Biochemical and Biophysical Research Communications 292, 1092–1097 (2002)

doi:10.1006/bbrc.2002.6773, available online at http://www.idealibrary.com on

The Adhesion Receptor CD-31 Can Be Primed to RapidlyAdjust the Neutrophil Cytoskeleton

Ivan Dimitrijevic, Lena Axelsson, and Tommy Andersson1

Experimental Pathology, Department of Laboratory Medicine, Lund University, U-MAS, SE-205 02 Malmo, Sweden

Received March 12, 2002

The adhesion receptor CD-31 is expressed on neutro-phils and endothelial cells and participates in trans-endothelial migration of neutrophils. Although neces-sary, information on CD-31-induced signaling and itsinfluence on the shape-forming actin network isscarce. Here, we found that antibody engagement ofCD-31 on suspended neutrophils triggered a promptintracellular Ca2� signal, providing the cells had beenprimed with a chemotactic factor. Inhibition of Src-tyrosine kinases blocked this Ca2� signal, but not afMet-Leu-Phe-induced Ca2� signal. Despite the abilityof fMet-Leu-Phe to activate Src-tyrosine kinases, it didnot per se induce tyrosine phosphorylation of CD-31.However, fMet-Leu-Phe did enable such a phosphory-lation following an antibody-induced engagement ofCD-31. This clustering also triggered a Ca2�-dependentdepolymerization of actin and, surprisingly enough, asimultaneous polymerization. The ability of CD-31 tosignal dynamic alterations in the cytoskeleton, partic-ularly the Ca2�-induced actin depolymerization, fur-ther explains how neutrophils can squeeze themselvesout between adjacent endothelial cells. © 2002 Elsevier

Science (USA)

Key Words: actin; calcium; CD-31; human neutrophils;Src tyrosine kinases; transendothelial migration.

Polymorphonuclear leukocytes are guided to and ac-cumulate at sites of inflammation, and this highly co-ordinated multi-step process involves cytokines, che-moattractants, and different types of cell adhesionmolecules (1). An essential part of this process is theextravasation of leukocytes, a passing through vascu-lar cell walls that comprises at least three distinct

Abbreviations used: DMSO, dimethylsulfoxide; ECL, enhancedchemiluminescence; fMet-Leu-Phe, N-formyl-L-methionyl-L-leucyl-L-phenylalanine; PMSF, phenylmethylsulfonyl fluoride; RAM, rab-bit anti-mouse antibodies.

1 To whom correspondence should be addressed at ExperimentalPathology, Lund University, Malmo University Hospital, U-MAS,Entrance 78, SE-205 02 Malmo, Sweden. Fax: �46 40 33 73 53.E-mail: tommy.andersson@exppat.mas.lu.se.

10920006-291X/02 $35.00© 2002 Elsevier Science (USA)All rights reserved.

integrin-dependent adhesion; and transendothelial mi-gration (1, 2). It has been suggested that the last ofthese events, when leukocytes squeeze through endo-thelial cell junctions, requires the presence of func-tional CD-31 molecules on the surface of both the neu-trophils and the endothelial cells (2). The CD-31molecule, also designated PECAM-1 (platelet endothe-lial cell adhesion molecule 1), is a 130-kDa adhesionmolecule that belongs to the immunoglobulin super-family, members of which are expressed on the surfaceof all vascular cells, except erythrocytes (3).

