Endothelial Light Chain Kinase Regulates Neutrophil Migration Across Human Umbilical Vein Endothelial Cell Monolayer

This information is current as Hajime Saito, Yoshihiro Minamiya, Michihiko Kitamura, Satoshi of October 2, 2021. Saito, Katsuhiko Enomoto, Kunihiko Terada and Jun-ichi Ogawa J Immunol 1998; 161:1533-1540; ; http://www.jimmunol.org/content/161/3/1533 Downloaded from References This article cites 45 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/161/3/1533.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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Endothelial Myosin Light Chain Kinase Regulates Neutrophil Migration Across Human Umbilical Vein Endothelial Cell Monolayer1

Hajime Saito,* Yoshihiro Minamiya,2* Michihiko Kitamura,* Satoshi Saito,* Katsuhiko Enomoto,† Kunihiko Terada,‡ and Jun-ichi Ogawa*

Although extravasation of neutrophils is a critical step in acute inflammation, the role of the endothelial in neutrophil transmigration has not been fully investigated. We used an in vitro model of neutrophil transmigration across a monolayer of HUVEC cultured on amniotic membrane. Human neutrophils were allowed to migrate across the HUVEC monolayer in response to a gradient leukotriene B4 and then the number of migrated neutrophils were counted microscopically. We also followed

endothelial F- and myosin filament formation using rhodamine-phalloidin and anti-myosin Ab staining. Myosin light chain Downloaded from (MLC) phosphorylation in endothelial cells was determined by immunoprecipitation of 32P-labeled HUVEC with anti-myosin polyclonal Ab. Normally, neutrophil migration induced F-actin formation, myosin filament formation, and MLC phosphorylation in HUVEC. When HUVEC was pretreated with the myosin light chain kinase (MLCK) inhibitor, ML-9, neutrophil migration was diminished and F-actin formation, myosin filament formation, and MLC phosphorylation were inhibited. Pretreatments of HUVEC with the intracellular calcium ion chelator, bis-(O-aminophenoxyl)ethane-N, N, N؅, N؅-tetraacetic acid acetoxymethyl

ester (BAPTA/AM), and the antagonist, trifluoperazine, had similar effects. These results indicate that a calcium/ http://www.jimmunol.org/ calmodulin-dependent MLCK in endothelial cells regulates neutrophil transendothelial migration. The Journal of Immunology, 1998, 161: 1533–1540.

critical step in acute inflammation is migration of the discontinuities at the tricellular corner where the majority of neu- circulating neutrophil from the vascular compartment trophils migrate across (14). A through a gap between adjacent endothelial cells into Phosphorylation of myosin II light chain (MLC) by a calcium/ surrounding tissue (1–3). Substantial evidence has accumulated calmodulin-dependent kinase is considered to be an essential step demonstrating that adhesion molecules, such as ␤2 integrin (4–7), in contraction of smooth muscle cells (15–18) and other cells (19– ICAM-1 (6–8), and platelet endothelial cell adhesion molecule-1 21) through interaction of actin and myosin (22, 23) and formation by guest on October 2, 2021 (PECAM-1)3 (9–11), are required for initiation of neutrophil of myosin II filaments (24–27). There have also been reports that transendothelial migration. Mechanisms involved after neutrophil the actin filament binds directly to the adherence junction-associ- adhesion have received less attention. In particular, signaling ated , ␣- (28, 29), and that ␣-, which is mechanisms triggered within endothelial cells during neutrophil cross-linked to the actin filament, binds to the tight junction asso- transmigration are not well understood. Huang et al. have provided ciated-protein, ZO-1 (30). Histamine and thrombin-induced phos- data indicating that cytosolic calcium-dependent endothelial sig- phorylation of MLC have been shown to initiate endothelial cell naling results in creation of gaps at junctions between adjacent retraction (31–35) and to result in disassembly of the adherence endothelial cells through which neutrophils are able to pass (12). junction complex (36). Taken together, these findings support a There are two kinds of endothelial cell junctions (ECJ) described hypothesis that phosphorylation of MLC induces opening gaps be- as tight junctions and adherence junctions (13, 14), both displaying tween endothelial cells associated with tricellular corners and with ECJ. In the present study we examine the hypothesis that MLCK in endothelial cells plays an active role in transendothelial migration † ‡ *Second Department of Surgery, First Department of Pathology, and First Depart- of neutrophils. To assess MLC phosphorylation during neutrophil ment of , Akita University School of Medicine, Hondo Akita City, Japan transmigration, we used an in vitro model consisting of a mono- Received for publication September 29, 1997. Accepted for publication April 8, 1998. layer of HUVEC cultured on amniotic membrane (37). We also The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance investigated the effect of inhibition of MLCK on migration of neu- with 18 U.S.C. Section 1734 solely to indicate this fact. trophils across a HUVEC monolayer. Our results are in accord 1 This work was supported by Grant-in-Aid for Scientific Research (C) from the with a calcium/calmodulin-dependent MLCK acting to regulate Ministry of Education, Science, Sports, and Culture of Japan 08671507 (Y.M.). transendothelial neutrophil migration. 2 Address correspondence and reprint requests to Dr. Yoshihiro Minamiya, Assistant Professor of Pulmonary Surgery, Second Department of Surgery, Akita University School of Medicine, 1-1-1 Hondo Akita City 010-8543, Japan. E-mail address: [email protected]. Materials and Methods 3 Abbreviations used in this paper: PECAM-1, platelet endothelial cell adhesion mol- Abs and reagents ecule-1; ECJ, interendothelial junction; MLC, myosin II light chain; MLCK, myosin light chain kinase; anti-M II pAb, anti-human platelet myosin II rabbit polyclonal Ab; Anti-human platelet myosin II rabbit polyclonal Ab (32) (anti-M II pAb) 2ϩ was kindly provided by Dr. Robert Wysolmerski (St. Louis University, St. LTB4, leukotriene B4; [Ca ]i, intracellular calcium ion; BAPTA/AM, bis-(O-amino- Ј Ј phenoxyl)ethane-N, N, N , N -tetraacetic acid acetoxymethyl ester; IC50, 50% inhib- Louis, MO). The anti-chicken myosin light chain mouse mAb MY-21, itory concentration. which cross-reacts with human MLC, was purchased from Sigma (St.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 1534 ENDOTHELIAL CELLS REGULATE NEUTROPHIL MIGRATION

