Transendothelial Migration Cleaved Annexin A1 in Neutrophil A

A Proinflammatory Role for Proteolytically Cleaved Annexin A1 in Neutrophil Transendothelial Migration

This information is current as Samantha L. Williams, Ian R. Milne, Christopher J. Bagley, of September 30, 2021. Jennifer R. Gamble, Mathew A. Vadas, Stuart M. Pitson and Yeesim Khew-Goodall J Immunol 2010; 185:3057-3063; Prepublished online 2 August 2010;

doi: 10.4049/jimmunol.1000119 Downloaded from http://www.jimmunol.org/content/185/5/3057

References This article cites 34 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/185/5/3057.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 30, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Proinﬂammatory Role for Proteolytically Cleaved Annexin A1 in Neutrophil Transendothelial Migration

Samantha L. Williams,*,† Ian R. Milne,*,1 Christopher J. Bagley,*,1 Jennifer R. Gamble,*,2 Mathew A. Vadas,*,2 Stuart M. Pitson,*,† and Yeesim Khew-Goodall*,†

Neutrophil extravasation, a critical component of innate immunity must be tightly regulated to prevent inadvertent or prolonged inflammation and subsequent tissue damage. We have shown previously that endothelial ERK1/2 signaling essential for neutrophil transendothelial migration is induced by a soluble factor produced by activated neutrophils. In this study, we demonstrate that the soluble neutrophil factor is a truncated form of annexin A1 (AnxA1) that can be generated by calpain 1 cleavage of the N terminus, thus identifying a novel proinflammatory function to AnxA1. In contrast, neither the full-length protein nor the N-terminal 26 aa peptide, previously shown to be antiinflammatory, were able to activate Erk. Our data suggest that two different fragments of

AnxA1 have opposing functions in inﬂammation. We also provide evidence that C-terminal AnxA1 functions by increasing ICAM1 Downloaded from clustering around adherent neutrophils to anchor them to the endothelium and promote transmigration through the transcellular route. The Journal of Immunology, 2010, 185: 3057–3063.

eutrophils are an important component of the innate mechanisms downstream of ICAM1 clustering leading to extrava- immune response, but the sustained presence of activated sation are being revealed (4, 5), little is known about the signal N neutrophils can result in tissue damage because of the transduction mechanisms regulating ICAM1 clustering. http://www.jimmunol.org/ release of factors, such as superoxides, tissue elastase or colla- In an earlier study, we identified a novel endothelial signaling genase, eicosanoids, and leukotrienes (1). Leukocyte extravasation pathway that regulates neutrophil TEM. We reported that the acti- involves initial tethering, rolling, and tight adhesion to the endo- vation of endothelial ERK 1/2 facilitated neutrophil TEM (10) and thelium prior to diapedesis or transendothelial migration (TEM), that endothelial ERK was activated in response to a soluble protein with each of these steps being tightly regulated (2, 3). The mo- produced by fMLP- or IL-8–stimulated neutrophils. In this study, we lecular mechanisms of TEM are understood less than those me- have identified the proinflammatory neutrophil protein as annexin diating the rolling and adhesion steps, but recent advances have A1 (AnxA1). The current literature pertaining to AnxA1 describes yielded insights into the molecular mechanisms of TEM—in the intact molecule and its N-terminal peptides as antiinflammatory, by guest on September 30, 2021 particular, the proactive role of the endothelium in this step (4, 5). with the ability to inhibit neutrophil accumulation in a number of Leukocytes can traverse the endothelium by the paracellular or well-established animal models of inflammation (11). In this study, transcellular routes. Most reports support the paracellular route, we describe a novel proinflammatory action of an N-terminally where leukocytes extend between adjacent cells of the endothelial truncated form of AnxA1 generated by calpain I proteolysis and monolayer (6, 7). However, recent studies have provided evidence its potential role in transcellular migration. for transcellular migration of leukocytes through the endothelial cells via the formation of transmigratory cups (8, 9). Whichever Materials and Methods route is used, clustering of ICAM1 around the transmigrating leu- Reagents and Abs kocyte appears to be crucial for TEM (4, 5). Although the signaling Abs to the following were sourced as follows: ERK and phosphorylated ERK (Promega, Madison, WI), annexin AI (mAb; BD Pharmingen, San *Division of Human Immunology, Centre for Cancer Biology, SA Pathology; and Diego, CA; polyclonal, AbCam, Cambridge, MA), ICAM1 (12). Calpain I †School of Molecular and Biomedical Science (Biochemistry Discipline), University was acquired from Sigma-Aldrich (St. Louis, MO) and Calpain inhibitor of Adelaide, Adelaide, South Australia, Australia XII was acquired from EMD Biosciences (San Diego, CA). Ac2-26 was synthesized by Auspep (Parkville, Australia). All other reagents and Abs 1Current address: Analytical Development, Hospira Adelaide, Thebarton, South Aus- tralia, Australia. used are as referenced in Ref. 10. 2Current address: Centenary Institute of Cancer Medicine and Cell Biology, The Fractionation of neutrophil-conditioned medium University of Sydney, New South Wales, Australia. Neutrophil (from healthy donors) purification, stimulation, and preparation Received for publication January 14, 2010. Accepted for publication June 24, 2010. of conditioned medium were as previously described (10). Conditioned This work was supported by the National Heart Foundation, Australia. S.L.W. was medium was concentrated and chromatographed sequentially through two funded by an Australian Post-Graduate Award. Y.K-G. is a Cancer Council of South Superdex 75 HR 10/30 columns (internal diameter, 10 mm; height of Australia Senior Research Fellow. S.M.P. is a National Health and Medical Research packed bed, 30–31 cm; total bed volume, ∼24 ml; Pharmacia Biotech, Council Australia Senior Research Fellow. Uppsala, Sweden). Chromatography was performed at 4˚C in PBS (pH 7.4) Address correspondence and reprint requests to Dr. Yeesim Khew-Goodall, Division at a flow rate of 0.5 ml/min. of Human Immunology, Centre for Cancer Biology, SA Pathology, Frome Road, Adelaide, South Australia 5000, Australia. E-mail address: yeesim.khew-goodall@ Mass spectrometry health.sa.gov.au Abbreviations used in this paper: AnxA1, annexin A1; CM, neutrophil-conditioned Proteins from the active fraction resolved by SDS-PAGE were excised and medium; Inh, calpain inhibitor; KLH, keyhole limpet hemocyanin; Nil, no conditioned digested with trypsin, and the resulting peptides desalted into a quadrupole- medium added; pERK, activated ERK; TEM, transendothelial migration. time-of-flight mass spectrometer through a C18 reverse-phase silica column. Automated MS/MS sequencing was performed using data-directed Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 analysis techniques. The data were analyzed using Protein Lynx Global www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000119 3058 PROINFLAMMATORY ROLE OF CLEAVED ANNEXIN A1