Several findings support the notion that CD-31 playsan intimate role in the transendothelial migration ofleukocytes. For instance, it has been found that CD-31is localized to the lateral junctions between two neigh-boring endothelial cells (4), and antibodies against theCD-31 molecules on leukocytes and endothelial cellsblock the transmigration of the leukocytes without af-fecting their adhesion to endothelial cells (2). Further-more, in experiments conducted by other investigators(5), it was observed that CD-31 expression on transmi-grating monocytes was enriched in the endothelial con-tact region but was not detectable when these cellswere in the extravascular tissue, which implies thatthe role of CD-31 is related solely to the junction zonesbetween endothelial cells, or, in other words, to thetransmigration process. In addition, studies in CD-31-deficient mice have demonstrated that the transmigra-tion of leukocytes is delayed in a cytokine-specific man-ner (6). It is reasonable to assume that, to be able toparticipate in leukocyte transmigration, the CD-31 ad-hesion molecule must have the capacity to generateintracellular signals and to modulate the leukocyteactin cytoskeleton. In human neutrophils, it has beenshown that antibody-induced engagement of CD-31cause both up-regulation (7) and activation (8) of �2integrins. These results suggest that CD-31 expressedon neutrophils is capable of intracellular signaling.Despite this, the only support for such a concept hasbeen reported by Pellegatta and coworkers (9), whodetected an association between CD-31 and the p85

sequential events: selectin-mediated rolling; tight,

subunit of phosphatidylinositol 3-kinase in neutrophilsthat had been plated on a surface coated with anti-CD-31antibodies for as long as 30 min. However, in endothelialcells and platelets, engagement of CD-31 has been foundto cause an influx of Ca2�, and various natural and arti-ficial stimuli can induce a Src tyrosine kinase-dependentphosphorylation of CD-31 (10, 11, 12).

Obviously, during transendothelial migration, neu-trophils, as well as other types of cells that undertakesuch movement, must force themselves between adja-cent endothelial cells. This process requires rapid anddynamic alterations of the mechanical characteristicsof the motile cells, and these features are highly de-pendent on the organization of the cellular actin net-work (13). The actin cytoskeleton helps generate theforce needed to achieve changes in shape and motion,hence a rapid and dynamic modulation of the neutro-phil cytoskeleton is clearly essential for the transendo-thelial migration of these cells. So far, evidence of aqualitative link between CD-31 and rearrangement ofthe actin cytoskeleton has been found only in a mor-phological study of human natural killer cells (14).

Chemoattractants can modify the properties of ad-hesion receptors by altering their binding affinity andnumber on the cell surface, and also by transientlyimpairing their signaling capacity, as in the case of �2integrins (15). Compared with up-regulation of inte-grins on the cell surface, inhibition of �2 integrin sig-naling is a more rapid process. It is known that pro-longed exposure to the chemotactic factor N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe)down-regulates the number of CD-31 adhesion recep-tors on the surface of neutrophils (7), but the shortterm effects of fMet-Leu-Phe on CD-31 function inthese cells have not been elucidated. Inasmuch as neu-trophils adhere firmly to the endothelial cells beforebeginning the transmigration to extravascular tissues,it is possible that chemoattractants and other solublefactors can also modify the signaling capacity and func-tion of CD-31 at that stage. Consequently, extracellu-lar factors can probably participate in short-term reg-ulation of the functional properties of CD-31, similar towhat has been observed regarding �2 integrin adhe-sion molecules.

To better understand the mechanisms involved inregulation of neutrophil behavior during the intricateprocess of transendothelial migration, we investigatedthe signaling characteristics of CD-31 and the ability ofthis receptor to participate in modulation of the actincytoskeleton in human neutrophils.

MATERIALS AND METHODS

Materials. The chemicals used and their sources were as follows:phenylmethylsulfonyl fluoride (PMSF), fMet-Leu-Phe, cytochalasin D,inhibitor cocktail II, and dimethyl sulfoxide (DMSO), Sigma ChemicalCo. (St. Louis, MO); 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2�-amino-5�-methylphenoxy)-ethane-N,N,N�,N�-tetra-acetic

acid/penta-acetoxymethyl ester (fura-2/AM), Molecular Probes Inc. (Eu-gene, OR); an enhanced chemiluminescence (ECL) kit, Amersham In-ternational (Amersham, Bucks, UK); leupeptin-O and pepstatin,Boehringer-Mannheim (Mannheim, Germany); protein A-Sepharose,dextran, and Ficoll-Hypaque, Pharmacia Fine Chemicals (Uppsala,Sweden); electrophoresis reagents, Bio-Rad (Richmond, CA); nitrocellu-lose membrane, Schleicher and Schuell (Dassel, Germany); the Srctyrosine kinase inhibitor PP1, Alexis Biochemicals (San Diego, CA). Allother chemicals used were of analytical grade.

