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The Journal of Immunology, 2005, 175: 3484-3491.
Copyright © 2005 by The American Association of Immunologists

Neutrophils Lacking Platelet-Endothelial Cell Adhesion Molecule-1 Exhibit Loss of Directionality and Motility in CXCR2-Mediated Chemotaxis1

Yue Wu, Paul Stabach, Michael Michaud and Joseph A. Madri2

Department of Pathology, Yale University Medical School, New Haven, CT 06520


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Time-lapsed videomicroscopy was used to study the migration of platelet-endothelial cell adhesion molecule-1-deficient (PECAM-1–/–) murine neutrophils undergoing chemotaxis in Zigmond chambers containing IL-8, KC, or fMLP gradients. PECAM-1–/– neutrophils failed to translocate up the IL-8, KC, and fMLP gradients. Significant reductions in cell motility and cell spreading were also observed in IL-8 or KC gradients. In wild-type neutrophils, PECAM-1 and F-actin were colocalized at the leading fronts of polarized cells toward the gradient. In contrast, in PECAM-1–/– neutrophils, although F-actin also localized to the leading front of migrating cells, F-actin polymerization was unstable, and cycling was remarkably increased compared with that of wild-type neutrophils. This may be due to the decreased cytokine-induced mobilization of the actin-binding protein, moesin, into the cytoskeleton of PECAM-1–/– neutrophils. PECAM-1–/– neutrophils also exhibited intracellularly dislocalized Src homology 2 domain containing phosphatase 1 (SHP-1) and had less IL-8-induced SHP-1 phosphatase activity. These results suggest that PECAM-1 regulates neutrophil chemotaxis by modulating cell motility and directionality, in part through its effects on SHP-1 localization and activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Platelet-endothelial cell adhesion molecule-1 (PECAM-1)3 is a 130-kDa membrane-spanning glycoprotein expressed on most vascular cells, including endothelial cells, platelets, and leukocytes (1, 2). Both in vitro studies using PECAM-1-blocking Abs and in vivo studies using PECAM-1–/– mice support the idea that PECAM-1 plays an essential role in neutrophil transmigration through the endothelial monolayer and the perivascular basement membrane in cytokine- and vascular bed-specific manners (3, 4, 5, 6, 7). Specifically, despite the increased vascular permeability observed in PECAM-1–/– mice after inflammatory stimuli (8), neutrophils of PECAM-1–/– mice were noted to be transiently arrested at the basement membrane region beneath the overlying microvascular endothelium in a chemical-induced peritonitis model (9). In addition, using a murine polyvinylacetyl sponge implantation model, we have observed decreased neutrophil infiltration in PECAM-1–/– mice 2 and 7 days after implantation (10). Recently, the most obvious effects of PECAM-1-blocking Abs on in vivo leukocyte transmigration models were shown in FVB/n and SLJ mouse strains, but not the commonly used C57BL/6 strain (11). These apparently discrepant findings raised the possibility of PECAM-1 having direct effects on neutrophil transmigration and directed migration, and prompted this study.

Transmigration is a multiple-step process comprised of three steps: selectin-mediated rolling, integrin-dependent tight adhesion, and transendothelial migration. Neutrophil-endothelial PECAM-1 homophilic ligation has been proposed to mediate the last step of this process by up-regulating adhesion molecules such as {alpha}6{beta}1 integrin and {beta}2 integrin (7, 12).

Once through the endothelial basement membrane, neutrophils exhibit cytokine-mediated directed migration toward the inflammatory focus. The directional cellular movement up a chemoattractant gradient, which is chemotaxis, requires a cell to develop polarity in which cytoskeleton components are differentially localized at two poles of the cell. Chemoattractant receptors are Gi protein-coupled receptors via which the extracellular shallow chemoattractic gradient can be transduced into intracellular polarized responses of multiple signaling molecules (reviewed in Ref.13). The roles of PECAM-1 in neutrophil chemotaxis-related signaling cascades remain unknown. Some ex vivo studies showed that engagement of CD31 modulates Ca2+ signaling and triggers Ca2+-dependent actin polymerization and deploymerization in neutrophils (14). Furthermore, homophilic oligomerization of CD31 on the human neutrophil surface has been shown to induce the association of the PI3K p85 subunit with CD31 (15).

To better understand the nature of potential PECAM-1 effects on neutrophil-directed migration, we used in vitro assays to assess neutrophil chemokine-mediated directed migration through endothelial cell monolayers and toward selected chemoattractants. Using videomicroscopy of neutrophils undergoing IL-8-, KC-, and fMLP-directed migration in Zigmond chambers to assess directionality and motility, we noted that PECAM-1–/– neutrophils exhibited a loss of directed migration, a rapid actin polymerization cycle, failure of Src homology 2 domain containing phosphatase 1 (SHP-1) and moesin localization to their leading edges, as well as decreased SHP-1 activity and moesin phosphorylation. These data indicate that beyond its function to regulate adhesion, PECAM-1 is spatially regulated during directed migration and acts as a modulator of signaling molecules, functioning to alter the inherent migration capability of chemotactic neutrophils by regulating SHP-1 localization, activity, and moesin localization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

