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Originally published online as doi:10.1189/jlb.0906553 on December 12, 2006

Published online before print December 12, 2006
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(Journal of Leukocyte Biology. 2007;81:686-695.)
© 2007 by Society for Leukocyte Biology

Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis

Anjelica L. Gonzalez*, Wafa El-Bjeirami*, Jennifer L. West{dagger}, Larry V. McIntire{ddagger} and C. Wayne Smith*

* Section of Leukocyte Biology, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA;
{dagger} Department of Bioengineering, Rice University, Houston, Texas, USA; and
{ddagger} Coulter Department of Biomedical Engineering, Georgia Institute of Technology/Emory University, Atlanta, Georgia, USA

1 Correspondence: Baylor College of Medicine, Leukocyte Biology, 1100 Bates, Suite 6014, Houston, TX 77030-2600, USA. E-mail: cwsmith{at}bcm.tmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transendothelial migration of neutrophils induces phenotypic changes that influence the interactions of neutrophils with extravascular tissue components. To assess the influence of transmigration on neutrophil chemokinetic motility, we used polyethylene glycol hydrogels covalently modified with specific peptide sequences relevant to extracellular matrix proteins. We evaluated fMLP-stimulated human neutrophil motility on peptides Arg-Gly-Asp-Ser (RGDS) and TMKIIPFNRTLIGG (P2), alone and in combination. RGDS is a bioactive sequence found in a number of proteins, and P2 is a membrane-activated complex-1 (Mac-1) ligand located in the {gamma}-chain of the fibrinogen protein. We evaluated, via video microscopy, cell motility by measuring cell displacement from origin and total accumulated distance traveled and then calculated average velocity. Results indicate that although adhesion and shape change were supported by hydrogels containing RGD alone, motility was not. Mac-1-dependent motility was supported on hydrogels containing P2 alone. Motility was enhanced through combined presentation of RGD and P2, engaging Mac-1, {alpha}Vß3, and ß1 integrins. Naïve neutrophil motility on combined peptide substrates was dependent on Mac-1, and {alpha}4ß1 while {alpha}6ß1 contributed to speed and linear movement. Transmigrated neutrophil motility was dependent on {alpha}vß3 and {alpha}5ß1, and {alpha}4ß1, {alpha}6ß1, and Mac-1 contributed to speed and linear motion. Together, the data demonstrate that efficient neutrophil migration, dependent on multi-integrin interaction, is enhanced after transendothelial migration.

Key Words: transmigration • ß1 integrins • inflammation • extracellular matrix • motility • ß2 integrin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During inflammation, neutrophils interact with proteins of the extracellular matrix (ECM) after migration through vascular endothelium to the site of tissue injury or infection. These interactions are mediated through integrins, which are heterodimers of noncovalently associated {alpha} and ß subunits belonging to members of the ß1, ß2, and ß3 families [1 ]. Here, we investigate neutrophil motility mediated by specific interactions between neutrophil integrins and active domains of ECM proteins and evaluate functional changes of the neutrophil as a result of transendothelial migration.

Neutrophil integrin affinity and/or avidity may be stimulated through endogenous and bacterial chemokines and chemotactic factors, including IL-8, leukotriene B4 (LTB4), formyl-Met-Leu-Phe (fMLF), and anaphylatoxin C5a. In the vasculature, endothelial-derived chemokines (e.g., CXCL8) may stimulate the neutrophil to arrest and transmigrate, and tissue-derived chemotactic factors (e.g., bacterial-released fMLF or C5a) induce the neutrophil to migrate through the ECM toward the source of infection. Neutrophil integrins interact with ECM proteins through binding, which not only allows cell adhesion but also initiates signal transduction cascades that influence shape change, chemotaxis, cell proliferation, and survival. These phenomena have only recently been linked to specific integrin-ligand interactions [2 3 4 5 ]. Recently, we investigated the ability of ECM protein sequences Arg-Gly-Asp (RGD) and TMKIIPFNRTLIGG (P2) to promote specific, integrin-mediated neutrophil adhesion and spreading [6 ]. That study used tissue-engineering techniques to evaluate interactions with the ECM peptides. Specifically, (poly)ethylene glycol (PEG) diacrylate derivatives were used to form hydrogels as a nonadhesive background for covalent attachment of bioactive moieties. Those experiments demonstrated that neutrophils show little ability to adhere to unmodified PEG hydrogels, but adhesion and spreading are robust on peptide-modified hydrogels. Incorporating RGD or P2, alone or in combination, has enabled recognition of differential functions of neutrophil integrins in adhesion and spreading. In this case, the PEG hydrogel serves as an inert background [7 ], rendered bioactive by including RGD-Ser (RGDS) [8 , 9 ] and P2 peptides [6 ]. Combined interactions result in adhesion that differs markedly from that seen with either integrin engaged independently.

