treatment with peptide Ac2-26 (10 M), AF2 (10 mM), LXA4 (100 nM), or fMLF (1 mM) caused significant changes in AnxA1 intracellular location, although they occurred Results to a different extent and in a different pattern (Fig. 1B). Over the Agonist-dependent mobilization of intracellular AnxA1 in time course, LXA4 rapidly (10 min) reduced AnxA1 cytosolic con- human PMN tent, and this was mirrored by an increase in the membrane pool Resting PMNs contained large amounts of AnxA1, which was, at 30 min (Fig. 1A,1B). AF2 provoked qualitatively similar—yet for the most part, intracellularly located, with a small proportion in slower—effects, with significance being reached only at the 30-min the membrane pool. This arrangement can be seen in Fig. 1A and time point. Peptide Ac2-26 seemed to provoke changes similar to the following figures, with densitometric values of cumulative those of LXA4, but its effects were lost by 60 min, with the pool of

FIGURE 4. CsH affects fMLF-elicited mobilization of AnxA1 in human PMNs. Human PMNs were pretreated with CsH (10 mM, 10 min), prior to addition of vehicle (CTR), Ac2- 26 (10 mM), or fMLF (1 mM) for an additional 30 min. Subcellular fractions were then processed and analyzed by Western blotting. A, Representative blots from three different experiments in the presence (+) or absence (2) of CsH. B–C, Densitometric analysis for AnxA1 expression in cytosol (B) and membrane (C). Data, mean 6 SEM, are calculated as percentage of vehicle-treated cytosolic AnxA1 expression taken as 100. *p , 0.05, **p , 0.01, ***p , 0.001 versus CTR (i.e., no CsH), xp , 0.05, xxp , 0.01, xxxp , 0.001 versus vehicle (i.e., in presence of CsH). fMLP, formyl-methionyl-leucyl-phenylalanine. The Journal of Immunology 4909

Characterization of AF2- and LXA4-induced AnxA1 mobilization Incubation of human PMNs with AF2 (1–10 mM) provoked a reduction of cytosolic AnxA1 and a simultaneous increase in either membrane fraction and cell supernatant (Fig. 2A) AnxA1. This effect was evident at 1 mM AF2 and was robustly observed at concentrations of 3–10 mM. Treatment of cells with LXA4 (10– 100 nM) induced changes in AnxA1 location similar to those made by AF2; notably, these effects were concentration dependent and optimal at ∼10 nM (Fig. 2B). Pretreatment of human PMNs with the antagonist WRW4 (10 mM, 10-min preincubation) affected both LXA4- and AF2- induced AnxA1 mobilization. In particular, the marked mobilization of AnxA1 on the cell surface induced by either FPR2/ALX agonist was essentially abolished by WRW4; representative blots are shown (Fig. 3A), with cumulative data for cytosolic, membrane, and supernatant pools of AnxA1, respectively (Fig. 3B–D). Peptide Ac2-26 was also tested in these experiments; however, its modulation of PMN AnxA1 localization was not affected by Downloaded from WRW4 (Fig. 3B–D). Experiments performed in the presence of the selective FPR1 antagonist CsH showed that AnxA1 mobilization induced by FIGURE 5. FACS analysis on human PMNs after treatment with LXA4 fMLF was genuinely due to engagement of this receptor (Fig. 4). m and AF2. Human PMNs were treated with vehicle (CTR), AF2 (10 M), A different scenario emerged for peptide Ac2-26, which retained m LXA (100nM), or fMLF (1 M) for 30 min prior to staining with a spe- http://www.jimmunol.org/ 4 its ability to mobilize AnxA1 (Fig. 4). Of interest, even when a ciﬁc mAb. PMA (100 nM) was used as positive control for PMN degranulation. A, MFI determination for cell surface staining for PMN combination of CsH and WRW4 was tested, peptide Ac2-26, used m granule markers CD35, CD66b, and CD63. B, MFI for FPR1 and FPR2/ at 10 M, yielded marked mobilization of AnxA1 (Supplemental ALX receptor surface expression upon treatment with FPR2/ALX agonists Fig. 1).