PECAM-1–/– mice were housed at Yale University Medical Center, and all experiments involving mice were approved by the Yale University Medical School animal care center committee. In each experiment, age- and gender-matched experimental and control mice were used. The following reagents were purchased: IL-8, KC, and rat anti-mouse CXCR2 from R&D Systems; fMLP from Sigma-Aldrich; Mec13.3 rat anti-mouse PECAM-1 from BD Pharmingen; rhodamine-phalloidin, FITC-phalloidin, Alexa Fluor 594-conjugated goat anti-rat IgG, and slow-fade regents from Molecular Probes; Percoll from Amersham Biosciences; SHP-1 and phosphotyrosine mAbs and SHP-1 Ab-conjugated agarose beads from Santa Cruz Biotechnology; universal tyrosine phosphatase assay kit from Takara Bio; and moesin and phosphomoesin Abs from Cell Signaling Technology.

Neutrophil transmigration assay

PECAM-1-negative and PECAM-1-positive lung endothelioma cells lines (8, 10) were plated onto collagen IV-coated, 24-well, 3-µm pore size Transwells (Biocoat; BD Biosciences) at a concentration of 1 x 104 cells/well and grown for 4 days until confluent. On day 4, mouse neutrophils were isolated from blood via cardiac puncture and isolated using the protocol in Current Protocols in Immunology (7.23.1). Approximately 1 x 106 neutrophils were isolated from four mice. The cells were resuspended in 1.2 ml of Opti-MEM-I. Each Transwell was washed with Opti-MEM-I, and 100 ng/ml IL-8 (Sigma-Aldrich) was added to the bottom of each well; 0.2 ml of the neutrophil suspension was added to each well (for the total of three PECAM-1-negative endothelioma and three PECAM-1-positive endothelioma wells). The transmigration was allowed to proceed for 4 h at 37°C, at which point the fluid from the bottom well was removed, the well was washed with PBS, and any remaining adherent neutrophils were trypsinized. The fluid was pooled and spun at 8000 rpm for 10 min. The cells were counted using a hemocytometer, and transmigration was expressed as the percentage of cells recovered from the bottom well to the cells plated in the top well.

Neutrophil purification and Zigmond chamber chemotaxis assay

Murine bone marrow neutrophils were prepared by centrifugation through Percoll gradients as described previously (16). Purified neutrophils were resuspended either in Hanks’ buffer (0.14 M NaCl, 5.4 mM KCl, 1 mM Tris, 1.6 mM CaCl2, and 1 mM HEPES (pH 7.2)) for immunoblots/immunoprecipitation or in Hanks’ buffer containing 1% BSA for migration assays. In chemotaxis assays, the cells were placed in the middle of coverslips and allowed to adhere for 10 min at 37°C. The coverslips were then flipped, and the cell-covered portion was centered above the bridge of Zigmond chambers. IL-8 (20 nM) in Hanks’ buffer was added to one groove along the bridge, and control buffer was added to the other groove. After a 10-min incubation at 37°C, the cells were fixed in the Zigmond chambers by the careful removal of chemotaxis buffers and replacement with 3% paraformaldehyde in PBS for 15 min at room temperature or –20°C in methanol/acetone (1/1) for 10 min. Paraformaldehyde fixation was followed by permeabilization with a solution of 0.5% Triton X-100 in PBS and blocking with 2% BSA for 1 h at room temperature. Abs to the proteins of interest were then added (anti-PECAM-1 Mec13.3, anti-Thr558-phospho-moesin, and anti-SHP-1). After overnight incubation at 4°C, the cells were washed, and secondary Abs were added. Finally, coverslips were mounted with a drop of antifade reagent containing 4',6-diamido-2-phenylindole hydrochloride (DAPI) to label cell nuclei. The samples were then examined using fluorescence microscopy (Olympus IX71 microscope equipped with an Optronics MicroFire camera using PictureFrame software).

Time-lapsed videomicroscopy of migrating neutrophils

In Zigmond chambers, purified neutrophils were recorded crawling in the presence of a IL-8 gradient (20 nM), a KC gradient (1 µg/ml), or an fMLP gradient (10 µM). In some experiments, purified neutrophils were preincubated with PD98059 (10 µM) for 15 min at 37°C or anti-CXCR2 blocking Ab (50 µg/ml) for 30 min at 37°C before the chemoattractant stimulation. The microscope was equipped with a x40 objective. Images were captured at 10- or 15-s intervals with a charge-coupled device camera and Openlab imaging software from Improvision. The recorded data were used for analyses of neutrophil chemotaxis by tracing the front edge of migrating neutrophils and determining cell motility as well as directionality. Motility was calculated using the net migration distance (from the origin to the destination) divided by the migration time. The directionality was shown by plotting the relative final positions of neutrophils vs a common origin. Neutrophils that moved at least 10 µm over the 10-min observation period and had negative x-coordinates were considered translocating toward the chemoattractant source.