A secondary goal of this study is to evaluate phenotypic changes of the neutrophil as a result of transendothelial migration. Although investigators have observed changes in integrin expression after transendothelial migration [10 , 11 ], little has been done to investigate the functionality of these integrins after transendothelial migration. In this study, we provide evidence that integrin-mediated motility is altered following neutrophil transendothelial migration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peptides
RGDS peptide was purchased from American Peptide Company Inc. (Sunnyvale, CA, USA). P2 [12 ] was synthesized using an automated peptide synthesizer (Model 431A, PE Applied Biosystems, Foster City, CA, USA) and standard fmoc chemistry [6 ].

Development of biomimetic hydrogels
The biomimetic PEG hydrogels were prepared as described previously [6 , 13 ]. Briefly, PEG diacrylate was prepared by combining 0.1 mmol/ml dry PEG (10,000 Da, Fluka, Milwaukee, WI, USA), 0.4 mmol/ml acryloyl chloride, and 0.2 mmol/ml triethylamine in anhydrous dichloromethane and stored under argon overnight. The resulting PEG diacrylate was then precipitated with ether, filtered, and dried. PEG diacrylate was dialyzed prior to use.

Peptides were conjugated to PEG monoacrylate by reacting the peptide with acryloyl-PEG-N-hydroxysuccinimide (3400 Da, Nektar, Inc., San Carlos, CA, USA) in sodium bicarbonate (pH 8.5) at a 1:1 molar ratio for 2 h. The coupled acryloyl-PEG peptide was lyophilized, and the conjugated PEG peptide was dialyzed prior to use. All polymers were characterized by proton nuclear magnetic resonance (Avance 400 Mhz, Bruker, Billerica, MA, USA) and gel permeation chromatography with an evaporative light-scattering detector using PEG standards (Polymer Laboratories, Amherst, MA, USA).

Hydrogels were prepared by combining 0.1 g/ml PEG diacrylate and 0.02 g/ml (5.2 µmol/ml) acryloyl-PEG-RGDS, 0.02 g/ml (4.0 µmol/ml) acryloyl-PEG-P2, or a combination of 0.01 g/ml acryloyl-PEG-RGDS and 0.01 g/ml acryloyl-PEG-P2 in PBS (pH 7.2) containing 0.1 g/L glucose. The solution was then sterilized by filtration (0.2 µm with 0.8 µm prefilter, Pall Corp., Ann Arbor, MI, USA). 2,2-Dimethoxy-2-phenylacetophenone (10 µl/ml) in n-vinylpyrrolidone (300 mg/ml) was added as the photoinitiator. The resulting solution was exposed to UV light (365 nm, 10 mW/cm2) for 30 s to convert the liquid polymer solution to a covalently cross-linked hydrogel. The polymerized gels were then incubated overnight in PBS to allow them to reach their equilibrium swelling. PEG diacrylate hydrogels without peptides were used as controls.

Endotoxin detection
Endotoxin assays were conducted on polymer solution prior to gelation. The Limulus amoebocyte lysate (LAL) assay was used to determine endotoxin presence in each component of the PEG hydrogel. Briefly, a 100-µl sample was placed in a sterilized glass tube with 100 µl LAL. This solution was then placed on a heating block for 1 h. Endotoxin-negative solutions resulted in no gelation, and endotoxin-positive solutions became gelated. Pyrogen-free water (Baxter Healthcare Corp., Deerfield, IL, USA) served as a negative control, and bacterial LPS served as an endotoxin-positive control. All samples of PEG hydrogel reagents were endotoxin-negative.

Neutrophil isolation
Neutrophils were obtained from the whole blood of healthy human volunteers. The method for isolation has been described previously [14 ]. Briefly, neutrophils were purified by 6% Dextran sedimentation to remove RBC and by Ficoll-Hypaqe gradient centrifugation to remove remaining leukocytes. The cells were finally suspended in PBS containing 0.1 g/L glucose to be used at a concentration of 1 x 106 cells/ml.

Antibodies and reagents
Neutrophil-stimulating agent fMLF was obtained from Sigma Chemical Co. (St. Louis, MO, USA) and used at 1 x 10–7 M concentration. Mouse antihuman membrane-activated complex-1 (Mac-1) antibody, R15.7, was received as a generous gift from Dr. Lora Whitehouse (Repligen, Cambridge, MA, USA) and used at a concentration of 10 µg/ml [14 ]. Anti-E-selectin mAb CL3 was used at a concentration of 10 µg/ml, as described previously [15 ]. Mouse antihuman integrin {alpha}vß3 mAb, LM609, was purchased from Chemicon International (Temecula, CA, USA) and was used at a concentration of 10 µg/ml [16 ]. Mouse antihuman antibodies against {alpha}4 (CD49d), {alpha}5 (CD49e), and {alpha}6 (CD49f) were purchased from BD Biosciences PharMingen (San Jose, CA, USA).