Louis, MO). Leukotriene B4 (LTB4) was kindly provided by Ono Phar- at room temperature with anti-M II pAb diluted with PBS containing 0.1% maceutical (Osaka, Japan). Rhodamine-phalloidin and bis-(O-aminophe- BSA (final Ab concentration: 0.75 mg/ml). After rinsing with PBS con- noxyl) ethane-N, N, NЈ, NЈ-tetraacetic acid acetoxymethyl ester (BAPTA/ taining 0.1% BSA, the membrane was incubated at room temperature for AM) were purchased from Molecular Probes (Eugene, OR). 1 h with secondary Ab, anti-rabbit IgG pAb conjugated with rhodamine. Trifluoperazine and ML-9 were purchased from Calbiochem (La Jolla, CA) After washing with PBS, several drops of 90% glycerol/10% PBS con- and Seikagaku Kogyo (Tokyo, Japan), respectively. taining 0.1 M N-propylgallate were added and the membrane was covered with a coverslip. Finally, the membrane was examined using a confocal Assay of transendothelial migration of neutrophils laser scanning microscope system (LSM 410, Zeiss, Oberkochen, Ger- many) coupled to a Axioverd 135 fluorescence microscope (Zeiss). HUVEC culture. HUVEC were harvested by perfusion of umbilical vein with 0.25% trypsin (Life Technologies, Grand Island, NY) according to the Myosin light chain phosphorylation modified method of Jaffe et al. (38). Cell preparations were transferred to 60-mm plastic tissue culture dishes coated with type I collagen (Sigma), To analyze myosin phosphorylation, myosin was immunoprecipitated as and HUVEC were grown in M199 medium (Life Technologies) supple- described by Wysolmerski and Lagunoff (35, 41) with minor modifications. mented with 20% FCS, 100 U/ml penicillin G, and 100 ␮g/ml streptomycin A confluent monolayer of HUVEC on an amniotic culture ring (diameter 32 while being maintained at 37°C in a humidified 5% CO2, 95% air atmo- 90 mm) was labeled with [ P]orthophosphoric acid (DuPont-NEN, Bos- sphere. HUVEC were then seeded onto acellular human amniotic tissue ton, MA) as follows. HUVEC were washed twice with low phosphate according to the method of Furie and McHugh (37). Briefly, human am- medium (DMEM, Life Technologies) and then incubated for3hat37°C ␮ 32 niotic tissue obtained by cesarean section was secured to a glass ring with (humidified 5% CO2, 95% air) with 75 Ci/ml of [ P]orthophosphoric a rubber band (amniotic culture ring: 10 mm diameter for neutrophil trans- acid in the same. After washing HUVEC with PBS to remove excess free endothelial migration assay; 90 mm diameter for immunoprecipitation). [32P]orthophosphoric acid, a neutrophil transendothelial migration assay 6 The amniotic epithelium was removed by lysis with 0.25 N NH4OH for 1 h was performed. Briefly, 2.0 ϫ 10 neutrophils were added to the upper 32 at room temperature. Amniotic culture rings were stored for up to 1 mo at compartment of P-labeled HUVEC on an amniotic culture ring and LTB4 4°C in PBS containing 200 U/ml penicillin G and 200 ␮g/ml streptomycin. was added to the lower compartment to a concentration of 10Ϫ7 M. After Downloaded from HUVEC were seeded onto the stromal surface (the upper compartment) of being incubated for 10, 30, or 60 min, HUVEC on the amniotic culture ring the amniotic culture ring at a density of 1.5 ϫ 105 cells/cm2. The medium were washed three times with cold PBS, lysed with 600 ␮l of the lysis bathing the amniotic culture ring was replaced twice weekly. HUVEC gen- buffer (25 mM Tris-HCl, pH 7.9, 250 mM NaCl, 100 mM Na4P2O7,75mM erally reached confluence within 4 days and were used for experiments NaF, 0.5% sodium deoxycholate, 1% Nonidet P-40, 5 mM EGTA, 5 mM beginning 7 days after plating. We confirmed confluency by measuring EDTA, 0.2 ␮M PMSF, and 10 ␮g/ml leupeptin), incubated on ice for 30 electrical resistance (12, 39) with the Epithelial Voltohmmeter (World Pre- min and scraped with a rubber policeman. After rinsing the amniotic cul- cision Instruments, Sarasota, FL). The electrical resistance of a confluent ture membrane with 200 ␮l of the lysis buffer, the soluble cell extract was http://www.jimmunol.org/ HUVEC monolayer was 8.6 Ϯ 1.6 ohm/cm2 and this value agrees with centrifuged at 132,000 ϫ g for 10 min and the supernatant was collected. previous reports (39). The pellet was extracted again by incubation for 20 min with 200 ␮lofthe Neutrophil isolation. Human neutrophil were isolated from normal hep- lysis buffer containing 600 mM NaCl. The insoluble materials were pel- arinized (10 U/ml) venous blood by dextran sedimentation followed by leted at 132,000 ϫ g. The supernatant was diluted with an equal volume of Lymphoprep (Ficoll-Paque, Pharmacia Biotech, Upsala, Sweden) gradient the