Moesin mobilization and F-actin polymerization assays

After incubation with or without IL-8 at 37°C for the indicated time, the neutrophils were pelleted and then resuspended with ice-cold cytoskeleton stabilization buffer (CSB; 138 mM KCl, 3 mM MgCl2, 2 mM EGTA, 0.32 M sucrose, and 10 mM MES (pH 6.1)) containing 0.5% Triton X-100 and protease inhibitor mixtures. Cell lysates were centrifuged at 14,000 x g for 10 min, and the pellets (insoluble cytoskeletal fractions) and supernatants (soluble cytosol fractions) were separated and analyzed by immunoblots for moesin. For F-actin polymerization assay, the simulated neutrophils were immediately fixed with CSB containing 3% paraformaldehyde for 10 min on ice. The fixed cells were then washed and permeabilized with CSB containing 0.5% Triton X-100 for 5 min on ice. After washing and incubation with blocking buffer (PBS containing 1% BSA) for 1 h, the cells were incubated with rhodamine-phalloidin in blocking buffer for an additional 40 min. The cells were then thoroughly washed using PBS buffer, and the cytoskeleton-bound rhodamine was extracted with methanol overnight. The extracted rhodamine fluorescence was measured with the Wallac 1420 Victor multilabel counter.

Pull-down immunosorbent assays to detect membrane CD31-associated SHP-1

The 96-well microplates were coated with 10 µg/ml anti-CD31 rabbit polyclonal Ab "sleet" (17). Plates were then blocked with 3% BSA in Dulbecco’s PBS at 4°C overnight. The next day the purified wild-type (WT) neutrophils were added to coated plates (105 cells/well) and incubated at 4°C for 20 min, followed by 30 min at 37°C. IL-8 or control buffer was then added for 2 min at 37°C. The remaining adherent cells were lysed with 50 mM Tris/0.5% CHAPS buffer. After washing, the plates were stained with primary mAbs (anti-SHP-1 and phosphotyrosine; 1/1,000 dilution), followed by HRP-conjugated secondary Ab (1/10,000). The samples were developed with tetramethylbenzidine, and OD450 was measured using a Wallac 1420 Victor multilabel counter.

SHP-1 pull-down and activity assays

Purified neutrophils were stimulated with IL-8 (20 nM) in Hanks’ buffer for 2 min. The cells were then lysed with PBS buffer (pH 7.4) containing 1% Nonidet P-40, 2 mM sodium vanadate, and protease inhibitor mixtures. The supernatant from lysate was collected and then incubated with anti-SHP-1 rabbit IgG agarose-conjugated beads overnight. The slurry containing the complex of the pulled down SHP-1 and the Ab was recovered after centrifugation (13,000 rpm, 10 s) and washed with PBS lysis buffer three times. The slurry was resuspended in phosphate reaction buffer (Takara Bio) containing 2-ME and then added to wells of phosphatase substrate-immobilized microplates (Takara Bio) for incubation. After 45 min, the solution was removed, and the wells were washed with PBS buffer containing 0.05% Tween 20 four times. The wells were then incubated with blocking solution for 30 min and further incubated with anti-phosphotyrosine (PY20)-HRP (Takara Bio) for 30 min. Finally, HRP substrate solution (tetramethylbenzidine) was added, and the OD450 nm of developed color was measured using a Wallac 1420 Victor multilabel counter. A standard curve of tyrosine phosphatase activity was constructed by measuring the activities of serially diluted CD45 phosphatase controls. The phosphatase activities of samples were calculated on the basis of prepared standard curves.

Neutrophil adhesion assays

IL-8 or control buffer was added to the purified bone marrow neutrophils, and then the cells (3 x 105 in 100 µl) were immediately incubated at 37°C in wells of a microplate previously coated with fibronectin (10 µg/ml), fibrinogen (100 µg/ml), or BSA (10 µg/ml). After 15 min, the unadherent cells were removed by inverting the plates and centrifuging on Whatman no. 3 filter paper. The remaining cell number was quantified by measuring the total cell DNA amount using the CyQuant Cell DNA Proliferation Kit (Molecular Probes). The 100% standard was determined by measuring the value of a sample of the original cell suspension.

Flow cytometric analyses of CXCR2 expression

Purified neutrophils were stimulated with control buffer or IL-8 (20 nM) and KC (1 µg/ml) for the indicated time. The reaction was stopped by placing the cells on ice. The cells were then pelleted and resuspended in FACS staining buffer (PBS with 3% FBS). FcR was blocked by preincubation with the 2.4G2 anti-FcR (BD Pharmingen). The cells were further incubated with rat anti-mouse CXCR2 (R&D Systems), followed by FITC-labeled goat anti-rat IgG secondary Ab (Jackson ImmunoResearch Laboratories). Data acquisition was performed with a FACSCalibur, and data were analyzed with FlowJo 6.0 software (TreeStar).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Analyses of neutrophil transmigration through endothelial cell monolayers

When either WT or PECAM-1–/– purified peripheral blood neutrophils were placed in the upper wells of 3-µm pore size, collagen type IV-coated Transwell culture inserts containing confluent cultures of either PECAM-1–/– (knockout; KO) or PECAM-1 reconstituted lung microvascular endothelial cells and stimulated to migrate by placing 100 ng/ml rIL-8 in the lower wells, we observed significantly less migration of the PECAM-1–/– neutrophils (Fig. 1; p < 0.01).