HUVEC culture
HUVEC harvest and culture have been described by Burns et al. [17 ]. Briefly, HUVEC were harvested from human umbilical veins by collagenase perfusion, according to Huang et al. [18 ] and pooled, plated, and pretreated with 0.2% gelatin (Difco, Detroit, MI, USA). Monolayers are cultured in a 1:1 mixture of M199 (Gibco BRL, Grand Island, NY, USA) and medium supplemented with 10% FBS and 10% bovine calf serum (Hyclone Laboratories, Inc., Logan, UT, USA), 1% penicillin-streptomycin (Gibco BRL), 1% fungizone (Gibco BRL), 1% HEPES buffer (Gibco BRL), 1 µg/ml heparin (Sigma Chemical Co.), and 50 µg/ml endothelial cell (EC) growth supplement (Collaborative Biomedical Products, Bedford, MA, USA).

Neutrophil transendothelial migration
Primary HUVEC were dissociated using trypsin/EDTA, seeded onto gelatin-coated, 3.0 µm pore polycarbonate Transwell® filters (Corning Inc., Corning, NY, USA), which were placed in matching six-well plates and cultured for 4 days. HUVEC monolayers were stimulated with IL-1ß (10 U/ml) for 4 h at 37°C. Following stimulation, the inserts were washed with HBSS, once transferred to six-well plates coated with a thin layer of 1% agarose, which facilitated removal of transmigrated neutrophils for further analysis and minimized additional activation by the plastic wells. Freshly isolated neutrophils from healthy volunteers were placed on top of the insert at a ratio of 4:1 (PMN:endothelial) and allowed to transmigrate through the activated HUVEC for 1 h. Nontransmigrated neutrophils were then collected from the top of the insert (referred to as EC contacted), and transmigrated neutrophils were collected from the bottom of the insert. Collected cells were counted and used for experimentation.

RNA isolation and RT
Total RNA was isolated from transmigrated and nontransmigrated neutrophils using Trizol reagent following the manufacturer’s instructions (Life Technologies/Invitrogen, Carlsbad, CA, USA). RT was performed in a 20-µl final volume containing 40 U RT AMV, 50 mA260 units primer random p(dN)6, 20 U RNase inhibitor, and 1 mM deoxy-unspecified nucleoside 5'-triphosphates (Roche Applied Sciences, Indianapolis, IN, USA). The mixture was subjected to 25°C for 10 min and 42°C for 60 min and inactivated at 95°C for 5 min. The cDNA was analyzed immediately or stored at –20°C until use.

TaqMan® assay-based real-time PCR
Primers and probes for human {alpha}4, {alpha}5, {alpha}6, and 18S were acquired from PE Applied Biosystems (Assays-on-Demand). These predesigned and preoptimized TaqMan gene expression human sequence-based assays are provided in a 20x format and used according to the manufacturer’s instructions. The preformulated assay consists of two unlabeled PCR primers (900 nM each final concentration) and a dye-labeled TaqMan® minor groove binder probe (250 nM final concentration). Real-time TaqMan PCR systems for {alpha}4, {alpha}5, or {alpha}6 were multiplexed with 18S (internal standard). Each cDNA sample (1 µL) was analyzed. All assays were run in triplicates in a 96-well format plate. Real-time fluorescent detection of PCR products was peformed using an ABI 7500 (PE Applied Biosystems) using the following thermocycling conditions: 1 cycle of 50°C for 2 min and 95°C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were analyzed by the sequence detection systems (SDS) software (PE Applied Biosystems). Subsequent analysis was performed on the data output from the SDS software using Microsoft Excel. Relative RNA expression was determined using the formula Rel Exp = 2–({Delta}{Delta}Ct), where, for example, {Delta}{Delta}Ct = (Ct {alpha}5–CT 18S). The ratio for {alpha}5/18S in transmigrated neutrophil samples was then normalized to the ratio seen in nontransmigrated samples and expressed as the means ± SEM.

Flow cytometric analysis
The following antibodies to human antigens were used: CD49d ({alpha}4, PE-conjugated, BD Biosciences PharMingen); CD49e ({alpha}5, PE-conjugated, BD Biosciences PharMingen); CD49f ({alpha}6, PE-conjugated, BD Biosciences PharMingen); CD11b (Mac-1, PE-conjugated, BD Biosciences PharMingen); av (BD Transduction Laboratories, Franklin Lakes, NJ, USA); Alexa Fluor® 546 (goat antimouse IgG, Molecular Probes, Eugene, OR, USA). PE-conjugated, mouse IgG1 was purchased from BD Biosciences PharMingen.

For flow cytometric analysis, neutrophils (1x106 in a volume of 100 µl PBS) were stained in one step with one of the above fluorochrome-labeled antibodies (1 µg). After incubation with the indicated antibodies for 15 min on ice, the cells were washed three times with cold PBS and finally resuspended in 400 µl PBS with 1% paraformaldehyde. Data were collected with a FACScan and analyzed with CellQuest software (BD Biosciences PharMingen).