LXA4 and AF2. Data, reported as MFI units, are mean 6 SEM from three To determine whether AnxA1 mobilization was secondary to, different experiments. *p , 0.05, **p , 0.01 versus CTR. fMLP, formyl- or accompanied by, granule mobilization, Abs specific for PMN methionyl-leucyl-phenylalanine. granules, such as CD35 (secretory vesicles), CD66b (specific granules), and CD63 (azurophil granules), were used. Flow cytometry intracellular AnxA1 apparently replenished. The FPR1 agonist analysis showed that, following 30 min of incubation, unlike PMA by guest on September 28, 2021 fMLF mobilized AnxA1 with kinetics similar to those produced (positive control), neither LXA4 nor AF2 induced changes in the by peptide Ac2-26: At 30 min significant reduction of intracellu- PMN cell surface expression of any of these markers (Fig. 5A). lar AnxA1 was observed, which was partially replenished by 60 Interestingly, both agonists caused internalization of FPR2/ALX min, possibly consequent to a reduction in the membrane pool by over the 30-min incubation time, with no effect on FPR1 (Fig. 5B). this time point (Fig. 1B). In these experiments, fMLF (1 mM) provoked significant upreg- Changes in AnxA1 concentrations in cell supernatants were de- ulation of CD66b (+60%; Fig. 5A). tected only after 30 min of treatment (data not shown), implying Because membrane AnxA1 did not appear to derive from in- some delay in the completion of the AnxA1 externalization process tracellular granules, we monitored AnxA1 phosphorylation status after its exposure on the plasma membrane (20, 36); normally, in the cytosolic pool (25, 24), using an Ab against phospho-Ser27- AnxA1 is not detectable in the culture medium of resting PMNs AnxA1 (25). Shorter time points were tested in this study, with (data not shown). From this set of experiments, we selected the Fig. 6 reporting data obtained at 10 min. In resting cells, there was 30-min time point and the two FPR2/ALX agonists AF2 and little membrane phosphorylated AnxA1; addition of LXA4 and LXA4 for further studies. AF2 caused a rapid increase in this species (Fig. 6A). Incubation

FIGURE 6. Phospho-Ser27-AnxA1 detection in PMN membrane fractions. Hu- man PMNs were treated with vehicle

(CTR), AF2 (10 mM), LXA4 (100nM), or fMLF (1 mM) for 10 min prior to recovery of membrane-bound AnxA1 by EDTA. A, Phospho-Ser27-AnxA1 with representative Western blotting and densitometric cumulative values. B, Total AnxA1 in the membrane pool with representative West- ern blotting and densitometric cumulative values. Data, reported as relative OD arbi- trary unit (AU), are mean 6 SEM from three experiments. **p , 0.01, ***p , 0.001 versus CTR. fMLP, formyl-methionyl- leucyl-phenylalanine. 4910 ANNEXIN A1 IS MOBILIZED BY FPR2/ALX AGONISTS

FIGURE 7. Detection of cell surface AnxA1 by flow cytometry. Human PMNs were treated with vehicle (CTR), AF2 (10 mM), LXA4 (100 nM), or fMLF (1 mM) for 30 min prior to staining with a specific anti- AnxA1 mAb. A, Representative histograms depicting the increase in fluorescence intensity as AnxA1 becomes externalized. B, Cumulative data for AnxA1 expression. Data, reported as MFI units, are mean 6 SEM from three different experiments. ***p , 0.001 versus CTR. fMLP, formyl- methionyl-leucyl-phenylalanine.

