Platelets enhance neutrophil transendothelial migration via P-selectin glycoprotein ligand-1

Abstract

Platelets are increasingly recognized as important for inflammation in addition to thrombosis. Platelets promote the adhesion of neutrophils [polymorphonuclear neutrophils (PMNs)] to the endothelium; P-selectin and P-selectin glycoprotein ligand (PSGL)-1 have been suggested to participate in these interactions. Whether platelets also promote PMN transmigration across the endothelium is less clear. We tested the hypothesis that platelets enhance PMN transmigration across the inflamed endothelium and that PSGL-1 is involved. We studied the effects of platelets on PMN transmigration in vivo and in vitro using a well-characterized corneal injury model in C57BL/6 mice and IL-1β-stimulated human umbilical vein endothelial cells (HUVECs) under static and dynamic conditions. In vivo, platelet depletion altered PMN emigration from limbal microvessels after injury, with decreased emigration 6 and 12 h after injury. Both PSGL-1^−/− and P-selectin^−/− mice, but not Mac-1^−/− mice, also had reduced PMN emigration at 12 h after injury relative to wild-type control mice. In the in vitro HUVEC model, platelets enhanced PMN transendothelial migration under static and dynamic conditions independent of firm adhesion. Anti-PSGL-1 antibodies markedly inhibited platelet-PMN aggregates, as assessed by flow cytometry, and attenuated the effect of platelets on PMN transmigration under static conditions without affecting firm adhesion. These data support the notion that platelets enhance neutrophil transmigration across the inflamed endothelium both in vivo and in vitro, via a PSGL-1-dependent mechanism.

the sole role of platelets was once thought to be for thrombosis, but an expanding body of evidence has shown them to be important in a number of inflammatory responses, including the recruitment of neutrophils to sites of inflammation (42). Trafficking of neutrophils is important in inflammation, which is composed of rolling, firm adhesion, and transendothelial migration. Previous works have shown the effects of platelets on the capture and rolling of neutrophils via Mac-1 (10), P-selectin (31), and P-selectin glycoprotein ligand (PSGL)-1 (32). Whether platelets contribute to transmigration is unknown.

The influence of platelets on leukocyte recruitment in inflammation has been assessed in several models. Work from our laboratory in a corneal injury model has demonstrated a role for platelet-neutrophil interactions in wound healing (25, 26). Furthermore, platelet-neutrophil interactions play an increasingly recognized role in clinical disease states, such as angina (30) and acute lung injury (1, 47). In a mouse model of acid-induced lung injury, platelet depletion before injury improves oxygenation and decreases the number of alveolar neutrophils (47). Thus, platelets are important for mediating inflammation.

One of the mechanisms by which platelets and leukocytes interact is via binding of P-selectin on platelets to PSGL-1 on leukocytes. PSGL-1 is a homodimer composed of ∼120,000 molecular weight (MW) subunits, which serves as counterligand for P-selectin (33). P-selectin-PSGL-1 interactions are important for platelet-leukocyte aggregation under shear (14, 23, 43) and static (15) conditions. Furthermore, blockade of PSGL-1 significantly reduces leukocyte capture and rolling by platelets under flow conditions (15). Whereas the effect of PSGL-1 on neutrophil adhesion is well known, its contribution to neutrophil transendothelial migration is unclear.

The purpose of this study was to test the hypothesis that platelets enhance neutrophil transendotheial migration via PSGL-1.

METHODS

We examined the role of platelets on neutrophil transmigration using both in vivo and in vitro models. All experimental protocols were approved by the Institutional Animal Care and Use Committee and the Institutional Review Board of Baylor College of Medicine. Written, informed consent was obtained from all human subjects.

In Vivo Experiments

Animals.

C57BL/6J male mice were purchased from Jackson Laboratory (Bar Harbor, ME) and studied at an age of 6–8 wk. Mice with targeted deletions of CD11b (Mac-1^−/−) (11) and P-selectin (P-sel^−/−) (12) were backcrossed at least 10 generations with C57BL/6J mice. PSGL-1^−/− mice were bred using mice (B6.Cg-Selp[tm1Fur]/J) backcrossed 14 generations with C57BL/6J mice (Jackson Laboratory). Platelet and leukocyte counts were performed on tail vein whole blood via a Bright-Line hemacytometer (American Optical, Buffalo, NY) or automated complete blood counter (Center for Comparative Medicine facility, Baylor College of Medicine).