Server (Waters, Milford, MA), and the sequence tags were used to search against the SwissProt (National Center for Biotechnology Information, Bethesda, MD) database, which is limited to human entries. HUVEC ERK-activation assay The assay for ERK activation in HUVEC was performed as described previously (10). Activated ERK was detected by Western blot using a phospho-ERK Ab specific for the activated form. Total ERK or b-tubulin was used as a loading control after stripping the membrane and reprobing. Boyden chamber neutrophil transmigration assay Boyden chamber neutrophil transmigration assay was performed as previously described (10) using Transwells (6.5-mm diameter, 3.0-mm pore size; Costar, Cambridge, MA). Purification of AnxA1 HEK293 cells stably expressing human AnxA1 were established, from which AnxA1 was purified as outlined previously (13). Proteolysis of AnxA1 with calpain I

Purified recombinant AnxA1 was treated with 0.2 U human calpain I per Downloaded from 5 mg total protein; 5 mM CaCl2 was added to initiate the reaction, which was then incubated for 20 min at room temperature. For samples in which calpain inhibitor was used, calpain inhibitor XII was incubated with calpain I for 10 min at room temperature before adding to purified AnxA1. Calpain I was not inactivated prior to use in ERK-activation assays. Slide assay for neutrophil TEM FIGURE 1. Purification and identification of ERK-activating component http://www.jimmunol.org/ The slide assay for neutrophil TEM was performed as described previously in fMLP-stimulated neutrophil conditioned medium. A, Protein elution (8). ICAM1 was detected by indirect immunofluorescence and quantitated profile of conditioned medium from 100 nM fMLP-stimulated neutrophils using the Olympus Analysis Research (Olympus Europa, Hamburg, Ger- fractionated by gel filtration. B, One fifth of each fraction eluted from the many) software. gel filtration column was added to resting HUVEC monolayers for 10 min prior to Western blotting with a phospho-ERK Ab showing activated ERK Results (upper panel) in HUVEC and was reprobed after stripping with total ERK AnxAI secreted by activated neutrophils is essential and Ab (lower panel). C, Coomassie brilliant blue-stained SDS polyacrylamide sufficient for endothelial ERK activation and neutrophil TEM (15%) gel showing proteins present in fractions 9 to 11 from panel A. Protein bands 1–4 from fraction 10 were excised for identification by by guest on September 30, 2021 To identify the endothelial ERK-activating factor we had previ- quadrupole-time-of-flight mass spectrometry. D, Amino acid sequence of ously shown to be secreted by activated neutrophils (10), cell- AnxA1 showing peptides detected by mass spectrometry (underlined). Al- free conditioned medium from fMLP-stimulated neutrophils was ternating bold and light sequences indicate theoretical peptides that would fractionated by size exclusion chromatography (Fig. 1A). Frac- be generated following tryptic digestion of full length AnxA1. Numbers tions eluted from the column were assayed for their ability to above the sequence indicate the first four predicted tryptic peptides. activate ERK in HUVECs. The ERK-activating factor was found to elute in fraction 10 (Fig. 1B), which is predicted from calibra- release a factor that activates endothelial ERK (10), the effect of tion standards to contain proteins of molecular mass between 30 AnxA1 Ab on IL-8–induced neutrophil-conditioned medium was and 40 kDa. Proteins comprising the active fraction and flanking also investigated. This finding showed that the Anx1 Ab also re- inactive fractions were resolved by SDS-PAGE, and four proteins duced the potency of IL-8–stimulated neutrophil conditioned unique to the active fraction were considered as candidates for the medium to activate ERK (Fig. 2B), further supporting AnxA1 as ERK-activating factor (Fig. 1C). The proteins were identified by the ERK-activating protein secreted by activated neutrophils. Endo- mass spectrometry to be monocyte–neutrophil elastase inhibitor thelial ERK activation by neutrophil-conditioned medium was also (Swiss-Prot P30740), AnxA1 (P04083; Fig. 1D), annexin A3 inhibited by a polyclonal Ab to AnxA1 (data not shown), indicating (P12429), and chloride intracellular channel protein (O00299). that the effect was not Ab-specific. Importantly, when added to AnxA1 (Fig. 1D, band 2) has previously been reported to be both fMLP- or IL-8–stimulated neutrophil conditioned media, the antiinflammatory (11), and its presence in the ERK-activating AnxA1 Ab reduced levels of active ERK to the level at which no fraction of neutrophil-conditioned medium was therefore unex- conditioned medium was added (Nil) (Fig. 2A,2B), suggesting pected. Thus, its effect on endothelial ERK activation and neutro- that AnxA1 is the main ERK-activating constituent of neutrophil phil TEM was examined, initially using Abs against AnxA1. The conditioned medium. Thus, the other unique proteins (bands 1, 3, addition of AnxA1 Ab to fMLP-stimulated neutrophil-conditioned 4) identified in the active fraction of neutrophil-conditioned medium medium markedly reduced endothelial ERK activation compared were not investigated further. In addition, recombinant monocyte– with conditioned medium alone or conditioned medium contain- neutrophil elastase inhibitor (band 1; a gift from Dr. P. Bird, Monash ing a control isotype-matched irrelevant Ab (Fig. 2A). Further- University, Clayton, Victoria, Australia) did not activate endothelial more, when AnxA1 was immunodepleted from the neutrophil- ERK when used from 2-600 ng/ml (data not shown). conditioned medium, the potency of the depleted neutrophil- As we have previously shown that endothelial ERK activation is conditioned medium to activate ERK was significantly reduced essential for neutrophil TEM (10), we investigated whether AnxA1 relative to untreated conditioned medium (Fig. 2A). This finding Abs could also inhibit neutrophil TEM. In an in vitro assay of suggests that AnxA1 present in the fMLP-stimulated neutrophil- neutrophil transmigration across a confluent monolayer of cul- conditioned medium is essential for activating endothelial ERK. tured HUVEC in a transwell (10), the AnxA1 Ab significantly Having previously shown that IL-8–stimulated neutrophils also reduced both fMLP- and TNF-a–mediated transmigration relative The Journal of Immunology 3059