Antibodies. The monoclonal antibody directed against CD-31(IgG2a) and used for receptor engagement was purchased from R&DChemicals (Abingdon, UK); the rabbit anti-mouse (RAM) immuno-globulins were from Dakopatts (Glostrup, Denmark); and the anti-phosphotyrosine monoclonal antibody (clone 4G10; IgG2bk) wasfrom Upstate Biotech Inc. (Lake Placid, NY).

Isolation of human neutrophils. Blood from healthy donors wascollected and neutrophils were isolated under endotoxin-free condi-tions, according to a previously described method (16). Briefly, aftererythrocyte elimination by dextran sedimentation and a short hypo-tonic lysis, the cells were centrifuged on Ficoll-Hypaque at 800 � gfor 30 min to separate the polymorphonuclear leukocytes from mono-cytes, platelets, and lymphocytes. The polymorphonuclear leuko-cytes were then finally washed twice with a calcium-containing me-dium (136 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.1 mM CaCl2, 0.1mM EGTA, 1.2 mM KH2PO4, 5.0 mM NaHCO3, 5.5 mM glucose, and20 mM Hepes; pH 7.4) and then resuspended in the same medium ata concentration of 1.0 � 107 cells/ml. This cell suspension consistedof approximately 97% neutrophils.

Engagement of CD-31 adhesion molecules. Neutrophils weresuspended at a concentration of 5 � 106/ml in the calcium-containing medium (described above) and then incubated for 20min at 37°C with a monoclonal antibody against CD-31 (5 �g/ml),in the absence or presence of fura-2/AM. The cells were frequentlyagitated during this incubation, which was terminated by centrif-ugation at 250 � g for 3 min at room temperature. The cells werethen resuspended at a concentration of 2.5 � 106/ml in prewarmedcalcium-containing medium, or in the same medium withoutCaCl2 but with 1.0 mM EGTA (here called calcium-free medium).The cells were subsequently pre-incubated for 5 min in the ab-sence or presence of 10�7 M fMet-Leu-Phe, and the CD-31 mole-cules were engaged by exposure to RAM (final dilution 1:50).Pre-exposure to fMet-Leu-Phe was a prerequisite of CD-31 signal-ing, which strongly indicated that Fc receptor clustering was notresponsible for any of our results. However, to further excludesuch a possibility, we performed additional control experimentswith F(ab�)2 fragments, as previously described (17).

Measurement of cytosolic free Ca2�. Isolated neutrophils (5 �106/ml) were incubated for 20 min at 37°C with 2 �M fura2/AM inCa2�-containing medium; the anti-CD-31 antibody was presentduring this incubation as described above. The cells were thenwashed and resuspended in a cuvette containing 2 ml of thecalcium-free medium. Fluorescence was measured on a SPEXspectrofluorometer equipped with a thermostated (37°C) cuvetteholder and a continuous stirring device. Excitation wavelengthswere set at 340 nm and 380 nm, and the emission wavelength wasset at 505 nm. Cytosolic free Ca2� concentrations were calculatedas previously described (18).

Analysis of right angle light scattering. Neutrophils were pre-pared and handled according to the protocol outlined for measure-ment of cytosolic free Ca2� but excluding fura-2/AM. Right anglescattering as an indicator of the cellular F-actin content was ana-lyzed as described elsewhere (19), employing a SPEX spectroflu-orometer as indicated above. Excitation and emission wavelengthswere set at 340 nm. All measurements were monitored continuouslywhile the cells were stirred in the cuvette at 37°C.