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  		FIGURE 1. PECAM-1 expression on neutrophils determines their rate of transmigration across the endothelium. Immortalized mouse lung endothelial cells (either PECAM-1 KO or KO reconstituted with the full-length PECAM-1 protein (RC)) were grown to confluence on Transwell inserts. Freshly isolated mouse neutrophils (WT or PECAM-1–/–) were seeded onto the monolayers and allowed to transmigrate for 4 h toward IL-8 chemoattractant. PECAM-1-deficient neutrophils were observed to transmigrate significantly slower across both RC and KO endothelial layers compared with PECAM-1-positive neutrophils. RC wells, n = 11; KO wells, n = 10. *, p < 0.01 for WT vs KO neutrophils through RC endothelial cell monolayers; **, p < 0.05 for WT vs KO neutrophils through KO endothelial cell monolayers.

 
Analyses of neutrophil chemotaxis in Zigmond chambers

To better understand these observed in vivo and in vitro differences in neutrophil migratory behavior, time-lapsed videomicroscopy was used to examine the migration behaviors of WT and PECAM-1–/– neutrophils in IL-8, KC, and fMLP gradients (indicated by arrows in Fig. 2). The overall directionality of the migrating neutrophils was demonstrated by plotting the final position of individual neutrophils to their common origins (Fig. 2A). In the absence of chemoattractant gradients, only 4.7% (n = 50) of WT neutrophils and 10% (n = 94) of PECAM-1–/– neutrophils demonstrated translocation (centroid movement >10 µm over a 10-min observation period). In the presence of the IL-8 gradient, 68.2 ± 7% (four movies; n = 300) of WT neutrophils showed cell translocation, and among them 90% were moving up the IL-8 gradient (Fig. 2B). In contrast, 34 ± 6% (three movies; n = 209) PECAM-1–/– neutrophils were motile, and of these only 40% were moving toward the gradient. This value is comparable to the expected value of 50% if cells were undergoing random migration. In the presence of a KC gradient, 82.1 ± 5% (three movies; n = 200) of WT neutrophils showed cell migration, and among them 69% were moving toward the KC source. In contrast, only 26% (n = 100) PECAM-1–/– neutrophils were motile, and these motile cells migrated in a random manner because only 45% of cells were moving toward the gradient. In the presence of an fMLP gradient, 94.2 ± 1.65% (three movies; n = 200) of WT neutrophils showed cell migration, and among them 89% were moving toward the fMLP source. However, 30 ± 5% (three movies; n = 150) PECAM-1–/– neutrophils were motile, and of these only 40% were moving toward the gradient. When comparing only the moving cells, fold increases in both IL-8- and KC-induced motility were significantly reduced in PECAM-1–/– neutrophils compared with WT (in IL-8 gradients: KO, 2.13 ± 1.1; WT, 15.77 ± 2; in KC gradients: KO, 1.58 ± 1.12; WT, 6.3 ± 0.81; Fig. 2C), but in fMLP gradients, WT neutrophils only showed slightly higher motility than that of PECAM-1–/– cells (WT, 7.55 ± 1.6; KO, 5.58 ± 2.8; p < 0.279).



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  		FIGURE 2. Analysis of WT and PECAM-1–/– neutrophil migration direction. A, Purified bone marrow neutrophils were placed in Zigmond chambers containing control buffer in the right groove and chemoattractant in the left groove (IL-8, 20 nM; KC, 1 µg/ml; fMLP, 10 µM). Time-lapse microscopy was used to record the cell position on the bridge at 10-s intervals for 10 min (in the IL-8 and fMLP gradients) or 15 min (in the KC gradient). The final positions of neutrophils relative to a common origin were plotted. Each unit in x/y-coordinates is 100 µm. B, The percentages of translocating cells showing migration toward the chemoattractant sources. The final position of individual motile neutrophils was compared with its origin. The cells that moved at least 10 µm over the 10- or 15-min observation and had negative x-coordinates were considered as migrating toward the chemoattractant source. The calculated percentage should be compared with an expected value of 50% if cells undergo random migration. C, Comparisons of the WT and PECAM-1–/– neutrophil motility in Zigmond chambers containing IL-8, KC, or fMLP gradients. The motility was determined as the rate of cell translocation over a 10- or 15-min observation period. Both WT and PECAM-1–/– neutrophils in chambers containing the control buffer were slightly motile, that is, they made centroid movements of <30 µm over 15 min. Shown is the fold increase in motility, using chemoattractant-induced motility divided by cell basal motility in control buffer. **, Statistical significance (p < 0.05).

 
The percentage of nonmotile PECAM-1–/– neutrophils was ~30–60% more than that of WT in chemoattractant gradients. These nonmotile neutrophils not only include a portion of cells that move, but with centroid movement <10 µm/over a 10-min observation period, but also comprise the cells that spin around the origin and do not make any centroid movement.