In vivo platelet depletion.

Twenty-four hours before corneal wounding, mice were injected intraperitoneally with rat anti-mouse glycoprotein Ib-α antibody (Xia.B2, Emfret Analytics, Würzburg, Germany) to deplete platelets, as previously shown (26), or its IgG_2a isotype control (R35–95, BD Biosciences) at a concentration of 0.1 mg in 0.2 ml PBS.

Mouse model for corneal epithelium abrasion.

Before corneal epithelium abrasion, mice were anesthetized with 50 mg/kg pentobarbital sodium (Nembutal, Ovation Pharmaceuticals, Deerfield, IL) injected intraperitoneally. After sedation, the central cornea was marked with by a trephine 1.5 mm (Miltex, York, PA) in diameter, and the epithelium was debrided using an Algerbrush II with a 0.5 mm burr (The Alger, Lago Vista, TX) under a dissecting microscope. This method of injury does not injure the corneal basement membrane or stroma (18, 38). Mice were then killed, and corneas were excised before and 6 and 12 h after wounding.

Immunohistology.

Excised corneas, including the limbus, were fixed, permeabilized, and incubated with labeled monoclonal antibodies as previously described (25, 26). The following antibodies were used to identify cells of interest: 5 μg/ml anti-Ly-6G/FITC (neutrophils, NIMP-R14, Hycult Biotechnology, Canton, MA, and 1A8, BD Biosciences), 1 μg/ml anti-CD41/phycoerythrin (PE; platelets, MWReg30, BD Biosciences), 1 μg/ml anti-CD31/allophycocyanin (APC; endothelium, Mec13.3, BD Biosciences), and 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; nuclear stain, Sigma-Aldrich). PSGL-1 has been reported to be on the surface of venules of the small intestine (37) and on human umbilical vein endothelial cells (HUVECs) (9). To determine if PSGL-1 is present on the limbal vascular endothelium and participates in neutrophil extravasation, corneas were labeled with 2 μg/ml anti-PSGL-1/PE (2PH1, BD Biosciences) or a IgG₁/PE isotype control (R3-34, BD Biosciences) in conjunction with anti-Ly6G/FITC, anti-CD31/APC, and DAPI. Labeled corneas were cut radially to allow flattening and then mounted on glass slides in Airvol mounting medium (Air Products and Chemicals, Allentown, PA).

Corneal whole mounts were viewed on an Olympus IX70 inverted microscope (Olympus America, Center Valley, PA), and digital images were acquired for analysis (DeltaVision, Applied Precision, Issaquah, WA). Twelve limbal fields per mouse were imaged, and neutrophils and platelets were counted in a 150 × 150 μm square area (Fig. 1A). The limbus was defined as the intervening zone between the cornea and sclera where the vessels reside.

Fig. 1.

A: fluroescence image of the mouse corneal limbal region 6 h after injury. Neutrophils (green, thin white arrow, Ly6G/FITC), platelets (orange, white arrowhead, anti-CD41/phycoerythrin), and limbal vessels (red, large white arrow, anti-CD31/allophycocyanin). Magnification: ×40. Scale bar = 20 μm. B: phase-contrast image of the static adhesion assay. Transmigrated polymorphonuclear neutrophils (PMNs; phase dark, large black arrow), nonmigrated PMNs (phase bright; black arrowhead), platelets (small discoid, thin black arrow), and red blood cells (large discoid) are shown. Magnification: ×40. Scale bar = 20 μm.

In Vitro Experiments

EC culture.