with fMLF augmented AnxA1 on the membrane surface (Fig. 6B), kocyte interaction with the postcapillary venule endothelium ob- but this did not appear to be phosphorylated. served in sham-operated animals (zero to two cells per vessel) (see Cell surface AnxA1 in stimulated PMNs could also be quanti- Supplemental Fig. 2). Downloaded from fied by flow cytometry. At the optimal 30-min incubation time, Injection of a single bolus of LXA4 (1 mg i.v., corresponding to +/+ both LXA4 and AF2 augmented AnxA1 expression at the single- 2.8 nmol) to AnxA1 mice provoked a marked (.50%) de- cell level (.2-fold increase in MFI units; Fig. 7A,7B) and a tachment of PMNs from the endothelium within the 10-min ob- marked increase in AnxA-positive PMNs was noted, with values servation period (Fig. 8A). This effect was drastically reduced in close to 70% versus control (resting PMN) levels of ∼15%. A AnxA12/2 mice, with no significant alteration in the fate of the similar pattern was seen following incubation with fMLF. adherent leukocytes (∼15%; Fig. 8C). A similar result was obtained with AF2 (1 mg i.v., corresponding to 1 nmol), which af- http://www.jimmunol.org/ Anti-inflammatory properties of LXA4 and AF2 are tightly forded significant detachment of adherent leukocytes in AnxA1+/+, coupled to AnxA1 but not AnxA12/2, mice (Fig. 8B,8C). Next, we tested whether the rapid nongenomic modulation of It should be noted that, in line with the higher inflammatory re- PMN AnxA1 might be of relevance to PMN trafficking in inflam- activity observed within the microcirculation of AnxA12/2 mice mation. To this end, we used a validated protocol whereby LXA4 (43), absence of AnxA1 was associated with a slightly higher de- (42) and AnxA1 itself exquisitely modulate the fate of adherent gree of adherent cells in both sets of experiments (e.g., Fig. 8A,8B). leukocytes, a biological effect referred to as “the detachment Finally, we investigated whether the crucial role of endogenous +/+ phenomenon” (41). The mesenteric microcirculation of AnxA1 AnxA1 in LXA4-induced effects was also evident in a more severe by guest on September 28, 2021 mice was inflamed with an ischemia/reperfusion procedure, al- type of inflammation. IL-1b injection into dorsal air-pouches of lowing the selection of postcapillary venules with approximately mice provoked an intense recruitment of Gr1+ cells at a 4-h time +/+ six to eight adherent leukocytes, in contrast to the minimal leu- point, which was inhibited by treatment with LXA4 in AnxA1 ,

FIGURE 8. LXA4 and AF2 provoke leukocyte detachment by mobilizing endogenous AnxA1. Intravital microscopy analyses of AnxA1+/+ and AnxA12/2 mouse mesenteric microcirculation in- ﬂamed by an ischemia-reperfusion pro-

cedure. A and B, Administration of LXA4 and AF2 [1 mg i.v. at time 0 (i.e., once the preparation is on stage and the postcapillary venule is selected)] induced detachment of adherent leukocytes in AnxA1+/+, but not in AnxA12/2,mice. **p , 0.01 versus time 0. C, Cumulative modulation of the detachment process, as recorded at the end of the 10-min observation period. Data are mean 6 SEM of six mice per group. xxxp , 0.001 versus AnxA1+/+. D, Dorsal air-pouches of AnxA1+/+ and AnxA2/2 mice were injected with IL-1b (10 ng in 0.5 ml) at time 0;