in and purified from a mammalian cell line: HEK293. Purified recombinant AnxA1 (Fig. 3A) induced a marked dose-dependent increase in ERK activation when added to HUVECs that was not observed when a vehicle control was added (Fig. 3B). This finding suggests that AnxA1 alone was sufficient for activating endothelial ERK.

A calpain I cleavage product but not full-length AnxA1 activates endothelial ERK Preparations of recombinant AnxA1 purified from HEK293 cells contained both full-length and an ∼33-kDa form that routinely cofractionated (Fig. 3A), with the ratio of the two forms often varying. To test each form separately for their ability to activate endothelial ERK, they were separated into adjacent fractions by anion exchange chromatography (Fig. 3C). Equivalent amounts of protein from each of the fractions were tested. Fraction 9, which FIGURE 2. AnxA1 secreted by activated neutrophils is essential and contained predominantly the smaller molecular mass form of sufficient for endothelial ERK activation and neutrophil TEM. A, Western AnxA1, activated endothelial ERK to an extent similar to the blot showing activated (upper panel) and total (lower panel) ERK in Nil, 0.25 Downloaded from mg/ml PMA, fMLP-stimulated CM, CM treated with 12.5 mg/ml Anx A1 unfractionated protein and to a much greater extent than fraction mAb (CM+AnxA1 Ab), CM depleted of AnxA1 by immunoprecipitation 10, which contained predominantly full-length AnxA1 (Fig. 3C). with an AnxA1 Ab (AnxA1 IP SN), or CM treated with 12.5 mg/ml irrelevant The lower molecular mass of the active AnxA1 suggests that it has isotype-matched anti-KLH mAb (CM+control Ab). B, Western blot showing most likely arisen by proteolytic cleavage of the full-length protein. activated ERK (upper panel) and tubulin (lower panel) in Nil, resting neu- Its molecular mass of ∼33 kDa compared with the that of full-length trophil conditioned medium (unstim CM), IL8 (100 nM)-stimulated neutro- AnxA1 of 37 kDa suggests that ∼30 aa have been removed. Con- phil conditioned medium without (IL8 CM) or with AnxA1 Ab (12.5 mg/ml; sistent with this finding, analysis of the peptides recovered from http://www.jimmunol.org/ IL-8 + AnxA1 Ab) and 100 nM fMLP-stimulated neutrophil conditioned mass spectrometry showed that the most N-terminal peptide recov- medium (fMLP CM). C and D, In vitro neutrophil TEM assays across either ered (denoted peptide 4 in Fig. 1D) began at aa 30. However, from resting HUVEC with an fMLP (10 nM) gradient (C) or a TNF-a–activated a prediction of resultant tryptic peptides (Fig. 1D), the peptide (4 ng/ml) HUVEC monolayer (D), expressed as a percentage of neutrophils added. Neutrophils (5 3 105 per well) were added to the top chamber of the (peptide 3) immediately preceding this peptide, because of its small Transwell either in the absence of Ab (2), in the presence of 12.5 mg/ml size, is likely to escape detection by mass spectrometry even if it AnxA1 Ab (AnxA1) or 12.5 mg/ml irrelevant isotype-matched mAb (anti- were present. Therefore, the N terminus of the active truncated KLH; control). Each experiment was performed in triplicate. A one-way AnxA1 could be between aa 27 and 30. AnxA1 has been shown ANOVA was performed comparing AnxA1 mAb to irrelevant mAb (n = previously to be cleaved at various sites in the N terminus by by guest on September 30, 2021 3). pppp , 0.0001. CM, neutrophil-conditioned medium. a number of proteases, including calpain I and plasmin after Lys-26 and Lys-29 (15), respectively. To test whether the active to an irrelevant isotype-matched control Ab (anti-keyhole limpet truncated AnxA1 could be generated by calpain cleavage, full- hemocyanin [KLH]) (Fig. 2C,2D). length recombinant AnxA1 was treated with human calpain I, ThereductioninbothERKactivationandneutrophilTEMby and the resulting product was assayed for its ability to activate anti-AnxA1 Abs strongly suggests that AnxA1 is the critical com- ERK in HUVEC. Recombinant AnxA1 treated with calpain 1 for ponent of neutrophil-conditioned medium that regulates neutro- various times activated endothelial ERK to a greater extent than an phil TEM through activating endothelial ERK. We therefore equivalent amount of untreated full length AnxA1, with a maximum investigated whether purified recombinant AnxA1 alone was suf- effect at 20 min of treatment, whereas longer treatment times ficient to activate endothelial ERK. Because human AnxA1 is resulted in loss of enhancement (Fig. 4A), presumably because of glycosylated in vivo (14), recombinant AnxA1 was expressed overdigestion. The increase in the ability to activate endothelial