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Immunoprecipitation and Western blot analysis. Neutrophilswere suspended in calcium-containing medium and treated as de-scribed in the figure legends (including incubation with a monoclonalantibody (5 �g/ml) against CD-31). The reactions were stopped byadding 5 ml of ice-cold calcium-containing medium supplementedwith (final concentrations) 0.1 mM PMSF and 0.4 mM Na3VO4. Thecells were then pelleted and resuspended in 1 ml of a buffer contain-ing 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 1%(v/v) Triton-X-100, 1.0 mM EGTA, 2.0 mM Na3VO4, 1 mM PMSF, 10�g/ml leupeptin, and 10 �g/ml aprotinin. Thereafter, the lysateswere left on ice for 30 min, stirring occasionally, and then centrifugedat 15,000 � g for 5 min at 4°C. The resulting supernatants were usedin subsequent immunoprecipitations. An aliquot (1 ml) of a super-natant was mixed with 40 �l of a 50:50% (v/v) slurry of proteinA-Sepharose and incubated at 4°C for 2 h under continuous rotation.The beads were then washed three times with a medium consistingof 20 mM Hepes (pH 7.5), 150 mM NaCl, 0.1% (v/v) Triton-X-100, and10% (v/v) glycerol, and the precipitated proteins were eluted byboiling for 10 min in Laemmli sample buffer. Electrophoresis wasperformed in 10% polyacrylamide gels, after which the resolvedproteins were electrophoretically transferred to nitrocellulose mem-branes. Specific monoclonal antibodies and ECL were used to detectthe presence of tyrosine-phosphorylated proteins and CD-31.

RESULTS

Ca2� Signaling by CD-31 in Suspended Neutrophils

Figure 1 outlines the initial cytosolic free Ca2� re-sponses (due to intracellular mobilization of Ca2�)caused by engagement of CD-31 or by the chemotacticpeptide fMet-Leu-Phe (10�7 M). Antibody engagementof CD-31 alone did not cause any detectable Ca2� signal(Fig. 1A, trace A), whereas receptor engagement after a5-min pre-incubation with fMet-Leu-Phe triggered aprompt CD-31-induced Ca2� transient after the fMet-Leu-Phe-induced Ca2� transient had declined (Fig. 1A,trace B). Prolonged exposure to fMet-Leu-Phe (�20min) abolished the subsequent CD-31-induced re-sponse (data not shown), which agrees with a reportindicating that fMet-Leu-Phe causes shedding ofCD-31 from the cell surface after approximately 20 min(7). Consequently, we could only investigate the CD-31-induced Ca2� signal in a calcium-depleted medium(used throughout our study), because in the presence ofextracellular Ca2�, the CD-31-evoked signal wasblunted by a prolonged fMet-Leu-Phe-induced signal,and after this signal had finally declined, the CD-31molecules were sloughed off.

In light of previous results showing that CD-31 canbe phosphorylated on tyrosine through an agonist-induced activation of a Src tyrosine kinase(s) in endo-thelial cells (10–12), we tested the effect of the Srcfamily tyrosine kinase inhibitor PP1 (5 �g/ml) on CD-31-induced Ca2� signaling in neutrophils. The cellswere exposed to 2 �M PP1 during loading with fura-2and also during subsequent recording of the fura-2fluorescent signal. The results show that PP1 com-pletely inhibited the CD-31-induced Ca2� signal buthad no effect on the fMet-Leu-Phe-induced Ca2� signal(Fig. 1B, traces A and B, respectively).