HUVECs were cultured as previously described (3). HUVECs were routinely harvested from 3 to 10 umbilical veins by collagenase perfusion according to Huang et al. (20) and pooled and plated in T75 Corning flasks pretreated with 0.2% gelatin (Difco, Detroit, MI). Monolayers were cultured with medium 199 (GIBCO-BRL) with 10% FBS (Sigma), 10% bovine calf serum (Sigma), Fungizone (250 μg/ml amphotericin B, GIBCO-BRL), penicillin G (5,000 μg/ml)-streptomycin (5,000 μg/ml, GIBCO), 1% heparin (Sigma), 1 M HEPES buffer (GIBCO), and 50 μg/ml EC growth supplement (BD Bioscience). Five days after being seeded, HUVECs were passed at confluence onto either 25- or 35-mm glass coverslips (Belco), which were pretreated with 1% gelatin (Difco) and cross-linked with 0.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA). All monolayers were then used at 4 days postconfluence, when transendothelial electrical resistance is maximal and stable (5). Four hours before adhesion and transmigration assays (static and dynamic), HUVECs were stimulated with 10 U/ml human recombinant IL-1β (R&D Systems, Minneapolis, MN).

Neutrophil isolation.

Human peripheral blood neutrophils [polymorphonuclear neutrophils (PMNs)] were isolated via venipuncture from healthy, nonpregnant adults as previously described (3). Volunteers who had been ill or had taken salicylate-containing products in the previous 2 wk, nonsteroidal anti-inflammatory drugs in the previous 48 h, or caffeine in the previous 8 h were excluded. Venous blood was anticoagulated in citrate phosphate dextrose (Abbott Laboratories, Abbott Park, IL) and sedimented in 6% dextran (250,000 MW, Spectrum, Gardena, CA). Leukocyte-rich plasma was then centrifuged over a 6.07% Ficoll (400,000 MW, Sigma Chemical)-10% Hypaque (Sanofi-Winthrop Pharmaceuticals, New York, NY) gradient to isolate PMNs, which were washed and resuspended in Dulbecco's PBS (D-PBS; Life Technologies, Grand Island, NY). Cells were counted on a Bright-Line hemacytometer (American Optical) and diluted to 2–5 × 10⁶ PMNs/ml. PMNs were kept at room temperature in D-PBS (containing 10 mM glucose, 1 mM CaCl₂, and 1 mM MgCl₂) for up to 3 h before being used in adherence and transmigration assays. The activation state of isolated neutrophils was determined by shape change as described by Ehrengruber et al. (13) in the presence and absence of N-formyl-Met-Leu-Phe (Sigma).

Platelet isolation.

Human platelets were isolated as previously described in detail (6). Human platelets were isolated from venous blood anticoagulated in acid citrate dextrose (100 mM trisodium citrate dehydrate, 71 mM citric acid monohydrate, and 111 mM dextrose) and centrifuged to separate the platelet-rich plasma, which was then centrifuged to form a platelet pellet. After removal of the platelet-poor plasma, the platelet pellet was resuspended in 10 U/ml heparin sodium (American Pharmaceutical Partners, Los Angeles, CA) in Tyrode's albumin buffer [137 mM NaCl, 2.7 mM KCl, 0.43 mM NaH₂PO₄, 11.9 mM NaHCO₃, 0.98 mM MgCl₂, 2 mM CaCl₂, 5.01 mM HEPES, 5.55 mM dextrose, 0.35% human serum albumin (pH 7.35), Baxter Healthcare, Deerfield, IL], washed, and counted on a hemacytometer. In between centrifugation cycles, platelets were kept quiescent using prostacyclin (0.5 μM PGI₂, Sigma). In the static adhesion experiments, isolated human platelets were exposed to either vehicle control (HEPES-buffered saline; “unstimulated”) or 35 μM thrombin receptor agonist peptide (“TRAP stimulated,” S7152, Sigma) for 5 min at room temperature before use. TRAP is a 14-amino acid peptide sequence that is similar to the amino-terminal extracellular domain of proteinase-activated receptor-1, which is unmasked by proteolytic cleavage via thrombin and has been shown to induce platelet activation and aggregation (7) with minimal, if any, effect on human neutrophils (22). To determine the activation state of isolated platelets, freshly isolated platelets were incubated with neutrophils at 37°C in the presence or absence of TRAP and then studied under flow cytometry for the formation of platelet-neutrophil aggregates. The formation of platelet-leukocyte aggregates is a sensitive marker of platelet activation (29).

