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(Journal of Leukocyte Biology. 2000;68:47-57.)
© 2000 by Society for Leukocyte Biology

Preferential sites for stationary adhesion of neutrophils to cytokine-stimulated HUVEC under flow conditions

Priya K. Gopalan*,{dagger}, Alan R. Burns*,{ddagger}, Scott I. Simon*, Scott Sparks*, Larry V. McIntire{dagger} and C. Wayne Smith*

* Speros P. Martel Section of Leukocyte Biology, and
{ddagger} Section of Cardiovascular Sciences, Baylor College of Medicine, Houston, Texas; and
{dagger} Cox Laboratory for Biomedical Engineering, Rice University, Houston, Texas

Correspondence: C. Wayne Smith, M.D., Section of Leukocyte Biology, Baylor College of Medicine, CNRC, Room 6014, 1100 Bates, Houston, TX 77030-2399. E-mail: cwsmith{at}bcm.tmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils form CD18-dependent adhesions to endothelial cells at sites of inflammation. This phenomenon was investigated under conditions of flow in vitro using isolated human neutrophils and monolayers of HUVEC. The efficiency of conversion of neutrophil rolling to stable adhesion in this model was >95%. Neither anti-CD11a nor anti-CD11b antibodies significantly altered the extent of this conversion, but a combination of both antibodies inhibited the arrest of rolling neutrophils by >95%. The efficiency of transendothelial migration of arrested neutrophils was >90%, and the site of transmigration was typically <6 µm from the site of stationary adhesion. Approximately 70% of transmigrating neutrophils migrated at tricellular corners between three adjacent endothelial cells. A model of neutrophils randomly distributed on endothelium predicted a significantly greater migration distance to these preferred sites of transmigration, but a model of neutrophils adhering to endothelial borders is consistent with observed distances. It appears that stable adhesions form very near tricellular corners.

Key Words: inflammation • adhesion molecules • transmigration


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocyte rolling on the endothelium of venules is a well-known phenomenon that increases markedly at sites of inflammation [1 ]. To emigrate into tissue, rolling leukocytes must arrest on the apical surface of the endothelium. The factors that induce leukocytes to stop rolling are poorly understood. There is evidence that surface-bound chemotactic factors such as interleukin-8 (IL-8) [2 ] stimulate CD18-dependent adhesion, and that intercellular adhesion molecule-1 (ICAM-1) (CD54) expressed on the endothelium can serve as a ligand for such adhesion [3 ]. Two members of the CD18 (ß2 integrin) family, Mac-1 (CD11b/CD18) and lymphocyte function-associated antigen-1 (LFA-1) (CD11a/CD18), apparently play important roles in the mobility of neutrophils. Although these integrins share the ability for adhesion to ICAM-1 [4 , 5 ], there are clear distinctions that are functionally significant. The ligands recognized by LFA-1 seem to be limited to members of the ICAM subfamily of the immunoglobulin (Ig) superfamily, which now includes several members [6 7 8 9 10 ]. Although Mac-1 binds to ICAM-1 [5 ], it recognizes an array of apparently unrelated molecules, including fibrinogen [11 ], denatured albumin [12 ], and keyhole limpet hemocyanin [13 ]. The adhesion of neutrophils to endothelial cells may involve LFA-1 and Mac-1 [5 ]. LFA-1 binds to ICAM-1 [6 ], and Mac-1 binds to ICAM-1 and at least one other undefined endothelial ligand [14 ]. The individual contributions of LFA-1 and Mac-1 to the arrest of rolling neutrophils have not been defined.

An in vitro model of neutrophil rolling using a parallel plate flow chamber has revealed that neutrophils interacting with IL-1-stimulated endothelium at venular shear rates roll for varying distances before arresting and transmigrating [3 ]. This rolling is selectin-dependent [15 , 16 ], and chemokines present at the endothelial surface (e.g., IL-8) appear to stimulate CD18 integrin adhesion [17 ]. Transendothelial migration of neutrophils may occur rapidly after the leukocyte stops rolling, and this process is almost completely inhibited in the presence of antibodies that block adhesive functions of LFA-1 and Mac-1. However, LFA-1 appears to be distinctly dominant, because antibodies to Mac-1 alone are marginally effective under static conditions in vitro [18 ] and in vivo [19 , 20 ]. The dominance of LFA-1 is most clearly demonstrated in mice with a targeted deletion of CD11b [21 ], where the rate of neutrophil influx into the peritoneal cavity in response to thioglycollate is not significantly reduced, and emigration is markedly inhibited by anti-CD11a antibodies. The factors that determine the site of transmigration are also poorly defined, and our recent studies [22 , 23 ] raise some potentially important questions. In experiments performed under static conditions in vitro, we found that ~75% of the transmigrating neutrophils moved through corners where three endothelial cells meet. We found these to be sites of discontinuity in some aspects of the junctional complexes between endothelial cells. Whether the mechanisms that focus neutrophils at tricellular corners function when neutrophil adhesion occurs under conditions of flow is unknown.