FIGURE 3. A calpain 1 cleaved form but not full-length AnxA1 activates ERK in endothelial cells. A, Coomassie blue-stained polyvinylidene difluoride membrane after transfer from an SDS-polyacrylamide gel showing recombinant AnxA1 purified from HEK293 cells stably expressing human AnxA1. The recombinant protein was purified using a MonoQ column. B, Western blot showing activated (upper panel) or total (lower panel) ERK in HUVEC incubated with 1.25 or 2 mg/ml purified recombinant AnxA1 (as shown in A), the vehicle control (an equivalent volume of 20 mM ethanolamine, pH 9.0), or 0.25 mg/ml PMA. C, Upper panel, Western blot developed with an AnxA1 mAb showing high (full-length) and low molecular mass forms of AnxA1 eluting in consecutive fractions from a MonoQ column. Lower panel, Western blot showing activated (pERK) and total ERK in HUVEC treated with 5 mg of unfractionated, fraction 9 or fraction 10 eluant from a MonoQ column (upper panel) of recombinant AnxA1. 3060 PROINFLAMMATORY ROLE OF CLEAVED ANNEXIN A1

FIGURE 4. Calpain-cleaved AnxA1 activates endothelial ERK. A, Forty micrograms partially puriﬁed AnxA1 (total protein concentration) was proteolysed by human calpain 1 for the indicated time periods and then incubated on resting HUVEC monolayers for 10 min. Forty micrograms uncleaved AnxA1 and vehicle control (vehicle) were also tested. HUVEC ERK activation was determined by Western blotting whole cell lysates with an Ab to phosphorylated ERK. B, Western blot showing pERK and tubulin in HUVEC incubated with vehicle control, untreated puriﬁed rAnxA1 (20 mg), 20 mg AnxA1 pretreated with calpain (2 U/ml; rAnxA1 + calpain), 20 mg AnxA1 treated with calpain in the presence of calpain inhibitor XII (1 mM, 10 min; rAnxA1 + calpain + Inh), vehicle control with calpain (calpain) and vehicle control with Inh. C, Western blot showing pERK and b-tubulin in HUVEC incubated with fMLP-stimulated neutrophil CM Downloaded from and CM from neutrophils preincubated with calpain inhibitor XII 10 min prior to fMLP stimulation (CM + inhibitor). As a control, the calpain inhibitor was added after fMLP stimulation of the neutrophils (CM + Inh after). D, Western blot showing pERK in HUVEC treated for 10 min with PBS (Nil), unstimulated neutrophil conditioned medium (unstim CM), 250 mg/ml Ac2-26, fMLP-stimulated neutrophil CM (CM + vehicle), or fMLP-stimulated neutrophil- conditioned medium with 250 mg/ml Ac2-26 (CM + Ac2-26). E, Upper panel, Western blot showing activated ERK in HUVEC treated with recombinant AnxA1 (20 mg), AnxA1 cleaved with plasmin (20 mg), plasmin alone or PBS (Nil) as indicated. Lower panel, Western blot using AnxA1 Ab to detect full-length and cleaved AnxA1 pretreatment and posttreatment with plasmin. Inh, calpain inhibitor; pERK, activated ERK. http://www.jimmunol.org/