In one set of experiments, TRAP-stimulated platelets were centrifuged at 1,900 g for 8 min. The resulting supernatant (releasates) was separated, and the platelet pellet was resuspended in Tyrode's albumin buffer. Neutrophil transmigration was then assessed in the presence of buffer, activated platelets, or platelet releasates as previously described.

PMN adhesion and transmigration assay under static conditions.

Neutrophil adhesion and transendothelial migration was assessed in Muntz static adhesion chambers (40) as previously described (5). Coverslips with IL-1β-stimulated HUVEC monolayers were rinsed in D-PBS, placed into the adhesion chamber, and covered with a plain glass coverslip that was separated from the lower coverslip by a rubber O-ring. Within this closed compartment, a suspension of diluted PMNs (1 × 10⁶ PMNs/ml) premixed with Tyrode's albumin buffer (control), unstimulated platelets (100 × 10⁶ platelets/ml), or 35 μM TRAP-stimulated platelets (100 × 10⁶ platelets/ml) for 15 min at room temperature was introduced via a 25-guage needle. This provided a platelet-to-neutrophil ratio of 100:1, which is within the range seen in circulating blood of healthy adult humans (16). All experiments were conducted at 37°C on a Nikon Diaphot inverted microscope (Nikon, Garden City, NY). Under phase-contrast optics, the number of PMNs that contacted and transmigrated across the endothelial monolayer during an initial 500-s period was determined, as shown in Fig. 1B. Transmigrated PMNs were identified by playback of video recordings obtained with phase-contract optics using criteria previously described and validated in our laboratory (4, 5, 40). Briefly, transmigrated neutrophils are extensively spread, very phase dark, and lack the distinct and continuous phase bright halo demonstrated by adherent neutrophils above the endothelial monolayer. The adhesion chamber was then inverted for an additional 500 s, with nonadherent PMN falling from the monolayer surface. The percentages of adherent and transmigrated PMN were then determined (1,000 s).

PSGL-1 blocking monoclonal antibody experiments.

Mouse anti-human PSGL-1 monoclonal antibody (5 μg/ml, KPL-1, BD Biosciences) and its IgG₁ isotype control (107.3, BD Biosciences) were used in blocking experiments to determine if the enhanced transmigration of neutrophils by platelets was attenuated by blockade of PSGL-1. This anti-PSGL-1 antibody has been shown to block the interaction between P-selectin and neutrophils (41). Isolated PMNs (1 × 10⁶ PMNs/ml) were incubated with either anti-PSGL-1 or isotype control antibody (5 μg/ml) for 10 min at 37°C. TRAP-stimulated platelets (100 × 10⁶ platelets/ml) or Tyrode's albumin buffer was added, and the suspension was split for static adhesion experiments (as described above) and flow cytometry.

Flow cytometry was performed on a BD LSR II (Becton Dickinson, Franklin Lakes, NJ) to examine platelet-neutrophil aggregates. Cell suspensions were incubated with anti-CD11b/AF288 (ICRF44, BD Biosciences) and anti-CD41a/PE (HIP8, BD Biosciences) for 20 min at room temperature and then fixed in 0.5% formaldehyde for analysis.

PMN adhesion and transmigration assay under dynamic conditions.

Neutrophil adherence and migration under hydrodynamic flow was examined using a parallel plate flow chamber maintained at 37°C as previously described (24). Neutrophils were diluted (1 × 10⁶ neutrophils/ml) and perfused premixed with Tyrode's albumin buffer (control) or with unstimulated platelets (100 × 10⁶ platelets/ml) over IL-1β-stimulated HUVEC monolayers cultured on 35-mm glass coverslips at a shear stress of 2 dyn/cm² for 10 min. Adhesion and transmigration of neutrophils were video recorded under phase-contrast optics and analyzed using Image-Pro Plus 5.1 software (Media Cybernetics, Bethesda, MD).