Cell motility calculations
Cells were determined adherent to specific peptide-containing hydrogels using a static adhesion assay described previously [6 , 14 , 19 ]. Neutrophils, naïve or transmigrated, stimulated with 10–7 M fMLF, were seeded on hydrogels containing various adhesive peptide sequences. Neutrophils were allowed to settle on the hydrogel and were videotaped for 15 min while viewed under an inverted microscope. Each videotape was analyzed, and frames were digitized at one frame every 30 s using Image Pro Plus Version 5.1 (Media Cybernetics, Inc., Silver Springs, MD, USA). Neutrophils were selected randomly in various viewing fields (n=4 fields per sample, approximately three cells per field). The analyzed frame dimensions were calibrated using a micrometer. Using the Image Pro Plus Track Object image analysis option with semiautomated tracking, we tracked the centroid of each cell over the 15-min time period. Cell centroid x, y coordinates, displacement from origin (DO), total accumulated distance (DTOT), and average speed of movement (VTOT), were calculated. DO was used to determine motility. Cells with DO less than 10 µm, the approximate diameter of one cell, were considered immobile. Cells with DO greater than 10 µm were considered mobile. VTOT was calculated as DTOT/15 min. The trajectory figures presented are representative of typical cell trajectories under specific cell treatment and substrate conditions.

Statistical analysis
Data were expressed as mean ± SE for n > 30. At least six individuals were used. Statistical significance was verified using one-way ANOVA calculations followed by Bonferroni post-test. Pair-wise differences were tested using Student’s t-test. Statistical significance is indicated by # or *, P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Our previous studies demonstrated that hydrogels containing 5.2 µmol/ml RGDS or 4.0 µmol/ml P2 peptides provided optimal neutrophil adhesion after 10–7 M fMLF stimulation [6 ]. In addition, gels containing combined 2.6 µmol/ml RGDS and 2.0 µmol/ml P2 provided ligand density for optimal adhesion. In this study, we used these concentrations to investigate the effect of the adhesive peptides on motility.

RGD alone does not support neutrophil motility
To examine whether adhesion to RGDS facilitated motility, stimulated neutrophils on hydrogels containing 2.6 or 5.2 µmol/ml RGDS were tracked over a 15-min period after contact. Although naive neutrophils (i.e., isolated blood neutrophils without contact with EC) were adherent and able to undergo shape change on RGD, they did not translocate over the surface of the hydrogel. Neutrophils were immobile when seeded on hydrogels containing 2.6 µmol/ml (data not shown) or 5.2 µmol/ml RGDS (Fig. 1 ). This failure of locomotion on RGD-containing hydrogels was also observed with neutrophils that had been exposed for 1 h to monolayers of activated EC. Neither the neutrophils collected from the lower compartment of the chambers (i.e., the transmigrated cells) nor those removed from the apical surface (i.e., the nontransmigrated cells) were migratory (Fig. 1A) . Motility in this instance was determined by displacement (DO) of the neutrophil from the point of initial contact. Neutrophils with DO < 10 µm over 15 min were considered immobile (Fig. 2A ). These observations were consistent among neutrophil samples and treatments, and <10% of all neutrophils on RGDS were mobile (Fig. 2B) . The consistency of these data indicates that the RGD peptide alone was not sufficient to support chemokinesis of naïve neutrophils or those after contact with activated EC.


Figure 1
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  		Figure 1. Neutrophil chemokinesis on hydrogels containing RGD, P2, or RGDS and P2 peptide. Neutrophils were collected 1 h after transmigration through monolayers of activated HUVEC (IL-1ß for 4 h prior to contact of neutrophils). The neutrophils were washed in balanced salt solution and then placed on hydrogels containing 5.2 µmol/ml RGDS (A), 4.0 µmol/ml P2 (B), or 2.6 µmol/ml RGDS + 2.0 µmol/ml P2 (C). Chemokinesis induced by addition of fMLF to the medium was tracked for 15 min.

 

Figure 2
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  		Figure 2. Analysis of neutrophil chemokinesis on hydrogels containing adhesive peptides. Naïve neutrophils (freshly isolated without contact with activated EC) and neutrophils following 1 h contact with IL-1ß-stimulated HUVEC monolayers were evaluated. (A) Mean distance from origin was used to determine neutrophil motility. Greater than 30 neutrophils—naïve, having contacted endothelium but not having undergone transmigration (EC contacted), or transmigrated cells—were allowed to interact with hydrogels containing RGD, P2, and combined RGD + P2. A threshold of 10 µm was set (the approximate diameter of the neutrophil) as a motility threshold. Cells traveling a distance less than one cell diameter were classified as immobile. Bars represent mean displacement from origin (DO) ± SEM. *, P < 0.05, when compared with their counterparts on P2 alone. #, P < 0.05, when compared with naïve cells on RGD + P2. (B) Mean percentage of motile neutrophils per substrate and treatment. Those with DO >10 µm were considered motile. Bars represent percent cells that were mobile ± SEM. *, P < 0.05 (C) Neutrophils—naïve, EC contacted, or transmigrated—were allowed to interact with hydrogels containing P2 and RGD + P2. Bars represent average velocity (VTOT) ± SEM. *, P < 0.05, when compared with their counterparts on P2 alone. #, P < 0.05, when compared to naïve cells on RGD + P2.