vehicle (100 ml) or LXA4 (1 mg/100ml) was injected i.v. via the tail vein immedi- ately after. In all cases, air-pouches were washed 4 h later, and cells were stained to determine the amount of recruited PMN (Gr1+ cells). Data are mean 6 SEM of 6 mice per group. **p , 0.01 versus vehicle, xxp , 0.01 versus AnxA1+/+. The Journal of Immunology 4911 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 FIGURE 9. Schematic model for AnxA1 mobilization in human PMNs. Proposed scheme of AnxA1 mobilization whereby cell–cell interaction (e.g., adhesion to endothelial cells) would externalize AnxA1 by mobilizing predominantly the granular pool (18–20), whereas the nongranular (cytosolic) pool seems to be mobilized in response to soluble compound addition (e.g., glucocorticoids, cromones, and inhibitors of the cystic fibrosis transducer factor) (22–24). The present study adds FPR2/ALX to the list of compounds known to activate the AnxA1 pathway in the neutrophil. The latter mode of mobilization seems to have, as a consistent prerequisite, phosphorylation of Ser27 on the AnxA1 N terminus. It is yet unclear whether PMN adhesion to endothelial cells (20) leads to externalization of phosphorylated AnxA1 species. but not AnxA12/2, mice (Fig. 8D). Fig. 9 summarizes the modes endogenous anti-inflammatory phase is now well accepted (2). As of AnxA1 mobilization in activated PMNs. an example, proinflammatory prostanoids induce LXA4 synthesis in, what has been proposed to be, a “class switching” from the Discussion proinflammatory to the proresolving phase of an acute experi- In this study we provide strong evidence that engagement of FPR2/ mental inflammation (49). However, the option that a network ALX by selective agonists would induce AnxA1 phosphorylation might be operative within anti-inflammatory mediators themselves and mobilization in human PMNs. We propose that this cellular has been inadequately explored: In this article, we show a func- response operates to control PMN recruitment to postcapillary tional link between LXA4 and endogenous AnxA1. Incubation of venules and migration to inflamed sites, as demonstrated with human PMNs with LXA4 induced a rapid translocation of AnxA1 2/2 proof-of-concept experiments in AnxA1 mice, in which LXA4 to the cell surface, with a time profile different from that provoked and AF2 lost their antimigratory properties. by peptide Ac2-26 or fMLF. The effect of LXA4 was mimicked by An effective inflammatory reaction relies on the concerted action the anti-inflammatory synthetic nonapeptide AF2. of several pathways that attract WBCs from the vasculature into It is intriguing that both LXA4 and AF2 inhibit neutrophil ac- specific tissue sites. A classical example is that of the synovial joint; tivation in vitro as well as recruitment of these cells in vivo. AF2 at this site, TNF-a initiates a cascade of mediator generation and reduces HL-60 cell adhesion to endothelial cells, suggesting that it release (e.g., IL-1 or IL-6) (44, 45), culminating in the recruitment dampens inflammation by blocking leukocyte trafficking and the of blood-borne leukocytes into the synovial tissue and joint space. subsequent eicosanoid production (29). Furthermore, AF2 is ef- Blocking TNF-a activity is therefore a very successful therapeutic fective in suppressing endotoxin-induced uveitis in rats (50). The strategy (44, 46). In recent years an innovative approach to anti- data presented in this article indicate that such an effect might inflammatory therapeutic development has emerged; this approach occur, at least partly, via endogenous AnxA1. Our new data and aims to capitalize on the action of endogenous anti-inflammatory ensuing hypothesis may provide a mechanistic explanation for the and proresolving mediators (47, 48). The evidence that the pro- observation that endogenous LXA4 and AnxA1 are both operative inflammatory phase is required for an appropriate induction of the in germ-free mice for optimal generation of IL-10 in the gut (51). 4912 ANNEXIN A1 IS MOBILIZED BY FPR2/ALX AGONISTS