Statistical Analysis

Data were analyzed using either Student's t-test, one-way ANOVA with Newman-Keuls pairwise multiple comparisons, or two-way ANOVA with the Bonferonni posttest, where appropriate, using Prism 4.03 (GraphPad Software, La Jolla, CA). P values of <0.05 were considered significant.

RESULTS

Platelets Mediate Neutrophil Emigration In Vivo

To determine whether platelets mediate neutrophil emigration in vivo, we quantified emigrated neutrophils after corneal wound injury in mice treated with either platelet-depleting or isotype control antibodies. Platelet depletion before corneal injury altered the accumulation of extravascular neutrophils in the limbal region (overall interaction: P < 0.05 by two-way ANOVA) with decreased accumulation at 6 and 12 h after injury (Fig. 2). Platelet depletion was effective and selective: circulating platelet counts in anti-platelet-treated mice were reduced by 94% relative to those of mice treated with isotype control antibodies (P < 0.0001), whereas leukocyte and neutrophil counts did not differ between the groups (Table 1).

Fig. 2.

Extravascular PMNs in the limbal region of the injured mouse cornea after the intraperitoneal injection of control or anti-platelet antibodies. Depletion of platelets before injury altered the accumulation of extravascular neutrophils (overall interaction: P < 0.01 by two-way ANOVA) with decreased extravascular neutrophil accumulation seen at 6 and 12 h after injury (†P < 0.01 by Bonferonni posttest). Values are means ± SE; n = 3–6 per group.

Table 1.

Complete blood counts of mice 12 h after corneal injury

Group	Platelets	Leukocytes	Neutrophils	Lymphocytes	Monocytes
WT mice with control monoclonal antibody	1,073 ± 72	4.07 ± 0.64	0.48 ± 0.10	3.94 ± 1.11	0.08 ± 0.04
WT mice with CD42b monoclonal antibody	63 ± 8*	3.14 ± 0.60	0.40 ± 0.11	2.76 ± 0.41	0.04 ± 0.00
WT mice	981 ± 152	3.81 ± 1.25	0.34 ± 0.12	3.17 ± 1.16	0.15 ± 0.12
PSGL-1−/− mice	1,305 ± 182	4.90 ± 0.07	1.00 ± 0.09	3.50 ± 0.29	0.11 ± 0.02
P-sel−/− mice	1,199 ± 58	7.19 ± 1.75	1.11 ± 0.23	5.50 ± 1.32	0.16 ± 0.05

Values are means ± SE (in ×10³ cells/ml). WT mice, wild-type mice; PSGL-1^−/− mice, P-selectin glycoprotein ligand-1-deficient mice; P-sel^−/− mice, P-selectin-deficient mice.

^*P< 0.0001 vs. WT mice with control monoclonal antibody.

Role of PSGL-1 in Platelet-Mediated Neutrophil Emigration In Vivo

Because of the importance of P-selectin-PSGL-1 interactions between platelets and neutrophils, we hypothesized that PSGL-1-deficient mice would have decreased extravascular neutrophil accumulation after corneal injury. Extravascular accumulation of neutrophils was blunted in PSGL-1-deficient mice (overall interaction: P < 0.05 by two-way ANOVA), with decreased accumulation in the corneal limbus 12 h after injury (Fig. 3). Because P-selectin is the counterligand for PSGL-1, P-sel^−/− mice were evaluated at 12 h; these mice had a decreased number of extravascular neutrophils compared with wild-type mice, similar to PSGL-1^−/− mice (Fig. 3). This reduction in extravascular neutrophils could not be explained by any difference between PSGL-1^−/− and wild-type control mice in circulating platelet, total leukocyte, or neutrophil counts (Table 1). Also, immunostaining for PSGL-1 was not detected on the limbal endothelium; only neutrophils were found to stain for PSGL-1 (data not shown). Since PSGL-1 may affect Mac-1 clustering (17), we compared extravascular neutrophil accumulation in Mac-1^−/− mice with wild-type mice; however, there were no differences in emigrated neutrophils between the groups (4,488 ± 542 vs. 4,986 ± 446 PMNs/mm², not significant) between the groups.