 
P2 alone supports Mac-1-dependent motility
We examined whether the P2 peptide, a Mac-1 ligand, was sufficient to support neutrophil motility. Roughly half of all neutrophils seeded on the hydrogels containing 4.0 µmol/ml P2, regardless of pretreatment, were able to move two-dimensionally (2D; Fig. 2 ). As seen in Figure 1B , the trajectories of cells on P2 were enhanced over those on the RGDS peptide, although the ligand densities were similar. No parameter of locomotion on P2-containing hydrogels was apparently influenced by contact with activated EC.

In combination, P2 and RGD support enhanced neutrophil motility
Hydrogels containing both peptides (2.6 µmol/ml RGDS and 2.0 µmol/ml P2) were used to evaluate the possible combined effects of {alpha}vß3, ß1, and Mac-1 ligation on chemokinesis. As seen in Figure 1C , neutrophil trajectories were enhanced on the hydrogel containing both peptides. Cell displacement (DO) was increased significantly on the combined peptides for transmigrated or naïve neutrophils (Fig. 2A) . A difference was also noted between the number of transmigrated and nontransmigrated cells that were motile. More transmigrated cells were motile (81.25%) versus the 61.91% and 58.82% of naïve and EC-contacted cells, respectively (Fig. 2B) . These results indicate that a combination of peptides leads to behavior in cells, which was enhanced after transmigration, a change that may have been a result of increases in integrin surface expression.

Average neutrophil speed was increased on hydrogels containing RGD and P2 peptides
The average speed of cell motility, VTOT, was calculated as a function of the total accumulated distance, DTOT, over the 15-min time period. Speeds increased significantly between naïve and transmigrated cells on P2, from 2.89 ± 1.63 to 4.31 ± 0.32 µm/min (Fig. 2C) . On the combined peptides, there was also a significant increase in the speed of transmigrated neutrophils (5.42±0.25 µm/min) over naïve or EC-contacted neutrophils (3.29±0.16 and 3.85±0.29, respectively; Fig. 2C ). Again, these results indicate that neutrophils exhibit enhanced functional behavior when presented with a combination of RGD and P2 simultaneously. This also suggests that transmigration enhances the motility of cells on combined peptide substrates. This change in activity may be explained by an increase in ß1 integrin expression as a result of transendothelial migration leading to increased interaction with ß1 ligands.

De novo synthesis of {alpha}5 and {alpha}6, ß1 integrin subunits after transmigration
The increase in percent motile cells after transmigration may be an indication that RGD-binding ß1 is expressed and activated increasingly after transendothelial migration. {alpha}-Subunits of ß1 integrins, {alpha}4, {alpha}5, and {alpha}6, synthesis and expression, were determined by relative RT-PCR and flow cytometry. RT-PCR results showed an increase in {alpha}5 and {alpha}6 mRNA, although flow cytometry did not yield results indicating that actual binding site numbers were increased significantly. This suggests that although de novo integrin synthesis may be occurring after 1 h transmigration, change in integrin surface expression is undetectable. {alpha}4 mRNA levels remained relatively unchanged, and no population percentage increased in protein-expressing cells or in binding site number detectable via flow cytometry. Expression of CD11b was used as a positive control for protein expression, showing a significant threefold increase in binding site number (Fig. 3A and 3B ). Results of {alpha}V flow cytometry were indeterminate, likely a result of the low numbers of available ligand-binding sites.


Figure 3
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  		Figure 3. Relative mRNA and protein expression in neutrophils following transendothelial migration. Neutrophils were added to stimulated (IL-1) HUVEC grown in transwell filters. Transmigrated and nontransmigrated neutrophils were collected after 1 h. (A) ß1 integrin subunits, {alpha}4, {alpha}5, and {alpha}6, levels in transmigrated samples, were normalized to levels seen in nontransmigrated samples. Results are the mean ± SEM of four experiments. *, P < 0.05, when compared with {alpha}4 fold increase. (B) Flow cytometry indicates changes in protein expression through mean fluorescence intensity shift after transmigration. The filled peaks represent IgG controls, and thin, solid lines represent naïve neutrophil samples incubated with anti-{alpha}4, -{alpha}5, -{alpha}6, or -{alpha}M mAb. The bold, gray lines represent transmigrated neutrophil samples incubated with the same mAb.

 
Mac-1 antibody prohibits naive neutrophil motility on hydrogels containing RGDS and P2 peptides
Blocking antibodies for specific integrins were used to assess integrin dependency of locomotion. Naive neutrophils were stimulated with fMLF and preincubated with blocking antibodies against integrin subunits, {alpha}vß3, {alpha}M, {alpha}4, {alpha}5, and {alpha}6. Anti-E-selectin was used as negative control. Figure 4F shows that anti-{alpha}M had a profound, inhibitory effect on cell chemokinesis compared with control (Fig. 4A) . Antibodies against, {alpha}4, {alpha}5, {alpha}6, and {alpha}vß3 did not produce the dramatic reduction in chemokinesis seen with anti-{alpha}M, known to block Mac-1 function (Fig. 4B 4C 4D 4E) . Figure 5A shows that stimulated, naïve neutrophils incubated with anti-{alpha}M exhibited significantly reduced values of DO < 10 µm. This confirms trajectory images (Fig. 4F) , showing that Mac-1 inhibition of naïve neutrophils inhibits 2D motility completely on the RGDS and P2 combined substrate. In addition, there was a significant reduction in the naïve neutrophil DO when the neutrophils were inhibited with {alpha}4 and {alpha}6 antibodies, indicating that these ß1 integrins participate in motility on combined RGDS and P2. Inhibiting the Mac-1 integrin of naïve neutrophils blocked chemokinesis completely, and inhibiting the {alpha}4 and {alpha}6 subunits reduced the average rate of locomotion below that of naïve cells on combined peptides (Fig. 5B) .