Effects of Pervanadate and fMet-Leu-Phe/CD-31 onTyrosine Phosphorylation of CD-31 in Neutrophils

To further elucidate involvement of a Src tyrosinekinase(s) in the signaling mechanism of CD-31, weinvestigated the tyrosine phosphorylation status ofCD-31 by immunoprecipitation and Western blottingwith an anti-phosphotyrosine mAb. The identity of the130-kDa protein band was verified by reprobing theblots with an anti-CD-31 mAb (Fig. 2, lower blots in Aand B). The phosphatase inhibitor pervanadate (Fig.2A, lanes 2 and 3) was used as a positive control forcomparison with the chemotactic factor fMet-Leu-Phe.The illustrated results clearly show that fMet-Leu-Phewas incapable of inducing any tyrosine phosphoryla-tion of the CD-31 molecule (Fig. 2A, lanes 4 and 5), andthe same applies to CD-31 engagement alone. How-ever, engagement of CD-31 on neutrophils that hadbeen prestimulated with fMet-Leu-Phe caused a tran-sient and readily discerned tyrosine phosphorylation ofthe CD-31 molecule (Fig. 2B). This finding is in accor-dance with the requirements for a CD-31-induced Ca2�

signal.

Effects of CD-31 Signaling on the F-Actin Contentin Suspended Neutrophils

We measured right angle light scattering as an in-dicator of F-actin dynamics (19) and found that engage-

FIG. 1. Antibody engagement of CD-31 on primed neutrophilstriggers a prompt release of intracellular Ca2�. Neutrophils wereincubated with fura 2/AM and a CD-31 monoclonal antibody for 20min in a calcium-containing medium in the absence (panel A, bothtraces, panel B trace B) or presence (panel B, trace A) of PP1. Thecells were subsequently washed and resuspended in a calcium-freemedium and then transferred to cuvettes, where they were notstimulated (panel A, trace A) or were stimulated with fMet-Leu-Phe(left arrows: panel A, trace B; panel B, both traces). In all four traces,the arrows to the right indicate engagement of CD-31 adhesionreceptors. Representative traces are shown.

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ment of CD-31 per se had no effect on this feature (Fig.3A). However, chemotactic stimulation of neutrophilswith fMet-Leu-Phe caused a rapid increase in the cel-lular content of F-actin, similar to that reported byother investigators (19; Fig. 3B, left arrow), and thistreatment also enabled a subsequent CD-31-elicited transient increase in F-actin (Fig. 3B, right arrow).

Addition of PP1 had no effect on the fMet-Leu-Phe-induced F-actin response (Fig. 3C, left arrow), whereasit abolished the subsequent increase caused by CD-31(Fig. 3C). To explore the role of the Ca2� signal in theF-actin response, we incubated the cells withMAPTAM-1 (10 �g/ml), which buffers any agonist-provoked alterations in the level of cytosolic free Ca2�.The presence of MAPTAM-1 had no effect on the fMet-Leu-Phe-induced F-actin response, but it caused a sus-tained increase in the CD-31-triggered F-actin re-sponse (Fig. 3D).

Effects of Cytochalasin on CD-31-Induced Ca2�

Signaling in Suspended Neutrophils

It has previously been demonstrated that the signal-ing capacity of neutrophil �2 integrin adhesion recep-tors is highly dependent on the cytoskeleton (20). Toascertain whether the same is true for CD-31-inducedsignaling, we pretreated neutrophils with cytochalsinD (10 �M), employing an approach identical to thatused for �2 integrins. Cytochalasin D had no obviouseffect on the fMet-Leu-Phe-induced Ca2� signal (Fig. 4,trace A in upper panel), whereas it completely blockedthe CD-31-induced signal (Fig. 4, trace B in upperpanel). This indicates that, as is the case for otheradhesion molecules on neutrophils, the signaling abil-