lysis buffer without NaCl and then combined with the initial sample. To centrifugation and hypotonic lysis of erythrocytes, as previously described avoid nonspecific binding, the sample was preincubated for 30 min at 4°C (40). Neutrophils were washed and resuspended in HEPES buffer contain- with 100 ␮l of 20% skim milk (pH 7.4) and 20 ␮l of 50% protein NG- ing 5 mg/ml of BSA and kept at 4°C until used in experiments. Sepharose, centrifuged for 10 min at 15,000 ϫ g, and the supernatant was Transendothelial migration assay. A total of 2.0 ϫ 106 purified neutro- removed. The supernatant was incubated overnight at 4°C with 20 ␮lof phils were added to the upper compartment of the amniotic culture ring anti-M II pAb (12.5 mg/ml). The next day the sample was incubated for 2 h covered by a confluent HUVEC monolayer and bathed in M199 medium at 4°C with 20 ␮l of 50% protein NG-Spharose and the immune complexes by guest on October 2, 2021 containing 20% FCS. LTB4 was added to the lower compartment to a bound to affinity media were collected by centrifugation for 5 min at concentration of 10Ϫ7 M and the apparatus was placed in a humidified 12,000 ϫ g. The pellets were washed first in 1 ml of lysis buffer, then incubator (37°C, 5% CO2, and 95% air) for 10 to 60 min. The amniotic washed once with a 1:1 dilution of lysis buffer/PBS, twice with PBS and, culture ring was then washed with cold PBS three times to remove non- finally, once with a 1:1 dilution of PBS/distilled water. Then pellets were adherent neutrophils and fixed with 2.5% glutaraldehyde in PBS (pH 7.3) boiled in Laemmli sample buffer (2% SDS, 10% glycerol, 100 mM DTT, for 24 h. The amniotic tissue was removed and embedded in paraffin. Cross-sections (4 ␮m thick) were prepared and stained with hematoxylin and eosin. Next, the number of neutrophils beneath the HUVEC monolayer (migrated neutrophils) and on the HUVEC monolayer (adherent neutro- phils) were counted under the microscope for a 5-mm length of monolayer. The values are divided by the length of the HUVEC monolayer examined so that we expressed our data as the number of neutrophils counted per millimeter of monolayer. F-actin and myosin II immunofluorescence HUVEC were cultured on 12-mm diameter (0.4 ␮m pore size) polycar- bonate membrane inserts (Millicell PCF, Millipore, Bedford, MA) in 24- well plates by incubation in M199 medium containing 20% FCS. Once HUVEC had reached confluency, 2.0 ϫ 106 neutrophils were transferred to the upper compartment above the insert and LTB4 was added to the lower compartment to a concentration of 10Ϫ7 M. The plates were placed in a humidified incubator (37°C, 5% CO2, and 95% air) for 10 to 60 min and, at the end of this incubation period, membranes were fixed in formalde- FIGURE 1. Design of neutrophil transendothelial migration assay. A 6 hyde. To assess actin polymerization (F-actin formation), HUVEC were total of 2.0 ϫ 10 neutrophils were added to the upper compartment of a stained with rhodamine-phalloidin. Briefly, the membrane was fixed in 3% HUVEC monolayer on the amniotic culture ring bathed in culture medium. buffered formaldehyde (pH 7.0) and then stained for 120 min at room To start neutrophil transendothelial migration, LTB4 was added to the temperature with a solution of 10 U/ml of rhodamine-phalloidin in PBS lower compartment to a concentration of 10Ϫ7 M and the amniotic culture containing 0.1% Triton X-100 and 1% BSA. After washing with PBS, ring was incubated in a humidified incubator at 37°C. At the end of this several drops of 90% glycerol/10% PBS containing 0.1 M N-propylgallate time, the amniotic culture ring was washed three times with PBS to remove were added and the membrane was covered with a coverslip. For myosin the nonadherent neutrophils. After that, a cross-section of the amniotic II staining, the membrane was fixed in 1% buffered formaldehyde (pH 6.5) tissue was cut and stained with hematoxylin and eosin. Panel a shows the for 1 min and incubated for 60 min with 2% formaldehyde in PBS con- Ϫ7 taining 0.2% Triton X-100 and 0.5% sodium deoxycholate. After washing cross-section of the amniotic tissue with 10 MofLTB4. We defined the with PBS three times, the membrane was incubated for 2 min with 10 mM neutrophil beneath HUVEC monolayer and the neutrophil on the HUVEC sodium borohydrate. This was followed by a 60-min incubation with PBS monolayer, as the migrated neutrophil and adherent neutrophil, respec- containing 1% BSA. Next, the membrane was immunolabeled for 120 min tively (panel b). EC, endothelial cell; arrowhead, neutrophil; bar, 25 ␮m. The Journal of Immunology 1535