Figure 4
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  		Figure 4. Antibody influence on naïve neutrophil chemokinesis on hydrogels containing combined RGD and P2. Naïve neutrophils were fMLF-stimulated and incubated with antibodies against (A) E-selectin (CL3), (B) {alpha}4 (anti-CD49d), (C) {alpha}5 (anti-CD49e), (D) {alpha}6 (anti-CD49F), (E) {alpha}vß3 (LM609), and (F) {alpha}mß2 (R15.7). Neutrophils were tracked over 15 min, and average trajectories are shown here.

 

Figure 5
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  		Figure 5. Antibody influence on naïve neutrophil displacement and velocity. fMLF-stimulated, naïve neutrophils were collected and incubated with antibodies against {alpha}4, {alpha}5, {alpha}6, {alpha}Vß3, and {alpha}Mß2. Blocking antibody for E-selectin was used as a negative control. Neutrophils were seeded on hydrogels containing RGD, P2, or combined RGD + P2. Underlines indicate that cells were seeded on combined RGD + P2. (A) Neutrophils were tracked over 15 min. Bars represent average displacement from origin (DO) ± SEM. Neutrophils with DO < 10 µm were considered immobile. (B) Average velocity (VTOT) was calculated for naïve neutrophils, which were considered mobile. Bars represent average VTOT ± SEM. *, P < 0.05, when compared with naïve control on RGD + P2.

 
{alpha}vß3 and {alpha}5 antibodies inhibit transmigrated neutrophil motility on hydrogels containing RGDS and P2 peptides
Transmigrated neutrophils were stimulated and preincubated with anti-integrin antibodies in the same manner as were naïve neutrophils. Trajectory images suggested that antibodies against {alpha}4, {alpha}5, {alpha}vß3 and Mac-1 reduced neutrophil motility (Fig. 6B 6C 6E and 6F ). Transmigrated neutrophils—fMLF-stimulated and preincubated with anti-{alpha}vß3 and -{alpha}5 antibodies—had significantly reduced DO (Fig. 7A ), confirming trajectory images. Inhibiting the {alpha}4 and {alpha}M integrin subunits had less effect on cell motility than {alpha}vß3 and {alpha}5, although their inhibition still resulted in significant DO reduction when compared with motility of uninhibited neutrophils on the RGDS and P2 combined substrate (Fig. 7A) .


Figure 6
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  		Figure 6. Antibody influence on transmigrated neutrophil chemokinesis on hydrogels containing combined RGD and P2. Transmigrated neutrophils were fMLF-stimulated and incubated with antibodies against (A) E-selectin (CL3), (B) {alpha}4 (anti-CD49d), (C) {alpha}5 (anti-CD49e), (D) {alpha}6 (anti-CD49f), (E) {alpha}vß3 (LM609), and (F) {alpha}mß2 (R15.7). Neutrophils were tracked over 15 min, and average trajectories are shown here.

 

Figure 7
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  		Figure 7. Antibody influence on naïve neutrophil displacement and velocity. fMLF-stimulated, transmigrated neutrophils were collected and incubated with antibodies against {alpha}4, {alpha}5, {alpha}6, {alpha}Vß3, and {alpha}Mß2. Blocking antibody for E-selectin was used as a negative control. Neutrophils were seeded on hydrogels containing RGD, P2, or combined RGD + P2. Underlines indicate that cells were seeded on combined RGD + P2. (A) Neutrophils were tracked over 15 min. Bars represent average displacement from origin (DO) ± SEM. Neutrophils with DO < 10 µm were considered immobile. (B) Average velocity (VTOT) was calculated for transmigrated neutrophils with displacement distances >10 µm. Bars represent average VTOT ± SEM. *, P < 0.05, when compared with transmigrated control on RGD + P2.