Flow cytometric analyses of CXCR2

To ensure the integrity of the receptors for IL-8 and KC on PECAM-1–/– neutrophils, the CXCR2 cell surface expression levels were quantitated by flow cytometric analyses in both control buffer and chemoattractant (IL-8 or KC)-stimulated WT and PECAM-1–/– neutrophils (Fig. 3). The CXCR2 expression levels before chemoattractant treatments were comparable between WT and PECAM-1–/– neutrophils. IL-8 stimulation induced a 10% loss of cell surface CXCR2 in WT neutrophils and only 6.4% loss in that of PECAM-1–/– cells. KC treatment resulted in an ~50% decrease of cell surface CXCR2 over 10 min in both WT and PECAM-1–/– neutrophils.



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  		FIGURE 3. Chemoattractant-induced CXCR2 turnover in WT and PECAM-1–/– neutrophils. A, Flow cytometric analysis of CXCR2 expression levels on WT and PECAM-1–/– neutrophils in control buffer. B, Purified neutrophils were stimulated with IL-8 (20 nM) or KC (1 µg/ml) for the indicated times. CXCR2 levels were then analyzed by flow cytometry. The mean fluorescence intensity (MFI) represents the amount of CXCR2 expressed on the cell surface. The ratio of MFI after to before chemoattractant treatment was calculated and plotted over the time course.

 
The reduction of cell surface CXCR2 levels correlates with the endocytosis or internalization of CXCR2 receptors, the role of which in chemotaxis is still controversial (18, 19, 20). From these data, we speculate that 20 nM IL-8 is the optimal concentration for controlling both neutrophil migration motility and directionality, whereas 1 µg/ml KC is above the physiological threshold and results in the clearance of cell surface CXCR2 leading to down-regulation of cell motility as well as directionality. In fact, we observed that KC-induced WT motility was significantly less than that of IL-8-induced motility (11.6 vs 29 ± 4.6 µm/min; n = 50). Therefore, we used IL-8 as the chemoattractant in the subsequent experiments.

Neutrophil migration tracing and adhesion assay in IL-8-induced chemotaxis

The leading edges of individual migrating neutrophils were traced to define the path of cell translocation in the IL-8 gradient. WT neutrophils were found to be translocating toward the IL-8 source in a persistent manner. PECAM-1–/– neutrophils did adhere and polarize, and would often exhibit an apparent spinning motion during which the cells would stretch and retract in different directions as if they could not identify the directionality (Fig. 4A, and supplemental movies).4 Cell tracings (Fig. 4A) reveal that the motile PECAM-1–/– neutrophils move only short distances in random directions before changing direction again and again (assessed at 15-s intervals). Thus, although their net directed movement (defined as motility of >10 µm/10 min) is modest, they exhibit similar aggregate distances moved compared with WT cells, albeit not directed. These findings were noted in both bone marrow-derived and peripheral blood-derived neutrophils.



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  		FIGURE 4. WT and PECAM-1–/– neutrophil migration in Zigmond chambers containing IL-8 gradient. A, Tracings of WT and PECAM-1–/– bone marrow and blood neutrophil migration in IL-8 gradient. Neutrophils were traced at 15-s intervals as described in Materials and Methods. {circ}, The starting points; lines represent the path of individual cells. B, IL-8-induced adhesion of WT and PECAM-1–/– neutrophils. Neutrophils were stimulated with 20 nM IL-8 for 15 min. The percentage of cells remaining adherent to the coated microtiter plates was determined as described in Materials and Methods. No significant difference was found.

 
To ensure that the adhesion strength was not affected in PECAM-1–/– neutrophils, both {beta}1 and {beta}2 integrin-mediated adhesiveness was quantitated with fibronectin and fibrinogen as substrates. Because migrating neutrophils were in buffer containing 1% BSA, the adhesiveness of neutrophils to BSA was also measured. Compared with WT, PECAM-1–/– neutrophils showed similar IL-8-induced adherence (Fig. 4B). In addition, FACS analyses showed that the {beta}1 and {beta}2 integrin expression levels were almost identical in IL-8-stimulated WT and PECAM-1–/– neutrophils (data not shown).

Morphological analyses of migrating PECAM-1–/– neutrophils

The morphologies of migrating WT and KO neutrophils were compared. WT neutrophils showed the conventional polarization with filapodia or lamellipodia at the leading edge and a uropod at the trailing area of the cells (Fig. 5A). Although the few migrating PECAM-1–/– neutrophils also exhibited polarized cell bodies, they exhibited less obvious membrane protrusion/ruffling and tail uropods (Fig. 5B) and significantly less cell spreading. PECAM-1–/– neutrophils were found to have approximately half the cell area as that of the WT (KO, 600 ± 34.76 pixels; WT, 1200 ± 119.45 pixels; n = 30; Fig. 5C) and a significantly reduced cell area to cell perimeter ratio (KO, 5.78 ± 0.28; WT, 7.24 ± 0.51; n = 30; Fig. 5D).