FIG. 2. Effects of fMet-Leu-Phe and CD-31 receptor engagementon the tyrosine phosphorylation status of the receptor. Panel A: theneutrophils were not stimulated at all (lane 1) or were treated with50 or 200 �M pervanadate (lanes 2 and 3, respectively), in compar-ison with these controls, we also stimulated cells with fMet-Leu-Phefor 2 or 4 min (lanes 4 and 5). Panel B: all samples came from cellsthat were first exposed to fMet-Leu-Phe for 5 min and subsequentlyhad their CD-31 receptors engaged for different periods of time (0, 2,4, 8, 10, and 20 min, shown in lanes 1–6, respectively). In bothpanels, after the described treatments, the cells were lysed on ice,and the lysates were used for immunoprecipitation of CD-31. Theimmunoprecipitated proteins were resolved on SDS-PAGE gel, andthen transferred to nitrocellulose membranes, which were blottedwith an antiphosphotyrosine antibody (4G10) and subsequently re-probed with an anti-CD-31 antibody. Representative blots areshown.

FIG. 3. The effect of CD-31 engagement on right angle lightscattering. Neutrophils were pre-incubated for 20 min in the absence(controls, designated A and B) or presence of PP1 (C) or MAPTAM-1(D). Subsequently, the cells were (B–D, left arrows) or were not (A,control) stimulated with fMet-Leu-Phe. Thereafter, the CD-31 recep-tors on the cells were engaged by antibody cross-linking (A–D, rightarrows), while continuously registering the right angle scatteringproperties. Representative traces are shown.

FIG. 4. The effects of cytochalasin D on the CD-31-induced Ca2�

signal in neutrophils and the right angle scattering response. Neu-trophils were incubated in the absence (panel A, trace A) or presence(panel A, trace B; panel B) of cytochalasin D. During on-line regis-tration of cytosolic free Ca2� (panel A) or right angle scattering(panel B), the cells were stimulated with fMet-Leu-Phe (left arrows),and their CD-31 receptors were subsequently engaged (right ar-rows). Representative traces are shown.

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ity of CD-31 depends on interaction with the cytoskel-eton. As a control of the impact of cytochalasin D, wefound that neither treatment with fMet-Leu-Phe norengagement of CD-31 had any effect on the cellularcontent of F-actin (lower panel in Fig. 4, left and rightarrows, respectively).

DISCUSSION

Previous studies have shown that CD-31 moleculesare essential for the extravasation of neutrophils (2,4–6). Complementary to those results, we found thatthe CD-31 adhesion molecule expressed on human neu-trophils can gain both signaling competence and theability to rapidly modulate the F-actin network. Inother words, CD-31 can rearrange the actin cytoskele-ton and thereby modify the shape of neutrophils duringtransendothelial migration.

We observed that a rapid CD-31-triggered Ca2� sig-nal and a subsequent and related modulation of theactin network in neutrophils were enabled only if thecells were subjected to a short pre-activation with thechemotactic factor fMet-Leu-Phe. This finding is incontrast to studies of endothelial cells showing thatantibody engagement of CD-31 alone readily evoked acytosolic calcium signal (10, 11), which indicates thatCD-31 signaling entails cell-specific mechanisms. How-ever, in regard to signaling properties, CD-31 resem-bles the �2 integrins on neutrophils, which can beexperimentally engaged by exposure to a fibronectin-coated surface, providing the cells have been pre-treated with either a cytokine (21) or a chemotacticfactor (22). It is logical to assume that the requirementfor pre-activation observed in these experimental mod-els mimics the situation that prevails in vivo whenneutrophils adhere to vascular endothelial cells. Ex-pression of CD-31 molecules on endothelial cells occursprimarily at the junctions between such cells, whichindicates that, after attaching to the vessel wall, neu-trophils crawl along the endothelial lining before theyapproach the tight junction zone and their CD-31 re-ceptors are engaged. This means that the neutrophilswould have ample time to be stimulated by cytokinesand/or chemotactic factors, such as fMet-Leu-Phe,prior to engagement of their CD-31 receptors. Conse-quently, the experimental procedures we found to benecessary for activation of CD-31 receptors can wellreflect a situation in vivo, in which neutrophils areprestimulated (primed for engagement) either beforeor after they adhere to the vessel wall.