60 mM Tris-HCl, pH 6.8, and 0.001% bromphenol blue) and subjected to polyacrylamide gel electrophoresis (12–15%). The gel was stained with Coomassie blue, dried using a gel drier, and exposed to Fuji RX-U x-ray film. The film was developed with a Konica automatic processor SRX-101. A 19-kDa protein in the immunoprecipitate was considered to be MLC (35, 41) and this was confirmed by immunoblotting with the mouse anti- chicken MLC mAb, MY-21 (data not shown). Inhibitory experiments

2ϩ To determine the effect of chelation of intracellular calcium ion ([Ca ]i) and of inhibition of calmodulin and MLCK on the endothelial cytoskeleton and on transmigration of neutrophils, a series of experiments were con- ducted. HUVEC on an amniotic culture ring or a polycarbonate membrane 2ϩ were preincubated at 37°C for 30 min with the [Ca ]i chelator, BAPTA/AM (50, 100, 200 ␮M), with the calmodulin inhibitor, trifluoper- azine (10, 50, and 100 ␮M), or with the MLCK inhibitor, ML-9 (5, 50, and 300 ␮M). After washing three times with PBS, assay of neutrophil trans- FIGURE 3. Effect of inhibitors on neutrophil transendothelial migra- endothelial migration, staining of actin and myosin II, and assessment of tion. The HUVEC monolayer on an amniotic culture ring was preincubated MLC phosphorylation were performed as before. 2ϩ with the [Ca ]i chelator, BAPTA/AM, the calmodulin inhibitor, triflu- Statistics operazine, or the MLCK inhibitor, ML-9. After washing three times with PBS, the standard transendothelial migration assay was conducted. All Values are expressed as the mean Ϯ SD. The significance of any difference Downloaded from three agents inhibited neutrophil migration across the HUVEC monolayer between groups was assessed by one-way analysis of variance with the Scheffe’s multiple comparison test. Differences between groups were con- in a dose-dependent manner. Blank bars, control without inhibitor; solid Ͻ ϭ sidered significant for any p Ͻ 0.05. bars, pretreated with inhibitors; *, p 0.05. n 5 for each group. Results Neutrophil transendothelial migration assay ing of the number of neutrophils beneath HUVEC monolayer after

staining a cross-section of amniotic tissue. To find the appropriate http://www.jimmunol.org/ Figure 1 shows the experimental design of the neutrophil migra- dose of LTB4, the transendothelial migration assay was performed tion assay. The number of neutrophils migrating across the using various concentrations of LTB4 (Fig. 2A). The maximum Ϫ HUVEC monolayer toward LTB4 was quantified by actual count- 7 number of migrated neutrophils was observed at 10 M LTB4. Figure 2B shows the time course of neutrophil transendothelial migration. The highest rate of neutrophil migration across the HUVEC monolayer was observed between 15 min and 30 min. After 30 min, numbers of migrated neutrophils increased only slightly. Therefore, we selected 10Ϫ7 M as the concentration of by guest on October 2, 2021 LTB4 and 30 min as the incubation time for later experiments. 2ϩ It has been reported that MLCK is a [Ca ]i/calmodulin-depen- dent kinase in endothelial cells (41). In an effort to investigate the role of MLCK in endothelial cells during neutrophil transmigra- tion, we determined the effects of inhibition of component mech- anisms contributing to MLCK activity. HUVEC were pretreated 2ϩ with the chelator of [Ca ]i, BAPTA/AM, the calmodulin inhib- itor, trifluoperazine, and the MLCK inhibitor, ML-9, respectively. After removing excess of inhibitors by washing with PBS, the