ERK after calpain 1 treatment was abolished when a calpain 1 the endothelial monolayer. HUVEC monolayer permeability was inhibitor was present during calpain treatment (Fig. 4B), suggesting assayed by monitoring the rate of dextran diffusion across the that the calpain 1 proteolytic activity was essential for activating HUVEC monolayer. The permeability of the HUVEC monolayer, AnxA1. Neither calpain nor its inhibitor alone increased the when measured in activated neutrophil-conditioned medium, was amount of active ERK in HUVEC compared with full length not significantly different from that measured in medium alone AnxA1. Furthermore, the degree of Erk activation mediated by (Fig. 5). Furthermore, the ERK activation inhibitor U0126 had no calpain-treated rAnxA1 over and above the unstimulated control effect on HUVEC permeability in the presence of neutrophil- is similar in extent to that mediated by IL-8 (Fig. 2B) or fMLP- conditioned medium, suggesting that the effect of endothelial by guest on September 30, 2021 stimulated (Fig. 4D) neutrophil conditioned medium. ERK activation by neutrophil-conditioned medium, which facili- To ascertain whether calpain I is the physiologic protease that tates TEM, was not to increase endothelial permeability. In contrast, cleaves AnxA1 into the active form during neutrophil activation, thrombin significantly increased HUVEC monolayer permeability a calpain I inhibitor was included during fMLP-stimulation of neu- compared with Nil or other treatments. trophils. Conditioned medium obtained from neutrophils that were Recent studies have shown that productive TEM after the initial stimulated in the presence of the calpain inhibitor showed re- attachment requires lateral clustering of ICAM1, VCAM1, or both, duced ability to activate ERK in HUVEC when compared with con- bound to the b2 integrins of leukocytes to form transmigratory ditioned medium without inhibitor or when the inhibitor was added cups (8, 9). We therefore examined whether the addition of the after harvesting (Fig. 4C). These data indicate that calpain I proteolysis of AnxA1 was essential for generating an active AnxA1 product that could activate ERK in HUVEC, and the molecular mass of the active form of ∼33 kDa suggests that the C-terminal fragment is the active peptide. To affirm this finding, we also tested the N-terminal AnxA1 peptide, Ac2-26, that would be generated by calpain 1 cleavage (15) and has been shown previously to mimic the known antiinflammatory functions of the full-length protein (16, 17). We found that it was not able to activate endothelial ERK (Fig. 4D). We also examined whether plasmin, which cleaves 3 aa C- terminal of the calpain cleavage site after Lys-29, could also activate AnxA1 and found that, unlike calpain 1, it did not enhance the amount of endothelial ERK activation beyond that which was ac- FIGURE 5. fMLP-stimulated neutrophil-conditioned medium does not tivated by untreated AnxA1 (Fig. 4E, upper panel) despite efficient alter HUVEC monolayer permeability to FITC-conjugated dextran. HUVEC cleavage of AnxA1 to the smaller molecular mass form by plas- monolayers grown in transwells were treated with the indicated samples min (Fig. 4E, lower panel). mixed with FITC-conjugated dextran in the apical chamber for 1 h. FITC- conjugated dextran present in the basolateral chamber was sampled at 10- AnxA1 plays a role in transmigratory cup formation during min intervals for the duration of the experiment. The figure shows one rep- neutrophil TEM resentative experiment of three, each performed in triplicate. Error bars indicate SEM. Lines of best fit were generated by linear regression analysis. To determine how AnxA1 and endothelial ERK facilitate neutrophil ppp , 0.01, one-way ANOVA, followed by Tukey’s multiple comparison TEM, we examined whether activated neutrophil conditioned me- post test, comparing the Thrombin slope to each of the treatments; the slopes dium, by activating endothelial ERK, altered the permeability of from other treatment groups were not significantly different. The Journal of Immunology 3061