Here we investigate neutrophil arrest and transendothelial migration under conditions of flow. We examine the relative contributions of Mac-1, LFA-1, and ICAM-1 to these events, and we investigate the site of transendothelial migration after neutrophils arrest on activated endothelial monolayers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of neutrophils
Neutrophils were obtained from healthy adult donors from heparin-anticoagulated (Elkins-Sinn, Cherry Hill, NJ; 10 U/ml) venous blood samples. Neutrophils were purified by centrifugation through a one-step Ficoll-Hypaque gradient (Mono-Poly Resolving Medium, ICN Biomedicals, Aurora, OH) [24 ] and were then washed and resuspended in Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4) (GIBCO BRL, Grand Island, NY) at 4°C, containing 0.1% human serum albumin (Alpha Therapeutic Corp., Los Angeles, CA) without Ca** or Mg**. Isolated neutrophils were maintained at 4°C for up to 4 h at a concentration of 107/ml.

Monoclonal antibodies
The blocking antibodies R3.1, R15.7, and R6.5 were provided by Dr. Robert Rothlein (Boehringer-Ingelheim Pharmaceuticals, Ridgefield, CT). R3.1 blocks CD11a [5 ], R15.7 blocks CD18 [25 ], and R6.5 maps to domain 2 of ICAM-1 and blocks the binding of LFA-1 and Mac-1 [26 , 27 ]. F(ab')2 fragments of R6.5, prepared with an ImmunoPure F(ab')2 preparation kit (Pierce, Rockford, IL), were used in the adhesion assay. The anti-CD11b monoclonal antibody (mAb), 60.1 [28 ], was provided as a F(ab')2 fragment by Dr. James Ruschie (Repligen Corporation, Cambridge, MA). TS2/4, a nonblocking mAb that binds to CD11a, was provided by Dr. Tim Springer (Center for Blood Research, Harvard Medical School, Boston, MA). F(ab')2 fragments, prepared with an ImmunoPure F(ab')2 preparation kit (Pierce), were used in the adhesion assay. R15.7, 60.1 F(ab')2, and TS2/4 F(ab')2 were used at 20 µg/ml, R3.1 was used at 10 µg/ml, and R6.5 F(ab')2 was used at 100 µg/ml. Cells were preincubated with the antibodies for 10 min at 37°C, and all mAbs were present in the perfusion buffer during the entire experiment.

Preparation of endothelial cell monolayers
Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase digestion as previously described [15 ]. Cells from 3 to 10 umbilical cords were pooled and plated on flasks coated for at least 30 min with 0.2% gelatin (Difco Laboratories, Detroit, MI). Monolayers were cultured in M199 (GIBCO BRL), containing 10% fetal bovine serum and 10% bovine calf serum (Hyclone Laboratories, Logan, UT), 1% penicillin-streptomycin (GIBCO BRL), 1% fungizone (GIBCO BRL), 1% HEPES buffer (GIBCO BRL), 1 µg/ml heparin (Sigma Chemical Co., St. Louis, MO), and endothelial cell growth factor (prepared as per Maciag and Weinstein [29 ]). Cultures were grown in a 37°C humidified atmosphere with 5% CO2 and reached confluence after 4–6 days. HUVEC were then passaged onto 35 mm tissue culture dishes (Corning Glass Works, Corning, NY), which were coated with glutaraldehyde crosslinked gelatin, as described by Burns et al. [22 ]. Two to four days later, HUVEC were stimulated with 10 U/ml recombinant human IL-1ß (Genzyme Diagnostics, Cambridge, MA) at 37°C for 4 h prior to the adhesion assays.