Table I. Effect of inhibitors on neutrophil adherence to HUVECa

Inhibitors Dose (␮M) No. Adherent Neutrophils (/mm)

BAPTA/AM 0 4 27.0 Ϯ 1.8 50 5 33.0 Ϯ 4.6 100 5 29.2 Ϯ 5.4 200 5 25.8 Ϯ 6.6 Ϯ FIGURE 2. Effect of concentration of LTB4 and time course of neutro- Trifluoperazine 0 3 26.0 2.7 Ϯ phil transendothelial migration. To find the optimal dose of LTB4 for later 10 5 30.0 4.7 experiments, the neutrophil transendothelial migration assay was per- 50 5 24.6 Ϯ 4.5 100 5 31.2 Ϯ 4.9 formed with different concentrations of LTB4 for an incubation period of 30 min. Neutrophils beneath the HUVEC monolayer were defined as mi- ML-9 0 4 28.0 Ϯ 3.4 grated neutrophils and were quantified as the number of neutrophils/mil- 5 5 29.6 Ϯ 5.9 Ϯ limeter monolayer. Maximal neutrophil migration was observed at a LTB4 50 5 33.2 5.6 concentration of 10Ϫ7 M(A). Panel B shows the time course of neutrophil 300 5 31.2 Ϯ 6.3 ϫ 6 migration. A total of 2.0 10 neutrophils were added to the upper com- a After HUVEC were pretreated with inhibitors (BAPTA/AM, trifluoperazine, and partment of the amniotic culture ring and LTB4 in the lower compartment ML-9) as before, neutrophils and LTB4 were added to the upper and lower compart- was 10Ϫ7 M. The amniotic culture ring was incubated for different periods ments of the HUVEC amniotic culture ring, respectively. Following an incubation at of time (5–120 min). The largest increase in neutrophils migrating across 37°C for 30 min, HUVEC were washed with PBS three times. The number of ad- ϭ herent neutrophils was then counted using a microscope. There was no significant the HUVEC monolayer was observed between 15 and 30 min. n 5 for difference in the number of neutrophils adhering to HUVEC pretreated with inhibitors each group. when compared with untreated HUVEC. 1536 ENDOTHELIAL CELLS REGULATE NEUTROPHIL MIGRATION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Immunofluorescence labeling of endothelial F-actin and myosin II during neutrophil transendothelial migration. Time course of redistri- bution of F-actin (a–f) and myosin II (g–l) is shown. Neutrophils (2.0 ϫ 106) were layered above a HUVEC monolayer on a polycarbonate membrane insert Ϫ7 and medium in the lower compartment of the well was made 10 M with respect to LTB4. Then, the 24-well plate was incubated at 37°C for different periods of time (10, 30, and 60 min). F-actin was visualized by rhodamine-phalloidin staining. Myosin II was visualized by indirect immunostaining with The Journal of Immunology 1537 Downloaded from http://www.jimmunol.org/

FIGURE 5. Effects of inhibitors on endothelial F-actin and myosin II redistribution. A monolayer of HUVEC on a polycarbonate membrane insert was 2ϩ ␮ ␮ ␮ pretreated with the [Ca ]i chelator, BAPTA/AM (200 M), the calmodulin inhibitor, trifluoperazine (100 M), or the MLCK inhibitor, ML-9 (50 M). After washing with PBS three times, neutrophils (2.0 ϫ 106) were layered above the HUVEC monolayer, and medium in the lower compartment of the well was made Ϫ7 10 M with respect to LTB4. Next, the 24-well plate was incubated for 30 min at 37°C. Then the polycarbonate membrane insert was fixed and stained with rhodamine-phalloidin (a–c) and anti-M II pAb followed by rhodamine-labeled anti-rabbit IgG Ab (d–f). Panels a and d, b and e, and, c and f depict HUVEC treated

with BAPTA/AM, trifluoperazine, and ML-9, respectively. The images were observed using confocal laser scanning microscopy. A rim of F-actin staining was by guest on October 2, 2021 present at cell margins with a few randomly disoriented stress fibers within the cytoplasm (a–c). Myosin II was diffusely localized within the cytoplasm and exhibited no obvious organization (d–f). These patterns were identical to those observed in controls (Fig. 4, a, g). Therefore, pretreatment with the inhibitors prevented the formation of actin and myosin II filaments in endothelial cells, changes induced by neutrophil transmigration (Fig. 4, b–d and h–j). neutrophil transendothelial migration assay was repeated. We also margins of cells, with a few randomly disoriented stress fibers investigated the effect of these inhibitors on neutrophil adhesion. within the cytoplasm (Fig. 4a). Myosin II was different in that Pretreatment with all three agents inhibited neutrophil migration distribution of this protein was localized diffusely through the cy- across the HUVEC monolayer in a dose-dependent manner (Fig. toplasm and exhibited no obvious organization (Fig. 4g). In the