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  		FIGURE 5. Morphology of chemotaxing WT and PECAM-1–/– neutrophils. A and B, Representative phase-contrast images of migrating bone marrow neutrophils from WT (A) and PECAM-1–/– (B) mice in response to IL-8 gradient (IL-8 source is toward the left) in Zigmond chambers. C and D, Comparison of cell spreading area and cell spreading area/perimeter ratio between WT (C) and PECAM-1–/– (D) neutrophils. Image J software (<http://rsb.info.nih.gov/nih-image>) was used to determine the area and perimeter of individual cells.

 
Immunostaining of PECAM-1 in WT neutrophils

The above data suggest that PECAM-1 regulates neutrophil chemotaxis. It is known that chemotaxis-controlling signaling molecules can be spatially regulated and asymmetrically distributed in polarized migrating cells (21, 22). Therefore, the intracellular localization of PECAM-1 in neutrophils was examined. PECAM-1 was found to be localized at the leading front of polarized WT neutrophils, whereas it was distributed uniformly around the cell membrane in unstimulated resting cells (Fig. 6B). To reduce nonspecific labeling, PECAM-1 Mec13.3 Abs were preabsorbed on PECAM-1–/– neutrophil whole cell lysates, and specificity was tested by immunoblotting; thus, although PECAM-1 staining was retained on WT neutrophils, no PECAM-1 staining was observed in the fluorescence images of PECAM-1–/– neutrophils. Furthermore, in the IL-8-stimulated WT neutrophils, PECAM-1 was found to colocalize with F-actin at the leading front of migrating neutrophils (Fig. 6C). However, the observed PECAM-1 polarization is apparently not required for F-actin polarization at the leading front, because IL-8-induced F-actin polarization appeared to be normal in PECAM-1–/– neutrophils (Fig. 6A).



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  		FIGURE 6. The colocalization of PECAM-1 and F-actin in WT neutrophils. A, Actin staining pattern of WT and PECAM-1–/– bone marrow neutrophils fixed in Zigmond chambers containing an IL-8 gradient. WT (upper panel) and PECAM-1–/– (lower panel) neutrophils were examined using confocal microscopy as described in Materials and Methods. B, PECAM-1 immunostaining of WT BM neutrophils in IL-8-induced chemotaxis (left panel) or control buffer (right panel). The IL-8 source was located to the right. Shown are PECAM-1 (red) and nuclear DAPI (blue) staining. C, PECAM-1 and F-actin colocalization in chemotaxing WT neutrophils. Shown are three individual WT bone marrow neutrophils, with PECAM-1 (red) on the left, F-actin (red) in the center, and dual color localization (yellow) as well as DAPI staining (blue) on the right. The IL-8 chemoattractant source was toward the right. D, IL-8-induced F-actin polymerization in WT and PECAM-1–/– neutrophils. Neutrophils were stimulated with IL-8 for the indicated time, and F-actin-bound phalloidin rhodamine fluorescence levels were determined. Shown is a representative experiment. PECAM-1–/– neutrophils had less F-actin polymerization, and the time of polymerization was remarkably shortened compared with WT cells.

 
F-actin polymerization and cycling in WT and PECAM-1–/– neutrophils

Our finding of markedly different directed migration rates of WT and PECAM-1–/– neutrophils is consistent with a failure of persistent lamellipodia formation and maintenance toward the direction of the chemotactic gradient in the PECAM-1–/– neutrophils (see supplemental movie 2). This lack of directed lamellipodial stability in the PECAM-1–/– neutrophils is reflected in the markedly shortened F-actin polymerization time exhibited by the PECAM-1-null neutrophils compared with WT cells after IL-8 stimulation (Fig. 6D). In WT neutrophils, F-actin polymerization occurred 10 s after stimulation and reached its peak at ~60 s, and then this polymerization was maintained for a period of time. In contrast, the extent of F-actin polymerization in PECAM-1–/– neutrophils was less, and at the 2 min point, ~80% less actin polymerization was observed.

Moesin localization and phosphorylation in WT and PECAM-1–/– neutrophils

Because the ezrin-radixin-moesin protein moesin, an actin cross-linking protein associated with neutrophil plasma membranes, is known to be bound with PECAM-1 in thrombin-stimulated platelets (23, 24) and a mediator of leukocyte signaling (25), we investigated its mobilization and localization in WT and PECAM-1–/– neutrophils in response to IL-8 stimulation. As illustrated in Fig. 7A, after IL-8 stimulation, there was a time-dependent moesin mobilization into the cytoskeleton from the cytosol, and appreciably more moesin was recruited to the cytoskeletal fraction of WT neutrophils compared with PECAM-1–/– neutrophils. In addition, analysis of immunofluorescence localization of phosphothreonine-moesin after IL-8-induced chemotaxis (at 10 min) revealed markedly different staining patterns. Namely, phospho-moesin (green) was localized to the leading fronts of the migrating WT neutrophils, although there appeared to be no appreciable staining in the PECAM-1–/– cells (Fig. 7B).