AnxA1 Ab affected the formation of transmigratory cups using an and KLH mAb-treated samples were significantly higher than in vitro assay based on one previously described (8). In pilot those from the AnxA1 mAb treatment (Fig. 6B). Away from experiments, it was determined that TEM was already underway the transmigratory cups there was no significant difference in 2–3 min after the addition of activated neutrophils to the HUVEC surface ICAM1 staining between the treatments (Fig. 6C). This monolayer (data not shown). Therefore, fMLP-stimulated neutro- finding suggests that AnxA1 did not affect overall ICAM1 ex- phils were incubated with TNF-a–activated HUVEC for 2 min pression levels, but that it was involved in the clustering of before being fixed and processed for visualization of trans- ICAM1 around the neutrophil to form the transmigratory cup to migratory cups. In agreement with data obtained using the Boy- secure the neutrophils to the endothelium. den chamber TEM assays (Fig. 2C,2D), a decrease in the total Because both transcellular and paracellular routes of TEM were number of neutrophils adhering to or transmigrating through the observed in this assay (Fig. 6D), we examined whether AnxA1 was HUVEC monolayer was observed in the presence of the AnxA1 required for both routes of TEM. In the absence of the Anx A1 Ab compared with no Ab treatment (data not shown). Although Ab, 68 6 2.5% of TEM occurred by the transcellular route (Fig. .90% of the neutrophils on the slide exhibited ICAM1 staining 6E; Nil), which was not significantly different from treatment with around the periphery of the cell, it was apparent that the overall an irrelevant isotype-matched Ab (data not shown). However, intensity of ICAM1 staining in the transmigratory cups of AnxA1 treatment with the AnxA1 Ab reduced total transmigration to Ab-treated samples was reduced compared with no treatment ∼50% that of untreated samples, with approximately one third (Fig. 6A). Quantifying the intensity of ICAM1 staining surround- of the neutrophils migrating through the transcellular route (Fig. ing the transmigrating neutrophils showed that the average in- 6E; AnxA1 mAb). Hence, compared with the untreated samples, tensity of ICAM1 staining in the transmigratory cups for Nil the total number of neutrophils transmigrating through the Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 6. AnxA1 plays a role in ICAM1 clustering around transmigratory cups and facilitates transcellular migration. A, Representative immunofluorescence micrographs (original magnification 3200) showing ICAM1 staining (green) around transmigrating neutrophils through an HUVEC monolayer using a slide assay (8) in the absence of Ab (Nil) or presence of AnxA1 Ab (AnxA1). Nuclei from HUVEC (large) and neutrophils (small) counter-stained with DAPI (blue). Arrows indicate ICAM1 clustering in transmigratory cups. B, Scatter plot of data from A quantifying ICAM1 intensity surrounding transmigrating neutrophils relative to surface ICAM1 intensity. Four intensity measurements were taken per neutrophil and averaged. Four hundred transmigratory cups were analyzed (except KLH mAb control) from 59 representative fields using HUVEC from six independent donors. The red bar denotes mean relative ICAM1 staining. pppp , 0.0001, unpaired t test comparing Nil and AnxA1 mAb treatments. C, Four measurements of HUVEC surface ICAM1 staining were taken and averaged from 59 representative fields of view where transmigratory cup intensity was quantified. The data from Nil, AnxA1 mAb, and KLH mAb treatments from three individual experiments were compiled. Each experiment was performed in duplicate using two lines of HUVEC obtained from different women. Error bars indicate SEM. ANOVA indicates no significant differences between groups (p . 0.05). D, Paracellular and transcellular modes of neutrophil transmigration across an HUVEC monolayer. HUVECs were seeded at high density onto fibronectin- coated slides and grown overnight. HUVEC monolayers were stimulated with TNF-a for 4 h, and fMLP-stimulated neutrophils were added for 2 min. Nonadherent neutrophils were washed away and then the monolayers were stained with 1% AgNO3 and fixed according to a method modified from Baldwin (34) to highlight the intercellular junctions as indicated. Neutrophils that have transmigrated at intercellular junctions were deemed to have taken the paracellular route, and those that have transmigrated away from intercellular junctions were deemed to have taken the transcellular route as indicated in the figure. Original magnification 3600. E, Quantitation of numbers of neutrophils transmigrating by the transcellular or paracellular routes in untreated (Nil) or AnxA1 Ab-treated samples relative to total number of neutrophils transmigrating in the untreated (Nil) sample. pppp , 0.0001, unpaired t test comparing number of neutrophils transmigrating by the transcellular route in Nil versus AnxA1 mAb treatments; p . 0.05 when comparing neutrophils transmigrating via the paracellular route. 3062 PROINFLAMMATORY ROLE OF CLEAVED ANNEXIN A1 paracellular route was not significantly altered, and the AnxA1 Ab Ac2-26 are proposed to signal through the family of fMLP receptors largely affected transmigration through the transcellular route FPR and FPRL-1/ALXR, which are expressed on endothelial cells (Fig. 6E). (32). Future studies could use siRNA approaches to knock down these receptors on endothelial cells to determine whether they also Discussion act as receptors for the calpain I-cleaved AnxA1. We have shown previously that activation of ERK 1/2 in HUVEC Having identified that truncated AnxA1 is crucial for activating by a soluble protein secreted from fMLP- or IL-8–stimulated neu- endothelial ERK to promote TEM, we investigated the mechanism trophils was an essential requirement for neutrophils to traverse the by which AnxA1 did this. Our in vitro studies (Fig. 5) showed that endothelial barrier (10). In this study, we have demonstrated that the the truncated AnxA1 did not simply increase endothelial perme- soluble protein from activated neutrophils is a 33-kDa N-terminally ability and thereby enhance passage across the endothelial truncated AnxA1 generated by calpain 1 cleavage. This proinflam- monolayer through loss of junctional integrity. Recent studies matory role is novel for AnxA1 and contrasts the antiinflammatory have shown that clustering of ICAM1 around the neutrophil after role of the full-length protein and the N-terminal Ac2-26 peptide binding to LFA1 is essential for TEM (8, 9). Importantly, Yang observed in earlier studies, including those using the AnxA1 knock- et al. (33) showed that ICAM1 expression levels influenced the out mouse, which shows a high propensity toward inflammation route of TEM, deducing that higher LFA1 occupancy and clus- when challenged (11, 18). The 33-kDa form of AnxA1 activated tering at high ICAM1 levels promoted rapid, nonjunctional arrest endothelial ERK, which we have shown previously to be essential and transcellular TEM. To obtain further insight into how AnxA1 for neutrophil TEM; consistent with this, neutralizing AnxA1 with and endothelial ERK activation can facilitate neutrophil TEM, we an Ab inhibited neutrophil TEM. examined the role of AnxA1 in the formation of transmigratory Downloaded from Truncated forms of AnxA1, in particular the 33-kDa form, have cups. We show here that inhibiting AnxA1 led to decreased been noted in bronchoalveolar lavage fluid of patients with lung dis- ICAM1 clustering around the transmigrating neutrophil, suggest- eases, such as cystic fibrosis, idiopathic pulmonary fibrosis, and in- ing that AnxA1 regulates the degree of ICAM1 clustering. Con- terstitial lung disease (19–21). Reports that the 33-kDa form has lost sistent with the hypothesis proposed by Yang et al. (33) that high the capacity to suppress inflammation (19, 20), and that it is induced levels of ICAM1 clustering is essential for the transcellular route