Fig. 3.

Extravascular PMNs in the limbal region of the injured mouse cornea in wild-type (WT) mice, P-selectin glycoprotein ligand (PSGL)-1-deficient (PSGL-1^−/−) mice, and P-selectin-deficient (P-sel^−/−) mice. In PSGL-1^−/− and P-sel^−/− mice, there was a significant decrease in extravascular neutrophils 12 h after injury compared with WT mice (^P < 0.050). Values are means ± SE; n = 3 per group.

Platelets Enhance Neutrophil Transendothelial Migration In Vitro

Based on a shape-change assay, neutrophils isolated with our protocol demonstrated a negligible degree of activation activated (98 ± 1% PMNs were spherical), and neutrophils were sensitive to activation by N-formyl-Met-Leu-Phe at 0.1 nM (40 ± 6% spherical, P < 0.01) and 100 nM (16 ± 4% spherical, P < 0.001). Similarly, using platelet-neutrophil aggregates as a measure of platelet activation (29), isolated platelets were minimally activated compared with 35 μM TRAP-stimulated platelets (19 ± 3% vs. 93 ± 1% CD41a⁺ PMNs, P < 0.0001).

To determine whether the changes in extravascular neutrophils shown in vivo reflect effects on neutrophil transmigration independent of changes in neutrophil adhesion, we used an in vitro static adhesion model. Isolated neutrophils were mixed with buffer (control), unstimulated platelets, or TRAP-stimulated platelets before injection into the static adhesion chamber. Stimulated platelets enhanced early (500 s) neutrophil transendothelial migration under static conditions (Fig. 4A) but did not affect firm adhesion (Fig. 4B) or late neutrophil transendothelial migration (Fig. 4C). To determine if the presence of TRAP affected neutrophil transmigration independent of platelets, neutrophils in the presence or absence of 35 μM TRAP were injected into the static adhesion chamber. In this assay, the addition of 35 μM TRAP (without platelets) to neutrophils did not affect neutrophil adhesion, early transmigration, or late transmigration (n = 4; data not shown).

Fig. 4.

Effect of platelets on neutrophil transmigration across IL-1β-stimulated human umbilical vein endothelial cells (HUVECs) under static conditions. A: the presence of thrombin receptor agonist peptide (TRAP)-activated platelets (+ Act Plt) increased the percentage of transmigrated PMNs at 500 s compared with PMNs without platelets (PMN Alone, *P < 0.001) and compared with PMNs in the presence of unstimulated platelets (+ Plt; ^P < 0.05). B and C: the presence of platelets did not affect PMN adhesion (B) or transmigration (C) at 1,000 s. Values are means ± SE; n = 5–8.

To determine whether soluble mediators released from activated platelets were sufficient to enhance PMN transmigration, we performed an additional set of in vitro experiments on IL-1β-stimulated HUVECs. ECs were exposed to either PMNs alone, PMNs with TRAP-stimulated platelets, or PMNs with the releasate derived from TRAP-stimulated platelets. Whereas TRAP-stimulated platelets enhanced PMN transmigration to a similar magnitude as shown above (26.8 ± 9% relative increase in transmigration, P < 0.05), the releasate derived from the same preparation of platelets (n = 3 donors) had no effect on PMN transmigration (0.01 ± 3% relative change in transmigration, not significant).

To determine whether the enhancement of neutrophil transmigration by platelets was present under dynamic conditions, neutrophils with or without platelets were flowed across a HUVEC monolayer at a venous shear stress of 2 dyn/cm² (24). Under these dynamic conditions, the addition of platelets to neutrophils resulted in faster transendothelial migration after firm arrest (time to complete transendothelial migration was 35 ± 3 vs. 51 ± 3 s, P < 0.001; Fig. 5). In our assay, the presence of platelets did not affect neutrophil rolling velocity (mean or maximum) or the distance rolled after capture (data not shown).

Fig. 5.

Duration of migration after firm arrest under a parallel-plate flow of 2 dyn/cm². The presence of platelets shortened the duration of PMN transendothelial migration across IL-1β-stimulated HUVECs (*P < 0.001). Mean: 8–85 PMN events/group; n = 4 donors.