3). By contrast, these agents did not influence neutrophil adhesion presence of neutrophils and LTB4, F-actin rapidly formed orga- (Table I). Therefore, inhibition of neutrophil migration across nized filamentous networks (Fig. 4, b, c, and d). Myosin II also HUVEC monolayer by any of these inhibitors was not due to the underwent redistribution to form organized filamentous networks inhibition of neutrophil adhesion to endothelial cells. These data (Fig. 4, h and i) but the changes were more progressive and peaked 2ϩ suggest that a [Ca ]i/calmodulin-dependent MLCK regulates at 60 min (Fig. 4j). Neutrophils alone and LTB4 alone caused a neutrophil migration across the HUVEC monolayer. slight increase in actin filament formation (Fig. 4, e and f), how- ever, neither had any effect on myosin II filament formation (Fig. F-actin and myosin II reorganization during neutrophil 4, k and l). These results indicated that neutrophil migration across transendothelial migration a HUVEC monolayer induced actin and myosin II filament for- To assess actin and myosin II reorganization during neutrophil mation in endothelial cells. transendothelial migration, HUVEC were stained with rhodamine- Since we showed that BAPTA/AM, trifluoperazine, and ML-9 phalloidin and anti-M II pAb, respectively. In the absence of neu- all inhibited neutrophil migration across a HUVEC monolayer in a trophils and LTB4, a rim of F-actin staining was present at the dose-dependent manner, we were interested in whether these anti-M II pAb followed by rhodamine-labeled anti-rabbit IgG pAb. The images were observed using confocal laser scanning microscopy. Panels a and g were images obtained in the absence of neutrophils and LTB4 (controls). Panels e and k show effects of neutrophils alone; f and l show effects of LTB4 alone. Panels b and h, c and i, d and j depict the image at 10, 30, and 60 min, respectively. A rim of F-actin staining was present at the margins of control cells with a few randomly disoriented stress fibers within the cytoplasm (a). Myosin II was diffusely localized within the cytoplasm and exhibited no organized pattern in control cells (g). Neutrophils alone and LTB4 alone caused a slight increase in actin filaments (e and f), but did not cause any change in the distribution of myosin II (k and l). During neutrophil transendothelial migration, both F-actin (b–d) and myosin II (h–j) underwent progressive redistribution forming organized filamentous networks. Bar, 10 ␮m. 1538 ENDOTHELIAL CELLS REGULATE NEUTROPHIL MIGRATION

center, resulting in inhibition of the catalytic activity of MLCK (42). Although, ML-9 inhibits the catalytic activities of the

other enzymes, the inhibition constant (Ki) values are at least an order of magnitude higher than for MLCK (42). For example,

the Ki values of cAMP-dependent protein kinase, protein kinase C, and Ca2ϩ phosphodiesterase are 32 ␮M, 54 ␮M, and 50 ␮M, ␮ respectively. The Ki value of ML-9 for MLCK was 3.8 M. In FIGURE 6. Endothelial MLC phosphorylation. Confluent HUVEC on the present study, incubation with 5 ␮M of ML-9 inhibited 50% 32 an amniotic culture ring were incubated with [ P]orthophosphoric acid of neutrophil transmigration (Fig. 3) and we feel that inhibition and washed with PBS to remove unincorporated label. Next, a neutrophil at this concentration would be specific for MLCK. The IC transendothelial migration assay was performed. Then cells were lysed as 50 (concentration producing a 50% inhibition) in a model of described in Materials and Methods and the lysates were immunoprecipi- ϩ Ϯ ␮ tated with anti-M II pAb. The time course of appearance of 32P-labeled smooth muscle contraction by high K was 17.5 0.4 M. In ␮ phosphorylated 19 kDa MLC is shown (a). Lane 1, basal phosphorylation; our experiments, we used 50 M of ML-9 and this concentra- lane 2, 10 min; lane 3, 30 min; and lane 4, 60 min. MLC was phosphor- tion almost completely inhibited the effects of neutrophil mi- ylated during neutrophil transendothelial migration in a time-dependent gration on mechanisms within the endothelial cells, including manner. To determine the effect of inhibitors, HUVEC were preincubated actin and myosin II filament formation and MLC phosphoryla- with BAPTA/AM (200 ␮M), trifluoperazine (100 ␮M), or ML-9 (50 ␮M) tion (Figs. 3, 5, and 6). Although this value was a three times as before, the neutrophil transendothelial migration assay was performed, higher concentration of the IC50, it still did not reach to IC50 for and cells were immunoprecipitated with anti-M II pAb. Pretreatment with other kinases estimated from Ki values. Therefore, we con- Downloaded from all three inhibitors almost completely inhibited endothelial MLC phosphor- cluded that endothelial MLCK regulates neutrophil migration ylation (b, lane 1, BAPTA/AM; lane 2, trifluoperazine; lane 3, ML-9; lane across a HUVEC monolayer. 4, positive control at 60 min). It has previously been reported that MLCK in the endothelial 2ϩ cell is [Ca ]i/calmodulin dependent (41). Therefore, to determine effects of chelation of [Ca2ϩ] and calmodulin inhibition in endo- agents also prevented changes in HUVEC F-actin and myosin II i