 
Inhibiting {alpha}M, {alpha}4, and {alpha}6 integrin subunits reduces the speed of transmigrated neutrophil motility on hydrogels containing RGDS and P2 peptides
Transmigrated neutrophils exhibited characteristics not seen in naïve neutrophils under the same conditions. Transmigrated neutrophils seeded on combined RGD and P2 had average speeds, which were significantly greater than those on P2 alone (Fig. 2C) . When transmigrated neutrophils were stimulated and preincubated with antibodies against {alpha}4 and {alpha}6, the result was a significant decrease in average rate of movement (Fig. 7B) . Blocking Mac-1 reduced cell motility significantly (Fig. 7B) , although this did not inhibit movement completely, as seen with naïve neutrophils under comparable conditions.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Integrins play a critical role in the inflammatory response of neutrophils. Integrins, including {alpha}vß3, {alpha}4ß1, {alpha}5ß1, {alpha}6ß1, and {alpha}Mß2, are responsible for binding ECM proteins and forming the adhesive traction forces necessary for migration. These molecules become activated and able to bind their ligands in response to conformational changes mediated through chemokines and chemotactic factors. fMLF, for example, stimulates the neutrophil through p38 MAPK to elicit Mac-1 activation and chemotaxis and {alpha}4ß1-dependent polarization [20 ]. Using fMLF as a stimulant, we were able to evaluate adhesive mechanisms that support chemokinesis by presenting specific integrin ligands. In addition, we were able to determine functional changes in neutrophil integrins following transmigration. The data presented support the hypothesis that neutrophil chemokinesis is mediated by specific interactions between neutrophil integrins and bioactive domains of ECM proteins. This motility is enhanced by neutrophil transendothelial migration. Specifically, we show that neutrophil 2D chemokinesis is supported by the Mac-1 ligand, P2, and is enhanced through multi-integrin interactions with RGD and P2 (Figs. 1 and 2) . The data also demonstrate that phenotypic changes as a result of transendothelial migration enhance chemokinesis on a multipeptide-presenting substrate (Fig. 2A 2B 2C) .

In this study we use PEG hydrogel as an inert substrate to eliminate the issues of multiple ligand-binding, nonspecific binding, and nonspecific activation. We previously published that neutrophil adhesion to PEG was induced through RGDS, supporting adhesion and spreading through {alpha}vß3 and ß1 integrins [6 ]. In the present study, we find that these firmly adherent and widely spread neutrophils are immobile. In this case, {alpha}vß3-mediated adhesion and ß1-mediated spreading on RGD are not sufficient to promote neutrophil motility (Figs. 1A and 2A and 2B) . Our observations coincide with others who observed that the mobility of fMLF-stimulated neutrophils was inversely related to adhesion and increased spreading on glass, polyurethane, and octadecyltrichlorosilane [21 ]. Additional studies showed that firm adhesions via {alpha}vß3 clusters in the rear of the cell rendered the neutrophil incapable of migration on vitronectin [22 ]. Clearly, RGD-mediated firm adhesion, whether {alpha}Vß3- or ß1-dependent, is not sufficient to support motility.

Unlike RGD, ligation of Mac-1 via P2 is sufficient for 2D motility (Figs. 1B and 2A 2B 2C) . Others similarly report that Mac-1-dependent motility is possible in the absence of secondary integrin involvement [2 , 12 , 23 , 24 ]. Such Mac-1-dependent migration has been seen in the lung [2 ], through intestinal epithelium [23 ] and through matrices of fibrinogen and collagen [12 , 24 ]. To the best of our knowledge, this is the first instance in which Mac-1-dependent motility has been shown using a single peptide under isolated conditions, as opposed to whole protein or antibody inhibition of secondary integrin ligation. Although Mac-1 integrin-dependent motility is possible, our results confirm that the addition of RGD to hydrogels containing P2 promotes the involvement of the {alpha}vß3 and ß1 integrins, prompting neutrophils to move at increased rates (Figs. 1C and 2A 2B 2C) . Integrin-dependent motility is a result of a balance between cyclical binding and release. This balance allows the cell to form adhesive traction forces to pull it, while releasing from the substrate to allow forward advancement. As we see, on combined RGD and P2 hydrogels, this balance is preserved, and on RGD alone, it is not, making motility impossible. Our results also indicate that engagement of the ß1 integrins is not inhibitory, demonstrated by increases in displacement from the origin on surfaces with RGD and P2 together (Figs. 2A and 5A) . Again, this suggests that ß1 and Mac-1 crosstalk provides optimum signaling for efficient motility. Ugarova and co-workers [25 ] support the idea of integrin crosstalk through observations of functional interplay between {alpha}5ß1 and Mac-1, in which {alpha}5ß1 was predominantly responsible for motility of transfected, nonmyeloid cells on fibronectin. Additional support for crosstalk between integrins regulating migration was shown through fMLF and LTB4-stimulated neutrophil regulation of {alpha}5ß1, which subsequently regulated Mac-1 [26 ]. Efficient neutrophil motility is highly dependent on synergistic signaling between RGD-binding integrins {alpha}vß3 and {alpha}5 [27 28 29 ], as blocking their function leads to a substantial change in the behavior of the cells (Fig. 7A) . A relationship between {alpha}5ß1 expression and {alpha}vß3-mediated adhesion and motility on fibrinogen has been confirmed [30 ]. Together, these results indicate that multi-integrin involvement is necessary for effective and efficient neutrophil motility.