In some adhesion experiments described below, canine jugular vein endothelial cell (CJVEC) monolayers were used. These cells were isolated as previously described [30 ] and grown as confluent monolayers on glutaraldhyde crosslinked gelatin.

Adhesion assay under flow conditions
Neutrophil adhesion under shear flow was performed as previously described [15 , 31 ]. Briefly, confluent IL-1-stimulated HUVEC monolayers were mounted in a parallel plate flow chamber, and DPBS was perfused over the monolayer for 2–3 min. The isolated neutrophils were then diluted to 106/ml in DPBS containing Ca++, Mg++, and 11 mM glucose (DPBS + Glu) and were perfused over the monolayer at a range of flow rates that resulted in shear stresses between 0.2 and 3.0 dynes/cm2. Temperature was maintained at 37°C. The interaction between the HUVEC and neutrophils from the time the neutrophils first passed over the monolayer was recorded on videotape for 11 min by phase-contrast video microscopy (Diaphot-TMD microscope, Nikon Inc., Garden City, NY; CCD Video Camera, Sony Corp., Park Ridge, NJ) and was quantified with Optimas image analysis software (BioScan, Edmonds, WA). For each experiment, a single field of view was recorded for the first 9.5 min, and five additional fields of view were taped during the last 1.5 min (i.e., 9.5–11 min after neutrophils first interacted with the monolayer).

ICAM-1 was blocked on some HUVEC monolayers by incubation with R6.5 Fab for 30 min in a 37°C, 5% CO2 environment. In some experiments at 2.0 dynes/cm2, adhesion molecules on neutrophils were blocked by incubation with R3.1, 60.1 F(ab')2, TS2/4, or R15.7 for 10 min at 37°C. Neutrophils were then diluted and perfused through the flow chamber. R15.7, R3.1, 60.1, R6.5, and TS2/4 were present in the neutrophil suspension during the entire experiment.

Adhesion assay under static conditions
For the static adhesion assays, confluent IL-1-stimulated HUVEC monolayers were mounted in a parallel plate flow chamber, and isolated neutrophils (106/ml in DPBS + Glu) were perfused over the monolayer at 2.0 dynes/cm2 for 2–3 min. The flow was stopped for 4 min and three fields of view and recorded under a 20x objective. The flow was resumed, and unbound cells were washed away at 2.0 dynes/cm2, and firmly adherent cells were determined in three fields were recorded under a 20x objective. Neutrophils were incubated with R3.1 or 60.1 (whole) for 20 min at room temperature in some of the static adhesion experiments. Neutrophils were then diluted and perfused through the flow chamber. R3.1 and 60.1 were present in the neutrophil suspension during the entire experiment.

Determination of the site of neutrophil adhesion to stimulated endothelial monolayers in vitro was determined under static conditions. HUVEC monolayers were stimulated for 4 h with IL-1, as described above, or CJVEC monolayers were stimulated with 30 ng/ml of endotoxin for 4 h. Isolated human or canine neutrophils were allowed to contact the appropriate species of monolayer for 4 min. The monolayers were then fixed and stained with silver nitrate, as previously described [22 ]. The position of attached neutrophils was then determined by light microscopy. Neutrophils overlying a stained border and attached neutrophils not overlying a border were counted.

Quantitation of adhesion
The accumulation of neutrophils on the monolayer was determined by counting the total number of rolling, arrested, and transmigrated neutrophils on one field of view at each minute of flow during the first 9 min. After the initial 9.5 min of interaction, the number of neutrophils interacting with the monolayer at 2.0 dynes/cm2 was determined by averaging the total number of rolling, arrested, and transmigrated neutrophils on the monolayer from five fields of view from the last 1.5 min of the experiment. For the experiments where shear stress was varied, the number of interacting neutrophils was calculated by averaging the number of neutrophils in three fields of view.

Arrested cells were defined as those that remained stationary during a 10-sec interval, and rolling cells were those that moved during the 10-sec interval. Transmigrating neutrophils included only those that had completely transmigrated at the beginning of the 10-sec interval. Rolling and arrested neutrophils appeared phase-bright and could be easily discerned from transmigrated cells, which appeared phase-dark [5 , 26 ]. Transmigrated cells were included in the adherent population, because they had arrested before they began to transmigrate.

For the static adhesion experiments, the average number of neutrophils remaining adherent (including transmigrated cells) in three fields of view after the flow was resumed was divided by the average number of neutrophils in three fields of view contacting the monolayer during the static interval.