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  		FIGURE 7. Moesin mobilization and localization in WT and PECAM-1–/– neutrophils. A, IL-8-induced moesin mobilization from the cytosol to cytoskeleton. Equal numbers of neutrophils derived from WT and PECAM-1–/– mouse bone marrow were used in the experiments. Cells were stimulated for the indicated times, then the cell lysates were separated into cytosol and cytoskeletal fractions. The moesin amount retained in the different fractions at individual time points was shown by immunoblots. Shown is a representative experiment. Moesin mobilization was greatly decreased in PECAM-1–/– neutrophils. B, Localization of phospho-Thr558-moesin in IL-8-stimultated WT and PECAM-1–/– neutrophils. Neutrophils were stimulated in Zigmond chambers containing the IL-8 gradient for 10 min. The cells were then fixed and immunostained with rabbit polyclonal Abs to phosphothreonine moesin (green color). Phosphomoesin was localized at the leading fronts of chemotaxing WT neutrophils, but was not detected in PECAM-1–/– neutrophils.

 
SHP-1 localization in WT and PECAM-1–/– neutrophils

Because the cytoplasmic domain of PECAM-1 contains binding sites for Src homology domain 2 proteins such as SHP-1/SHP2 (26), and SHP-1 is known to play an important role in neutrophil migration (27), we investigated SHP-1 subcellular localizations in WT and PECAM-1–/– neutrophils before and after IL-8 stimulation. The localization of SHP-1 differed in WT and PECAM-1–/– neutrophils after IL-8 stimulation (Fig. 8A). Specifically, although SHP-1 appeared localized to the leading fronts of WT neutrophils and colocalized with F-actin, it showed a more diffuse localization over the PECAM-1–/– neutrophils and without colocalization with F-actin.



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  		FIGURE 8. SHP-1 intracellular localization in IL-8-stimulated WT and PECAM-1–/– neutrophils. A, Immunostaining of SHP-1 in WT (upper two panels) and PECAM-1–/– (KO; lower two panels) neutrophils. Both control buffer and IL-8-stimulated WT and PECAM-1–/– neutrophils were fixed in Zigmond chambers and stained with rabbit polyclonal anti-SHP-1, followed by FITC-conjugated anti-rabbit IgG (green (G)) as well as rhodamine phalloidin for F-actin (red (R)). The long dashed arrows indicate the dislocalization of SHP-1 in PECAM-1–/– neutrophil cytosol. B, The association of membrane PECAM-1 with SHP-1 in IL-8-stimulated WT neutrophils. The association between PECAM-1 with SHP-1 or phosphotyrosine was examined using immunoabsorbant assays. Shown is a representative experiment. SHP-1 was shown to be bound by membrane PECAM-1 after IL-8 stimulation.

 
Because both SHP-1 and PECAM-1 were localized at the leading front of migrating WT neutrophils, we hypothesized that in IL-8-stimulated WT neutrophil membrane, PECAM-1 could be associated with SHP-1. This was shown by pull-down immunosorbent assays using PECAM-1 Ab-coated microplates (Fig. 8B). The coated microplates were incubated with cell suspensions, followed by an IL-8 stimulus, followed by cell lysis, such that only the membrane portion that contains PECAM-1 and its associated molecules was retained for analysis. The results suggested that membrane-associated PECAM-1 can recruit and bind SHP-1 upon IL-8 stimulation. Additionally, with IL-8 stimulation there was an increased level of tyrosine phosphorylation associated with PECAM-1.

Because we found that moesin mobilization, phosphomoesin polarization, and SHP-1 polarization were all abolished in PECAM-1–/– neutrophils, we also examined whether SHP-1 activity was dysregulated. As illustrated in Fig. 9, PECAM-1–/– neutrophils exhibited a blunted SHP-1 phosphatase activity compared with WT neutrophils after IL-8 stimulation.



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  		FIGURE 9. SHP-1 activity is blunted in PECAM-1–/– neutrophils after IL-8 stimulation. Quantification of IL-8-induced SHP-1 phosphatase activity in WT and PECAM-1–/– neutrophils. PECAM-1–/– neutrophils, after immunoprecipitation of lysates with anti-SHP-1 and assessed for phosphatase activity, revealed a 21% reduction in SHP-1 activity compared with that of WT (p < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies support the concept that endothelial-leukocyte PECAM-1 homophilic ligation drives neutrophil transendothelial migration (3). Most studies are focused on the interaction between neutrophils and endothelial cells. It is unclear whether PECAM-1 deficiency elicits intrinsic neutrophil defects in migration speed or directionality.

In this report we demonstrate that compared with age- and sex-matched WT animals, neutrophils harvested from PECAM-1–/– animals exhibit loss of directionality and have significantly reduced directed migration speed using time-lapse videomicroscopy analysis of neutrophils in Zigmond chambers containing an IL-8 gradient. To ensure that IL-8 exerts its effects only through CXCR2 on murine neutrophils, we incubated WT neutrophils with anti-CXCR2 blocking Ab (50 µg/ml; 30 min at 37°C) before time-lapse videomicroscopy analysis and found almost 100% inhibition of IL-8-induced neutrophil migration (data not shown). The decreased chemotaxis motility was also illustrated using another CXCR2 agonist, KC-stimulated PECAM-1–/– neutrophils. However, as mentioned in Results, the motility and directionality changes in KC gradients were less profound compared with those observed in IL-8 gradients. This may be due to the significant difference in cell surface CXCR2 turnover rate between IL-8 and KC treatments. In addition, significantly decreased directionality, but only slightly decreased motility, were shown in fMLP-stimulated PECAM-1–/– neutrophils. Thus, despite the fact that the receptors for IL-8 and fMLP belong to the family of G protein-coupled, seven-transmembrane-spanning domain receptors, the mechanisms through which chemotaxis is achieved may differ (28).