by allergens (19), together with observations that its presence in of TEM, inhibition of AnxA1 resulted in the loss of transcellular http://www.jimmunol.org/ bronchoalveolar lavage fluid closely correlated with the severity TEM without affecting paracellular TEM. Furthermore, the effect of disease and neutrophil infiltration with no detectable full length of AnxA1 inhibition on clustering and transcellular TEM is sim- protein present in some patients (21), further support our thesis that ilar to the effect of loss of the intracellular domain of ICAM1 (33). the C-terminal 33-kDa AnxA1 is proinflammatory. Because the intracellular domain of ICAM1 is important for AnxA1 is a protein that is highly abundant in neutrophils. Upon linkage to the actin cytoskeleton, we propose that AnxA1 acti- neutrophil stimulation and adherence to the endothelium, AnxA1 is vation of endothelial ERK is needed for signaling events driving externalized to the plasma membrane, the majority of which is the the higher order clustering of ICAM1, which may be dependent on intact form (22). Although AnxA1 does not possess classical linkage to the actin cytoskeleton through the intracellular domain signal sequences to target the protein for export, both the full- of ICAM1. Future studies will aim to address this hypothesis. by guest on September 30, 2021 length and truncated forms are often observed in extracellular fluids and inflammatory exudates (19, 23–25). It appears that pro- Acknowledgments teolytic cleavage of AnxA1 is required for it to be secreted, be- We thank Jenny Drew and Anna Sapa for the preparation and culture of cause the majority of AnxA1 released from neutrophils is N- HUVEC and the staff members of the Women’s and Children’s Hospital terminally cleaved (26, 27). and Burnside Hospital (Adelaide, South Australia) for the collection of Ourdatasuggestthat,ofanumberofproteasesincludingcathepsin umbilical cords. D, calpain 1, and plasmin (15) that have previously been shown to cleave AnxA1 within the first 30 aa, only calpain 1 which cleaves Disclosures after lys-26 was able to generate a truncated form that can activate The authors have no financial conflicts of interest. endothelial Erk (data not shown for cathepsin D). Our data also show that calpain 1 is the physiologic protease for generating the ERK-activating form of AnxA1 during neutrophil activation. Cal- References 1. Kunkel, S. L., N. Lukacs, T. Kasama, and R. M. Strieter. 1996. The role of pain 1 is a ubiquitously expressed protein, and its expression and chemokines in inflammatory joint disease. J. Leukoc. Biol. 59: 6–12. pleiotropic role in neutrophils ranging from extravasation to apo- 2. Springer, T. A. 1994. Traffic signals for lymphocyte recirculation and leukocyte ptosis is well documented in the literature (28–30). It is of particular emigration: the multistep paradigm. Cell 76: 301–314. 3. Butcher, E. C. 1991. Leukocyte-endothelial cell recognition: three (or more) interest to note that plasmin, which cleaves 3 aa C-terminal of steps to specificity and diversity. Cell 67: 1033–1036. calpain 1, was not able to convert AnxA1 to the form that activates 4. Muller, W. A. 2009. Mechanisms of transendothelial migration of leukocytes. endothelial ERK, suggesting that aa 27–29, which are removed Circ. Res. 105: 223–230. 5. Wittchen, E. S. 2009. Endothelial signaling in paracellular and transcellular by plasmin cleavage, must be crucial for the proinflammatory leukocyte transmigration. Front. Biosci. 14: 2522–2545. activity of AnxA1. It is also interesting to note that the residues 6. Allport, J. R., W. A. Muller, and F. W. Luscinskas. 2000. Monocytes induce reversible focal changes in vascular endothelial cadherin complex during that mediate the proinflammatory functions of AnxA1 are juxta- transendothelial migration under flow. J. Cell Biol. 148: 203–216. posed against the residues that are important for its antiinflamma- 7. Shaw, S. K., P. S. Bamba, B. N. Perkins, and F. W. Luscinskas. 2001. Real-time tory activity, identified by other studies (31) as residues 19–26. imaging of vascular endothelial-cadherin during leukocyte transmigration across endothelium. J. Immunol. 167: 2323–2330. Hence calpain 1 is unique in that it cleaves in the junction between 8. Carman, C. V., and T. A. Springer. 2004. A transmigratory cup in leukocyte residues crucial for the antiinflammatory and proinflammatory diapedesis both through individual vascular endothelial cells and between them. functions of AnxA1, thus removing the antiinflammatory residues J. Cell Biol. 167: 377–388. 9. Shaw, S. K., S. Ma, M. B. Kim, R. M. Rao, C. U. Hartman, R. M. Froio, L. Yang, from the full-length protein while maintaining its proinflammatory T. Jones, Y. Liu, A. Nusrat, et al. 2004. Coordinated redistribution of leukocyte residues. Further study will be needed to address how the calpain LFA-1 and endothelial cell ICAM-1 accompany neutrophil transmigration. J. Exp. Med. 200: 1571–1580. 1-cleaved AnxA1 interacts with the endothelium and initiates 10. Stein, B. N., J. R. Gamble, S. M. Pitson, M. A. Vadas, and Y. Khew-Goodall. ERK signaling. Full-length AnxA1 and its N-terminal peptide 2003. Activation of endothelial extracellular signal-regulated kinase is essential The Journal of Immunology 3063