The time and distance that individual neutrophils rolled from capture until arrest at 2.0 dynes/cm2 were measured during the first 9 min of the experiment (Fig. 1A ). The field of view was 500 µm long, and only those cells that initially interacted directly with the monolayer in the first 100 µm of the field of view were counted. The distance for cells that did not stop in the field of view was denoted as >400 µm. The rolling velocity of each neutrophil was calculated by dividing the distance by the time that the neutrophil rolled from its capture until it stopped or rolled off the field of view. The data from at least three experiments for each condition, representing at least 40 total neutrophils, were pooled for the distance and velocity histograms. The transmigration time—the interval from arrest until the neutrophil had completely transmigrated—was also observed in a frame-by-frame analysis of the video tapes. Transmigration was judged to be complete when the phase-bright uropod became phase dark. The distance that a neutrophil moved from arrest (i.e., after it was stationary for 10 sec) to where it transmigrated was measured (Fig. 1B) . The location of transmigration was determined, as previously described [22 ], to be the position of its uropod above the endothelial monolayer as it transmigrated. The resolution of movement possible using a 20x objective with a numerical aperture of 0.4 was ~0.8 µm. The data from at least three experiments of at least 30 neutrophils were pooled for the transmigration time and distance.



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  		Figure 1. Measurement of neutrophil rolling (distance and velocity) and migration after stationary adhesion. (A) The distance that individual neutrophils rolled until they arrested or rolled out of the field of view. The flow adhesion assays were performed on HUVEC monolayers as described in Materials and Methods. Neutrophils captured within the first 100 µm of the field of view were observed until they stopped rolling. Rolling velocity was determined for these individual neutrophils. Neutrophils that immediately stopped without rolling (*) were denoted as having zero rolling distance and velocity, and those that rolled off the field of view had a rolling distance >400 µm. (B) The distance that individual neutrophils migrated from the point of arrest to the point of transendothelial migration was measured from the center of the spherical arrested cell to the tip of the phase-bright uropod just prior to the cell moving beneath the monolayer. The elapsed time for this migration was also recorded.

 
Determination of the site of neutrophil transmigration
HUVEC monolayers were stained with silver nitrate as described by Burns et al. [22 ]. Briefly, immediately after a flow experiment, the dish with the monolayer was removed from the flow chamber and fixed with 1 ml of 0.05% glutaraldehyde for 10 min at room temperature. The monolayer was then washed three times with DPBS and left in 2 ml DPBS until the completion of the entire set of experiments. The DPBS was then aspirated, and 2 ml of 0.25% silver nitrate (in distilled water) was added to each dish for 30–40 sec with swirling. They were then washed three times with 2 ml DPBS, left under ultraviolet light for 5 min, and then coverslipped.

Neutrophil transmigration between two adjacent endothelial cells or between three endothelial cells at tricellular corners (the site where the margins of three endothelial cells meet) was quantitated from the silver-stained HUVEC monolayers under the 20x objective of the Nikon Diaphot phase contrast microscope. One hundred cells that were in the process of transmigrating (i.e., a phase-bright and a phase-dark portion could be observed) were counted, and the number that transmigrated between two cells or through tricellular corners was noted. The data are reported as the average of three separate experiments.

Statistical analysis
Results are presented as mean ± SEM. For the analysis of adhesion under different shear stresses, the effects of different interventions on the percent of abruptly arresting neutrophils and the comparison of the actual and predicted transmigration distances, one-way analyses of variance (ANOVA), were performed using InStat (GraphPad Software, San Diego, CA). The probability of statistical significance between two interventions was determined by the Tukey test. For the velocity and distance analyses, Kruskal-Wallis tests were performed, and Dunn’s tests determined significance between two interventions. P values < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Neutrophil accumulation on HUVEC monolayers at 2.0 dynes/cm2.
Several recent studies demonstrated that neutrophils adherent to a planar surface were able to capture neutrophils from the free stream and thus amplify the number of cells recruited to the substrate [32 33 34 35 ]. The extent to which this phenomenon occurs when neutrophils are interacting with endothelial cells in the model used in our studies was uncertain and would potentially alter the interpretation of the data using this experimental model. In the present studies, total neutrophil recruitment (the sum of rolling, arrested, and transmigrated leukocytes) on IL-1ß-stimulated HUVEC was linear over the observation period (Fig. 2 ). This finding is inconsistent with a significant contribution of neutrophil homotypic interactions. Homotypic interactions have been shown previously to increase nonlinearly the number of cells recruited to purified substrates on artificial planar surfaces.