It should also be noted that a few examples of crosstalk in ment of this cell type to a variety of tissue sites and in response to resolution are beginning to emerge, with resolvin E1 promoting several distinct inflammogens (6). The same is partially true for exudate expression of LXA4, although the specific cell targets of AF2, although this nonapeptide has been used more often as an this mediator were not identified (52). In addition, there is an inhibitor of PMN activation in vitro (27, 29) rather than of cell indication that endogenous epi-LXA4, synthesized upon aspirin’s recruitment in vivo (65). For instance, AF2 inhibits PMN adhesion unique blockade of cyclo-oxygenase 2, can mobilize NO from the to endothelial cells (28) and leukocyte chemotaxis (66). Both endothelium (constitutive NO synthase activation) and macro- compounds were tested in the intravital microscopy protocol, phages (inducible NO synthase activation) to elicit anti-inflam- which we reported being sensitive to LXA4 treatment (42). The matory effects in a model of pleurisy as well as in the microcir- results we present in this paper indicate a crucial role for endoge- culation (53). We cannot exclude that an involvement of endog- nous AnxA1 in the detachment phenomenon promoted by either enous NO might also be occurring in our settings, although the FPR2/ALX agonist. experiments of leukocyte detachment reported in this article Peptide Ac2-26 was not tested in the leukocyte detachment took place within 10 min. In any case, the recent involvement of phenomenon in our study, in view of the in vitro profile we obtained rapidly produced endothelial NO and prostacyclin in the anti- with human PMNs. However, it has been previously shown that migratory effects of resolvin D2 (54) would suggest also keeping administration of this peptide to mice can activate this process in an this option viable upon the acute application of LXA4 and AF2. Fpr1-independent fashion (42), and peptide Ac2-26 also retains its Finally, the positive association between endogenous AnxA1 and antimigratory effects in AnxA1-null mice (21). In the present expression of mouse Fpr2 during resolving colitis should be in vitro settings, this AnxA1 mimetic induces AnxA1 mobilization reported (55). in a different manner with respect to LXA4 and AF2 (and fMLF) Downloaded from Most of the studies summarized above begin to propose the because it does not appear to evoke involvement of either FPR1 and existence of positive nongenomic loops in resolution; however, FPR2, as shown by experiments performed with FPR1 or FPR2/ evidence for genomic associations is also emerging. One classical ALX selective antagonists. It should be noted that PMN-activating example is that linking glucocorticoids to overexpression of effects associated with AnxA1 N-terminal–derived peptides (Ac9- AnxA1, with increments in gene promoter activity being measu- 25) do not require activation of FPR1 or FPR2/ALX (67). red with a reporter gene (14). In addition, LXA4 could induce two Finally, it should be noted that the inhibitory properties exhib- http://www.jimmunol.org/ 2/2 specific signaling molecules, NGFI-A binding protein 1 (56) and ited by LXA4 in the air-pouch model were lost in AnxA1 mice. suppressor of cytokine signaling 2 (57), to reprogram the pheno- With data from the recent observation of lack of effect of LXA4 in type of PMNs and dendritic cells, respectively. Among the bi- Fpr22/2 mice, using the same experimental model and inflamm- ological effects elicited by LXA4, its analogs, and other ogen (68), we propose a model whereby intravascular activation of proresolving lipid mediators, it is worth highlighting the aug- PMN Fpr2, in response to the agonists tested in our study, would mented expression of CCR5 on apoptotic leukocytes, a response bring about AnxA1 mobilization. This event would, in turn, act that facilitates removal of migrated cells and tissue resolution in a juxtacrine/autocrine fashion (6) to reinforce the overall at- (58). De novo gene synthesis has clearly been ascribed to gluco- tenuation of the process of leukocyte recruitment. Clearly, other corticoids; in this context, it must be noted that they will not only intracellular pathways (69, 70) and mediators might also act by guest on September 28, 2021 augment AnxA1 synthesis and release but would also increase downstream of LXA4 application (5, 66); nonetheless, the data expression of anti-inflammatory FPR2/ALX (59, 60). presented and discussed in this article point to the nongenomic The observation that exogenously applied LXA4 mobilizes mobilization of AnxA1 as one of the major downstream effectors AnxA1 and that this contributes, at least in part, to its inhibitory of LXA4, and AF2, actions that negatively modulate the cell-traf- properties on PMN trafficking, adds another layer of complexity to ficking process. Networks of proresolving mediators and re- the molecular and functional interrelationship between these two ceptors are an emerging feature of the endogenous homeostatic endogenous effectors of anti-inflammation and resolution. More- response; once more loops are discovered and dissected, these over, it is intriguing how a pattern of distinct inducers of AnxA1 “anti-inflammatory networks” are likely to become an accepted externalization is emerging. Processes such as cell adhesion and paradigm in the area of inflammatory resolution. activation, which induce rearrangement of intracellular organelles within the PMN, would mobilize preferentially the pool of AnxA1 Acknowledgments contained in granules and vesicles (20, 61) (see Fig. 9 for a sche- We thank Dr. Egle Solito for providing the AnxA1-Ser27 phospho-specific matic summary). In contrast, the typical mobilization induced by Ab. glucocorticoids using a receptor-dependent nongenomic pathway (25, 62, 63) seems to be replicated by other classes of drugs, in- Disclosures cluding cromones (23, 24) and, as described in this paper, LXA 4 R.J.F. and M.P. are Board Directors of William Harvey Research Limited. and AF2. 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