thelial cells, we repeated experiments after pretreating HUVEC http://www.jimmunol.org/ filament formation. HUVEC monolayers were pretreated with 200 with BAPTA/AM or trifluoperazine. Pretreatment with inhibitors ␮M of BAPTA/AM, 100 ␮M of trifluoperazine, or 50 ␮Mof inhibited neutrophil migration across the HUVEC monolayer as ML-9, respectively, and F-actin and myosin II staining were re- well as endothelial actin and myosin II filament formation, and peated. A rim of F-actin staining was still present at cell margins MLC phosphorylation. These results indicate that a [Ca2ϩ] /cal- with a few randomly disoriented stress fibers within the cytoplasm i modulin-dependent MLCK in endothelial cells regulates neutro- of endothelial cell (Fig. 5a–c), and myosin II staining remained phil transendothelial migration. diffusely localized within the cytoplasm of cells and exhibited no It has been well described that phosphorylation of MLC induced obvious organized pattern (Fig. 5, d–f). Therefore, pretreatment actin polymerization, endothelial cell centripetal retraction, and a with all three agents inhibited endothelial actin and myosin II fil- subsequent increase in the endothelial permeability (31–35). We by guest on October 2, 2021 ament formation. These patterns were identical to those observed speculate that the same mechanism regulates neutrophil trafficking in previous HUVEC experiments in the absence of neutrophil mi- across the endothelial barrier. The fact that actin and myosin II gration (Figs. 4a and g). filament formation, which we observed in endothelial cells during Endothelial MLC phosphorylation during neutrophil neutrophil migration, were similar to cytoskeletal changes ob- transendothelial migration served by other investigators in thrombin-stimulated endothelial cells (32) supports our speculation. On the other hand, other mech- To further investigate the role of endothelial MLCK, we studied anisms capable of regulating MLC phosphorylation, such as pro- MLC phosphorylation during neutrophil transmigration. Endothe- tein kinase C (43), cAMP-dependent protein kinase A (43), and lial MLC was progressively phosphorylated in a time-dependent myosin phosphatase inhibition (44) by Rho-kinase (45–47) were manner reaching near maximal levels at 30 min (Fig. 6a). We also recently reported. Thus, it is necessary to further investigate mech- determined the effects of inhibitors on this process. BAPTA/AM, anisms contributing to endothelial MLC phosphorylation during trifluoperazine, and ML-9 all almost completely inhibited endo- neutrophil transmigration and to examine the relationship between thelial MLC phosphorylation (Fig. 6b). these enzymes and MLCK. The present study and previous studies provide evidence that Discussion endothelial cell-dependent mechanisms regulate neutrophil trans- In this study we used HUVEC cultured on amniotic membranes to migration. Huang et al. demonstrated that cytosolic-free calcium in investigate the role of endothelial MLCK during neutrophil trans- the endothelial cell regulates neutrophil transendothelial migration migration. Isolated human neutrophils were allowed to migrate (12). This study suggests that some cytosolic calcium-dependent