We observed that PECAM-1 was colocalized with F-actin at the leading front of migrating WT neutrophils, which suggests that the spatially constrained PECAM-1 in polarized neutrophils may contribute to producing a gradient of directionality-sensing molecules. Tyrosine-phosphorylated PECAM-1 has been shown to recruit the tyrosine protein phosphatases SHP-1/SHP2 in hemopoietic cells and recruit, activate, and modulate SHP-2 activities in endothelial cells (26, 29). Indeed, we showed that in IL-8-stimulated WT neutrophils, membrane-associated PECAM-1 can recruit tyrosine phosphatase SHP-1, and SHP-1 was polarized at the leading front of migrating cells. Although PECAM-1 has been shown to have an association with the p85 subunit of PI3K upon PECAM-1 ligation in human neutrophils (15), we did not observe such physical association of PECAM-1 with PI3K p85 in IL-8-stimulated neutrophils (data not shown). However, directionality-controlling molecules, such as the p110{gamma} subunit of PI3K, the lipid product of PI3K activation, phosphotidyl inositol 3,4,5-triphosphate, and the downstream AKT/protein kinase B as well as Rac, are all known to be recruited to the leading fronts of migrating cells, forming persistent directional signaling cascades and facilitating actin polymerization (22, 30, 31). Therefore, we speculate that in IL-8-induced chemotaxing neutrophils, PECAM-1 is polarized to the leading front and recruits SHP-1, which may optimize the activities of several directionality-controlling molecules in close proximity by modulating their phosphorylation levels and further regulate the directional migration.

F-actin polarization still occurs in the poorly migrating PECAM-1–/– neutrophils (Fig. 6A); however, morphologically most PECAM-1–/– neutrophils could not exhibit a persistent lamellipodia/filapodia formation in IL-8 gradients, and few cells showed polarized shapes (Fig. 5B, and supplemental movies). This suggests that PECAM-1 deficiency may affect F-actin polymerization/stabilization and F-actin cycling. Earlier studies of PECAM-1–/– neutrophils demonstrated that PECAM-1 engagement followed by fMLP stimulation can trigger Ca2+-dependent actin polymerization/depolymerization (14). Thus, it is presumed that F-actin polymerization dynamics may be altered in PECAM-1–/– neutrophils. As a matter of fact, the F-actin polymerization cycle time was markedly shortened in PECAM-1–/– neutrophils. Consistent with this are the decreased IL-8-induced moesin cytoskeletal mobilization and the relative loss of moesin threonine phosphorylation at the leading front lamellipodia in PECAM-1–/– neutrophils (Fig. 7).

We hypothesize that PECAM-1 deficiency in IL-8-stimulated neutrophils causes SHP-1 dislocalization and dysregulated activity. Supporting this hypothesis is our immunoprecipitated SHP-1 activity assays, which revealed that in PECAM-1–/– neutrophils, SHP-1 activity was blunted (Fig. 9), and published studies in which murine SHP-1–/– neutrophils have been reported to exhibit diminished motility, similar to what was observed in PECAM-1–/– neutrophils in our studies (27).

In summary, our results indicate that PECAM-1 has a major role in modulating inherent neutrophil-directed migration by localizing at the leading front of migrating cells during chemotaxis. The PECAM-1–/– neutrophils are much less polarized and spreading in response to an IL-8 gradient. They are unable to migrate toward the chemoattractant source and exhibit significantly reduced directed migration and loss of directionality. The abnormal migration phenotypes observed in PECAM-1–/– neutrophils may be explained by the dysregulation of SHP-1 activity, localization, and scaffolding functions, affecting moesin localization and phosphorylation, which, in turn, affects F-actin cycling.


   Acknowledgments
 
We would like to thank Dr. Chi-Kuang Huang at University of Connecticut Health Center for providing the Zigmond Chambers used in chemotaxis assays and helpful discussions and Drs. Mark Mooseker and Jon Morrow at Yale University medical school for helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by U.S. Public Health Service Grants R37-HL28373 and PO1-KD55389 (to J.A.M.) and a Reed Foundation Fellowship in Vascular Biology (to Y.W.). Back

2 Address correspondence and reprint requests to Dr. Joseph A. Madri, Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06520. E-mail address: joseph.madri{at}yale.edu Back

3 Abbreviations used in this paper: PECAM-1, platelet-endothelial cell adhesion molecule-1; CSB, cytoskeleton stabilization buffer; DAPI, 4',6-diamido-2-phenylindole hydrochloride; KO, knockout; SHP-1, Src homology 2 domain containing phosphatase 1; WT, wild type. Back

4 The online version of this article contains supplemental material. Back

Received for publication February 14, 2005. Accepted for publication June 30, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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