Role of PSGL-1 on Platelet-Enhanced Neutrophil Transmigration

Analogous to the in vivo experiments, we examined the effect of PSGL-1 on platelet-enhanced neutrophil migration in vitro. Blocking antibody to PSGL-1 or its isotype control was added to a mixture of neutrophils and TRAP-stimulated platelets. Blockade of PSGL-1 attenuated the effect of platelets on PMN transmigration (Fig. 6A) without affecting firm adhesion (Fig. 6B). The presence of anti-PSGL-1 blocking antibodies blocked platelet-neutrophil aggregation, as detected by flow cytometry (Fig. 6C), suggesting that platelets enhance neutrophil transmigration via PSGL-1.

Fig. 6.

Static adhesion assay in the absence (left groups) or presence (right groups) of TRAP-activated platelets with either isotype control or anti-PSGL-1 monoclonal antibody (MAb). A and B: the presence of anti-PSGL-1 mAb (KPL-1) attenuated the effect of increased PMN transmigration by platelets (^P < 0.05) at 500 s (A) without affecting firm adhesion (B). Values are means ± SE; n = 3. C: flow cytometric analysis of isolated PMNs in the presence or absence of TRAP-activated platelets with either anti-PSGL-1 mAb or isotype control. The presence of anti-PSGL-1 mAb (KPL-1) blocked the formation of platelet-neutrophil aggregates (*P < 0.001). Values are means ± SE; n = 4.

DISCUSSION

The main finding of our study is that platelets promote neutrophil transendothelial migration both in vivo and in vitro. Of interest, these effects were evident despite using distinct models of inflammation in tissues from different species (IL-1β for HUVECs and corneal injury for mouse limbal vessels), consistent with a conserved effect of platelets in neutrophil transmigration. In vivo, depletion of platelets decreased the accumulation of extravascular neutrophils at 6 and 12 h, as shown in Fig. 3. In the absence of either PSGL-1 or P-selectin, the decreased accumulation of extravascular neutrophils was only seen at 12 h, suggesting the role of other mechanisms in platelet-enhanced neutrophil extravasation. A number of other platelet molecules may contribute to these interactions, including glycoprotein Ib-α, glycoprotein IIb/IIIa, and ICAM-2, among others (46). While our in vivo data revealed that platelets mediate neutrophil emigration in this model of inflammation, whether this effect is independent of neutrophil adhesion cannot be determined solely based on these observations. However, the in vitro model enabled us to distinguish effects of platelets on neutrophil adhesion and emigration. In the static model in vitro, the data demonstrated that platelets enhance neutrophil transendothelial migration, independent of adhesion (Fig. 4). Furthermore, this effect was attenuated with the addition of anti-PSGL-1 antibodies (Fig. 5). In a model of dynamic adhesion, the presence of platelets reduced the time needed for neutrophil transmigration across the endothelium after firm arrest. Taken together, these data suggest that platelets play a role in inflammation via the enhancement of neutrophil transmigration via P-selectin-PSGL-1 interactions.

While PSGL-1 has been shown to participate in intracellular signaling of leukocytes, augmenting slow rolling and adhesion, how PSGL-1 augments enhanced neutrophil transendothelial migration by platelets is unclear. Many studies have shown the signaling properties of PSGL-1 (14) via Src kinase (44), MAPK (39), and spleen tyrosine kinase (45) pathways. Specifically, the binding and cross-linking of PSGL-1 by P-selectin or E-selectin augments the adhesion of neutrophils, via β₂-integrins, to fibrinogen (27) and ICAM-1 (2). Green et al. (17) demonstrated that E-selectin binding to L-selectin and PSGL-1 on human neutrophils results in the clustering and colocalization of L-selectin and PSGL-1. This, in turn, leads to a shift toward a high-affinity state of neutrophil CD18 and clustering. Similarly, Hidalgo et al. (19) demonstrated that E-selectin binding to E-selectin ligand-1 on mouse neutrophil induces the polarization and clustering of Mac-1 at the leading edge of crawling neutrophils. Mac-1 plays an important role in neutrophil transendothelial migration (5, 21). Thus, clustering of Mac-1 on the leading edge of neutrophils by PSGL-1 activation may lead to enhanced transendothelial migration. Although we did not observe a decrease in neutrophils between Mac-1^−/− and wild-type mice at 12 h, this observation is not entirely unexpected, based on published reports. For example, Ding et al. (11) described increased extravasation of neutrophils in TNF-α-stimulated subcutaneous air pouches of Mac-1^−/− mice. This has been proposed to be due to the decreased apoptosis seen in Mac-1^−/− mice (8). Whether the effect of PSGL-1 on neutrophil transmigration demonstrated in the present study involves the clustering or activation of Mac-1 remains to be determined.