Freshly isolated neutrophils have been used traditionally for experimentation. However, except for rare circumstances, neutrophils in vivo must transmigrate across the endothelium before they interact with the ECM. Multiple changes in integrin expression as an effect of transendothelial migration have been recognized [10 , 11 , 31 ]. Although these reports show phenotypic changes following transendothelial migration, there are few studies evaluating cellular functionality. Here, we show that after transmigration, neutrophils are able to move at faster rates on hydrogels with combined RGD and P2 than are naïve cells (Fig. 2A 2B 2C) . Our results suggest that increased ß1 integrin expression as a result of transendothelial migration provides the neutrophil with a means to bind the RGDS peptide, promoting the signals necessary for cyclical binding and release mechanisms of cell motility [32 ]. This is supported by data showing a significant decrease in average speeds after inhibition of {alpha}4 and {alpha}6 (Fig. 7B) —{alpha}4 possibly being an RGD-binding integrin [33 , 34 ] and {alpha}6 being implicated as an integrin that becomes highly activated and up-regulated following transmigration [31 ]. Although debate remains about {alpha}4 binding to RGD [35 , 36 ], we show here that {alpha}4 does play a role in motility as a signaling or binding molecule, as blocking reduced the rate of movement effectively.

The expression of ß1 integrins has been shown to be up-regulated on the neutrophil surface as a result of transmigration across the endothelial layer in the pleural cavity [10 ]. Reinhardt et al. [11 ] reported an increase in expression of {alpha}4ß1 on the rat neutrophil following transmigration and an additional fivefold increase following stimulation. However, our data do not reveal increases in {alpha}4 surface expression nor increased signal for synthesis as a result of transmigration. In this report, we demonstrate increases in {alpha}5ß1 and {alpha}6ß1 synthesis in neutrophils after transendothelial migration using relative RT-PCR (Fig. 3A) . Flow cytometry did not confirm an increase in surface expression of stimulated neutrophils after transmigration (Fig. 3B) . These results are surprising, as Dangerfield et al. [31 ] noted an increase in {alpha}6ß1 on transmigrated neutrophils in mice. Our inability to confirm {alpha}4, {alpha}5, {alpha}6, and {alpha}v increases in expression using flow cytometry may be a result of the extremely low numbers of binding sites available on the cell surface or the inability of the assay to detect small changes in receptor number. It is also likely that there are low levels of protein up-regulation within the 1-h time period of our observations. Although mRNA is increased within the time period we are observing, protein expression is not evident within this time. Temporal restrictions seem a plausible explanation when we consider reports that neutrophil integrins become endocytosed, shed, or recycled under stimulated conditions. In particular, Pierini et al. [37 ] found that {alpha}5ß1 integrins can be internalized and later recycled to the cell surface for use in adhesion and motility. It is also possible that an increase in integrin function is a result, not only solely of up-regulation but also of integrin redistribution and increased activation following transmigration. Sorokin and co-workers [35 ] detected the presence of ß3 integrin on the neutrophil surface, and although there was not a substantial up-regulation in protein, they were able to detect an increase in {alpha}vß3-dependent adhesion following neutrophil stimulation. Redistribution of LFA-1, a neutrophil-presenting ß2 integrin, was noted during and following transmigration, altering the functional ability of the neutrophil to migrate [38 ]. Studies also suggest that {alpha}6ß1 up-regulation, along with an altered activation state, functionally alters the ability of the neutrophil to move through a perivascular basement membrane following transmigration [31 ].

Our studies closely simulate the results of others who have observed neutrophils on a HUVEC monolayer moving at rates of ~6 µm/min, although their observation of transmigrated cells below the HUVEC monolayer was at a highly increased rate of 14 µm/min [39 ]. The diminished velocities observed in our experiments with transmigrated cells could be a result of the texture of the hydrogel surface. The choice of PEG as the presenting substrate was based on its inert characteristic, resistance to protein adsorption, and relatively easy chemical manipulation. As suggested by Tan et al. [40 41 42 ], however, the topographical geometry and chemistry of a substrate can have a dramatic effect on the rate of movement of neutrophils. As our polymer did not vary from study to study, this effect does not hinder our ability to observe the differential effects of motility as a result of specific integrin and combined integrin interactions.

In summary, the results of this study demonstrate that RGD, as a single substrate for adhesion, is insufficient to support leukocyte chemokinesis, but the peptide P2 as a single substrate for adhesion is sufficient to support Mac-1-dependent chemokinesis. When both peptides are present, neutrophil locomotion is enhanced significantly and dependent on a combination of leukocyte integrins. In addition, the results demonstrate that neutrophil chemokinetic motility is increased significantly within 1 h of transendothelial migration, and on substrates containing RGD and P2 peptides, the increased locomotion is dependent on a combination of leukocyte integrins, predominately {alpha}vß3 and {alpha}5ß1.


   ACKNOWLEDGEMENTS
 
This work was supported by funding from the National Institutes of Health (HL070537, HL007939, HL18672, USDA 6250-51000-046). The writers acknowledge the nursing staff at the Labor and Delivery Units of The Woman’s Hospital of Texas and the Ben Taub General Hospital for aid in acquiring umbilical cords.

Received September 6, 2006; revised October 6, 2006; accepted November 8, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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