across the HUVEC monolayer in response to LTB4. Using this endothelial signaling mechanisms open a gap between adjacent model system, we demonstrated that endothelial MLC phosphor- endothelial cells through which neutrophils can pass. Alloport et ylation, endothelial actin, and myosin II filament formation all oc- al. (48) reported that neutrophil endothelial adhesion triggers the curred during neutrophil migration across HUVEC monolayer. We disruption and degradation of the VE-cadherin complex at the ad- also demonstrated that pretreatment of HUVEC with the MLCK- herence junction. These endothelial cell-dependent changes then specific inhibitor, ML-9, inhibited neutrophil migration across regulate neutrophil transendothelial migration. On the other hand, HUVEC monolayer as well as MLC phosphorylation and filament Burns et al. recently demonstrated that 75% of neutrophils migrate formation in endothelial cells. across at locations where the borders of these endothelial cells The most critical finding in this investigation was that ML-9 intersect, at tricellular corners. Here, tight junctions and adherence inhibited neutrophil migration across a HUVEC monolayer. junctions have discontinuities (14). The relationship between tri- ML-9, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4- cellular corners and the cytoskeleton of endothelial cells has not diazepine, binds at or near the ATP-binding site of the active been investigated. The present study demonstrated that endothelial The Journal of Immunology 1539 cytoskeleton regulates neutrophil transendothelial migration. molecule-1 inhibit chemoattractant-stimulated neutrophil transendothelial migra- Taken together, we speculate that tricellular corners may also be tion in vitro. Blood 78:2089. 7. Smith, C. W., R. Rothlein, B. J. Hughes, M. M. Mariscalco, H. E. Rudloff, regulated by the endothelial cytoskeleton. F. C. Schmalstieg, and D. C. Anderson. 1988. Recognition of an endothelial 2ϩ determinant for CD 18-dependent human neutrophil adherence and transendo- We have demonstrated an active role for endothelial [Ca ]i/ calmodulin-dependent MLCK in neutrophil transendothelial mi- thelial migration. J. Clin. Invest. 82:1746. 8. Furie, M. B., M. J. Burns, M. C. Tancinco, C. D. Benjamin, and R. R. Lobb. gration. However, “the switch” that opens gaps between adjacent 1992. E-selectin (endothelial-leukocyte adhesion molecule-1) is not required for endothelial cells at tight junctions, adherence junctions, and tricel- the migration of neutrophils across IL-1-stimulated endothelium in vitro. J. Im- ␤ munol. 148:2395. lular corners remains unclear. Neutralization with Abs to 2 inte- 9. Romer, L. H., N. V. McLean, H. C. Yan, M. Daise, J. Sun, and H. M. DeLisser. grin on the neutrophil (4–7), ICAM-1 on the endothelial cell (6– 1995. IFN-␥ and TNF-␣ induce redistribution of PECAM-1 (CD31) on human 8), PECAM-1 on the endothelial cell, and the neutrophil (9–11) or endothelial cells. J. Immunol. 154:6582. 10. Bogen, S., J. Pak, M. Garifallou, X. Deng, and W. A. Muller. 1994. Monoclonal CD47 on the neutrophil (49) all inhibited neutrophil transendothe- antibody to murine PECAM-1 (CD31) blocks acute inflammation in vivo. J. Exp. lial migration. Therefore, ICAM-1, PECAM-1, and the unknown Med. 179:1059. ligand for CD47 on the endothelial cell are candidates for being 11. Vaporciyan, A. A., H. M. DeLisser, H. C. Yan, I. I. Mendiguren, S. R. Thom, M. L. Jone, P. A. Ward, and S. M. Albelda. 1993. Involvement of platelet- “the switch” on the endothelial cell required to open a gap between endothelial cell adhesion molecule-1 in neutrophil recruitment in vivo. Science adjacent endothelial cells. We speculate that neutrophils may stim- 262:1580. ulate “the switch” and that “the switch” may then release a signal 12. Huang, A. J., J. E. Manning, T. M. Bandak, M. C. Ratau, K. R. Hanser, and 2ϩ S. C. Silverstein. 1993. Endothelial cell cytosolic free calcium regulates neutro- to activate [Ca ]i/calmodulin-dependent MLCK. In turn, MLCK phil migration across monolayers of endothelial cells. J. Cell Biol. 120:1371. catalyzes myosin II filament formation (24–27) and myosin-actin 13. Walker, D. C., A. MacKenzie, and S. Hosford. 1994. The structure of the tricel- lular region of endothelial tight junctions of pulmonary capillaries analyzed by interaction (22, 23), and induces actin polymerization. It was re- Downloaded from freeze-fracture. Microvasc. Res. 48:259. ported that actin filament binds directly to the adherence junction- 14. Burns, A. R., D. C. Walker, E. S. Brown, L. T. Thurmon, R. B. Bowden, associated protein, ␣-catenin (28, 29), and that the tight junction- C. R. Keese, S. I. Simon, M. L. Entman, and C. W. Smith. 1997. Neutrophil associated protein, ZO-1, binds directly to a cross-linking protein transendothelial migration is independent of tight junctions and occurs preferen- tially at tricellular corner. J. Immunol. 159:2893. of the actin filament, ␣-spectrin (30). Therefore, actin polymeriza- 15. Corson, M. A., J. R. Sellers, R. S. Adelstein, and M. Schoenber. 1990. Substance tion may directly open these junctions. In fact, Rabiet et al. (36) P contracts bovine tracheal smooth muscle via activation of myosin light chain kinase. Am. J. Physiol. 259:C258.

demonstrated that thrombin, which phosphorylates MLC (31–35), http://www.jimmunol.org/ 16. Gerthoffer, W. T. 1991. Regulation of the contractile element of airway smooth induced disassembly of the adherence junction complex (36). The muscle. Am. J. Physiol. 261:L15. relationship between the tricellular corner and the cytoskeleton of 17. Haeberle, J. R., T. A. Sutton, and B. A. Trockman. 1988. Phosphorylation of two sites on smooth myosin effects on contraction of glycerinated vascular smooth endothelial cells has not been investigated. It is possible that the muscle. J. Biol. Chem. 263:4424. tricellular corner may be regulated by endothelial signal transduc- 18. Hai, C. M., and R. A. Murphy. 1989. Ca2ϩ, crossbridge phosphorylation, and tion related to MLCK. Although there are many signaling steps to contraction. Annu. Rev. Physiol. 51:285. 19. Cande, W. Z., and R. M. Ezzell. 1986. 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inhibition of catalytic activity of smooth muscle myosin light chain kinase. Transendothelial migration of neutrophils involves integrin-associated protein Downloaded from J. Biol. Chem. 262:7796. (CD47). Proc. Natl. Acad. Sci. USA 92:3978. http://www.jimmunol.org/ by guest on October 2, 2021