Under static conditions, we observed an increase in early, but not late, neutrophil transendothelial migration in the presence of TRAP-stimulated platelets. This finding is not entirely surprising, as it may reflect an increased speed in transmigration, as seen in our dynamic adhesion assay, combined with a finite supply of neutrophils. In this scenario, the neutrophils that would eventually transmigrate completed the process earlier but reached a similar end point after a set amount of time. This explanation is also consistent with our observations in vivo, where the absence of platelets decreased extravascular accumulation over time. These observations at later time points may be due to a gradual accumulation in the extravascular region surrounding the limbal vessels, where the difference becomes greater over time. Future experiments using intravital microscopy with time-lapse monitoring of neutrophil adhesion and transmigration would help test this mechanism in vivo.

In our in vitro models, although there was a difference in neutrophil transendothelial migration in the presence of platelets, we did not observe an increase in firm adhesion under static or dynamic conditions. Others (31) have described platelets as enhancing leukocyte adhesion. While the reasons for the discrepancy in results are not entirely clear, differences in experimental conditions may limit a direct comparison of these findings. Regardless, the neutrophils that migrated under dynamic conditions in our model did so more rapidly in the presence of platelets, thus suggesting a role for platelets in neutrophil migration that is independent from firm adhesion. Under conditions of physiological shear stress in our parallel-plate model, platelets promoted transmigration in the absence of stimulation by TRAP. In fact, preliminary experiments with TRAP-stimulated platelets revealed neutrophil-platelet-neutrophil aggregates with reduced interactions with ECs, which precluded accurate counts of transmigration. Such aggregates can modify the rheology of neutrophils and alter potential binding sites. These observations are consistent with a previous study (36) of neutrophils flowing over platelet monolayers resulting in homotypic aggregates, leading to unstable adhesion and increased velocity. Besides direct platelet-leukocyte influences on leukocyte adhesion and migration, activated platelets have also been shown to produce microparticles (34); a potential contribution of platelet-derived microparticles in promoting neutrophil transmigration remains to be determined.

Of note, activation of platelets by TRAP results in the release of a broad range of growth factors, cytokines, and other inflammatory mediators (35), well known to increase endothelial permeability (28). Although it is conceivable that increased endothelial permeability induced by TRAP-activated platelets contributed to the enhanced PMN transmigration, two observations tend to argue against this mechanism. First, if enhanced PMN transmigration was primarily a result of platelet-induced increased endothelial permeability, then anti-PSGL-1 antibodies would not be expected to inhibit PMN transmigration in the presence of TRAP-activated platelets, as shown in Fig. 6A. Furthermore, releasates from TRAP-activated platelets did not enhance PMN transmigration in our study. However, subsequent studies quantifying platelet-induced changes in endothelial permeability relative to changes in neutrophil transmigration would be necessary to fully exclude this mechanism.

In summary, our data demonstrate that platelets enhance neutrophil transendothelial migration in inflammation both in vivo and in vitro. The findings are consistent with a mechanistic role for PSGL-1 in this response. The events downstream from PSGL-1 influencing neutrophil transmigration remain to be clarified.

GRANTS

This work was supported by National Institutes of Health (NIH) Grants EY-018239, EY-017120, and HL-079368 and by a Department of Veterans Affairs merit review grant. F. W. Lam was supported by NIH Grant T32-HL-07747.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).