for neutrophil transmigration: potential involvement of a soluble neutrophil fac- 22. Perretti, M., J. D. Croxtall, S. K. Wheller, N. J. Goulding, R. Hannon, and tor in endothelial activation. J. Immunol. 171: 6097–6104. R. J. Flower. 1996. Mobilizing lipocortin 1 in adherent human leukocytes 11. Lim, L. H., and S. Pervaiz. 2007. Annexin 1: the new face of an old molecule. downregulates their transmigration. Nat. Med. 2: 1259–1262. FASEB J. 21: 968–975. 23. Vishwanatha, J. K., R. G. Davis, I. Rubinstein, and A. Floreani. 1998. Annexin I 12. Gamble, J. R., Y. Khew-Goodall, and M. A. Vadas. 1993. Transforming growth degradation in bronchoalveolar lavage fluids from healthy smokers: a possible factor-beta inhibits E-selectin expression on human endothelial cells. J. Immu- mechanism of inflammation. Clin. Cancer Res. 4: 2559–2564. nol. 150: 4494–4503. 24. Vergnolle, N., C. Come´ra, and L. Bueńo. 1995. Annexin 1 is overexpressed and 13. Rothhut, B., C. Comera, B. Prieur, M. Errasfa, G. Minassian, and F. Russo- specifically secreted during experimentally induced colitis in rats. Eur. J. Bio- Marie. 1987. Purification and characterization of a 32-kDa phospholipase A2 chem. 232: 603–610. 25. Tsao, F. H., K. C. Meyer, X. Chen, N. S. Rosenthal, and J. Hu. 1998. Degra- inhibitory protein (lipocortin) from human peripheral blood mononuclear cells. dation of annexin I in bronchoalveolar lavage fluid from patients with cystic FEBS Lett. 219: 169–175. fibrosis. Am. J. Respir. Cell Mol. Biol. 18: 120–128. 14. Goulet, F., K. G. Moore, and A. C. Sartorelli. 1992. Glycosylation of annexin I 26. Perretti, M. 1997. Endogenous mediators that inhibit the leukocyte-endothelium and annexin II. Biochem. Biophys. Res. Commun. 188: 554–558. interaction. Trends Pharmacol. Sci. 18: 418–425. 15. Wang, W., and C. E. Creutz. 1994. Role of the amino-terminal domain in regulating 27. Rescher, U., V. Goebeler, A. Wilbers, and V. Gerke. 2006. Proteolytic cleavage interactions of annexin I with membranes: effects of amino-terminal truncation of annexin 1 by human leukocyte elastase. Biochim. Biophys. Acta 1763: 1320– and mutagenesis of the phosphorylation sites. Biochemistry 33: 275–282. 1324. 16. Perretti, M., A. Ahluwalia, J. G. Harris, N. J. Goulding, and R. J. Flower. 1993. 28. Pontremoli, S., E. Melloni, G. Damiani, F. Salamino, B. Sparatore, M. Michetti, Lipocortin-1 fragments inhibit neutrophil accumulation and neutrophil- and B. L. Horecker. 1988. Effects of a monoclonal anti-calpain antibody on dependent edema in the mouse. A qualitative comparison with an anti-CD11b response of stimulated human neutrophils. J. Biol. Chem. 263: 1915–1919. monoclonal antibody. J. Immunol. 151: 4306–4314. 29. Nuzzi, P. A., M. A. Senetar, and A. Huttenlocher. 2007. Asymmetric localization 17. Gavins, F. N., S. Yona, A. M. Kamal, R. J. Flower, and M. Perretti. 2003. of calpain 2 during neutrophil chemotaxis. Mol. Biol. Cell 18: 795–805. Leukocyte anti-adhesive actions of annexin 1. Blood 101: 4140–4147. 30. van Raam, B. J., A. Drewniak, V. Groenewold, T. K. van den Berg, and 18. Roviezzo, F., S. J. Getting, M. J. Paul-Clark, S. Yona, F. N. E. Gavins, T. W. Kuijpers. 2008. Granulocyte colony-stimulating factor delays neutrophil M. Perretti, R. Hannon, J. D. Croxtall, J. C. Buckingham, and R. J. Flower. apoptosis by inhibition of calpains upstream of caspase-3. Blood 112: 2046– Downloaded from 2002. The annexin-1 knockout mouse: what it tells us about the inflammatory 2054. response. J. Physiol. Pharmacol. 53: 541–553. 31. Walther, A., K. Riehemann, and V. Gerke. 2000. A novel ligand of the formyl 19. Chung, Y. W., H. Y. Oh, J. Y. Kim, J. H. Kim, and I. Y. Kim. 2004. Allergen- peptide receptor: annexin I regulates neutrophil extravasation by interacting with the FPR. Mol. Cell 5: 831–840. induced proteolytic cleavage of annexin-1 and activation of cytosolic phospho- 32. Hayhoe, R. P., A. M. Kamal, E. Solito, R. J. Flower, D. Cooper, and M. Perretti. lipase A2 in the lungs of a mouse model of asthma. Proteomics 4: 3328–3334. 2006. Annexin 1 and its bioactive peptide inhibit neutrophil-endothelium inter- 20. Smith, S. F., T. D. Tetley, A. Guz, and R. J. Flower. 1990. Detection of lipocortin actions under flow: indication of distinct receptor involvement. Blood 107: 1 in human lung lavage fluid: lipocortin degradation as a possible proteolytic 2123–2130. mechanism in the control of inflammatory mediators and inflammation. Environ.

33. Yang, L., R. M. Froio, T. E. Sciuto, A. M. Dvorak, R. Alon, and http://www.jimmunol.org/ Health Perspect. 85: 135–144. F. W. Luscinskas. 2005. ICAM-1 regulates neutrophil adhesion and transcellular 21. Kurosu, K., Y. Takiguchi, O. Okada, N. Yumoto, S. Sakao, Y. Tada, Y. Kasahara, migration of TNF-alpha-activated vascular endothelium under flow. Blood 106: N. Tanabe, K. Tatsumi, M. Weiden, et al. 2008. Identification of annexin 1 as 584–592. a novel autoantigen in acute exacerbation of idiopathic pulmonary fibrosis. J. 34. Baldwin, A. L. 1999. Modified hemoglobins produce venular interendothelial Immunol. 181: 756–767. gaps and albumin leakage in the rat mesentery. Am. J. Physiol. 277: H650–H659. by guest on September 30, 2021