Tyrosine phosphorylation of the cytoplasmic domainof CD-31 is believed to initiate the signaling cascade ofthis adhesion molecule. However, the CD-31 receptordoes not possess endogenous tyrosine kinase activity.Therefore, it was not surprising when we found that aSrc tyrosine kinase(s) participates in the regulation ofCD-31 functions, in light of their ability to phosphory-

late the cytoplasmic domain of CD-31 on tyrosine resi-dues in reconstitution experiment in COS-1 cells (23). Inendothelial cells, homophilic oligomerization of CD-31induces not only a Ca2� signal, but also tyrosine phos-phorylation of the cytoplasmic domain of this receptor(10). In contrast to these findings, we were unable todetect any tyrosine phosphorylation of CD-31 in eitherresting neutrophils, which agrees with results reportedby Skubitz et al. (24), or neutrophils whose surface CD-31molecules had been engaged. Moreover, by pre-incubating neutrophils with PP1, an inhibitor of tyrosinekinases of the Src family, we found that a Src tyrosinekinase(s) was essential for the CD-31-induced signalingthat occurred in these cells upon pre-activation with thechemotactic factor fMet-Leu-Phe. The effect of PP1 couldnot have been due to unspecific influence on the Ca2�

signal, since this inhibitor had no impact on the fMet-Leu-Phe-triggered Ca2� signal. Despite this finding andthe fact that fMet-Leu-Phe treatment activated Src-family tyrosine kinases in the neutrophils, we failed todetect any tyrosine phosphorylation of CD-31 when thecells were stimulated solely with fMet-Leu-Phe. This iscontrary to reports that tyrosine phosphorylation ofCD-31 is induced by thrombin alone (12) and by lysophos-phatidylcholine alone (25) in platelets and endothelialcells, respectively. Conversely, in neutrophils the fMet-Leu-Phe-induced activation of Src tyrosine kinases has tobe accompanied by an oligomerization of CD-31 to allowphosphorylation of these receptors, which is consistentwith the prerequisite of the CD-31-effected Ca2� signal inthese cells. It has been demonstrated that PLC-�1 canassociate with the cytoplasmic domain of CD-31 in THP-1cells, albeit in response to the unphysiological stimuluspervanadate (26), and this suggests that there is a linkbetween the CD-31-induced Ca2� signal and its depen-dence on Src tyrosine kinase activity observed in ourstudy.

We found that CD-31 exhibits an intracellular sig-naling capacity in neutrophils, which implies that thisadhesion receptor could also be involved in regulationof the actin network, ultimately affecting the shape ofthese cells as they transmigrate through the junctionbetween two endothelial cells. Depolymerization of ac-tin is particularly interesting in this context, since it iswell known that this process is triggered by a rise incytosolic free Ca2� (13), and it can enable neutrophils tosqueeze through the minute gap that exists betweentwo adjacent endothelial cells. However, our dataclearly demonstrate that engagement of CD-31 doesnot reduce, but instead causes a net increase in thecellular content of F-actin. By chelating the CD-31-induced Ca2� signal, we discovered that a depolymer-ization of actin occurred in response to CD-31 engage-ment, but it was hidden by a simultaneous and largerpolymerization of actin. This might be beneficial invivo, since the depolymerization would enable neutro-phils to pass between endothelial cells, while the poly-

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merization would allow these cells to form and extendprotrusions into the tissues surrounding the blood ves-sels from which they are emigrating. Since our analysisare made on a population of cells these two differentactin responses cannot be separated in time and in-stead we only register the sum of the two differentresponses.

ACKNOWLEDGMENTS

This work was supported by grants from the Swedish CancerFoundation, the SSF Inflammation Program, the U-MAS ResearchFoundations, the Osterlund Foundation, and the King Gustaf VMemorial Foundation. The authors are indebted to Ms. Patty Odmanfor linguistic revision of the manuscript.

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