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  		Figure 2. Neutrophil accumulation on HUVEC monolayers stimulated for 4 h with IL-1ß under a shear stress of 2.0 dynes/cm2. The flow adhesion assays were performed on HUVEC monolayers as described in Materials and Methods. The number of neutrophils was counted at each minute during the first 9 min of flow in the same field of view. The total number of neutrophils includes all interacting neutrophils (i.e., rolling, arrested, and transmigrated cells). A linear regression was performed with R2 = 0.984.

 
Assessments of the contributions of LFA-1 and Mac-1 to stable adhesion and transmigration were performed at the end of a 9.5-min observation period. Baseline observations in the absence of blocking antibodies revealed a total of 775 ± 46 neutrophils/mm2 were interacting with the monolayer at this time point. Among these neutrophils, 31.0 ± 3.4% were stationary on the apical surface of the monolayer, 57.8 ± 3.9% had transmigrated, and 11.2 ± 3.9% were rolling. A nonblocking anti-CD11a mAb, TS2/4, had no effect on any parameter. Blocking LFA-1 or Mac-1 alone also did not significantly affect the number of interacting neutrophils or the percentage of these cells that had arrested or migrated. However, blocking LFA-1 and Mac-1 with a combination of anti-CD11a and CD11b or with anti-CD18 mAbs greatly reduced the total number of neutrophils arrested and transmigrated over the observation period (Fig. 3 ). A decrease in the number of adherent cells resulted in a marked rise in the number of rolling cells at all time points. Blocking CD18 and ICAM-1 in combination did not decrease adhesion beyond anti-CD18 alone.



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  		Figure 3. Neutrophil arrest at 2.0 dynes/cm2 on stimulated HUVEC monolayers. The flow adhesion assays were performed on HUVEC monolayers as described in Materials and Methods. Neutrophils were untreated or preincubated with 10 µg/ml of the anti-CD11a blocking mAb, R3.1; 20 µg/ml of the anti-CD11b mAb, 60.1 F(ab')2; 20 µg/ml of anti-CD11, a nonblocking mAb TS2/4 (@); or 20 µg/ml of the anti-CD18 mAb, R15.7. HUVEC monolayers were untreated or preincubated for 30 min at 37°C with 100 µg/ml of the anti-ICAM F(ab')2, R6.5. All mAbs were present in the perfusion buffer during the entire experiment. The percent of control adhesion refers to the total arrested and transmigrated neutrophils after 9.5 min of flow. Data are expressed as the mean ± SEM from at least three separate experiments. **P < 0.01 compared with untreated neutrophils on untreated HUVEC. *P < 0.05 compared with untreated neutrophils on untreated HUVEC.

 
Anti-LFA-1 mAbs alone inhibit neutrophil adhesion to IL-1ß-stimulated HUVEC under static conditions [5 ]. This was confirmed in the present studies (Fig. 4A ), because anti-LFA-1 mAb produced 47.0 ± 4.6% inhibition (n=3, P<0.01). Anti-Mac-1 mAb alone was without significant inhibitory effect (95.2 ± 7.4% remaining, n=3, ns) under static conditions. The only effect of anti-Mac-1 was seen in combination with anti-LFA-1, where adhesion under static conditions was reduced by 75.6 ± 4.8% (n=3, P<0.001 compared with untreated neutrophils; P<0.01 compared with neutrophils treated with anti-LFA-1 antibody R3.1 alone). Because anti-LFA-1 did not reduce the ability of neutrophils to stop rolling at a wall shear stress of 2.0 dynes/cm2 over IL-1-stimulated HUVEC, a range of shear stress was employed to determine if a contribution of LFA-1 could be revealed. However, no inhibitory effects of this mAb alone were seen at wall shear stresses ranging from 0.2 to 3.0 dynes/cm2 (Fig. 4B) .



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  		Figure 4. Effects of anti-CD11a antibody on neutrophil adhesion to stimulated HUVEC. (A) Static adhesion assay was performed as described in Materials and Methods. Neutrophils were untreated or preincubated with 10 µg/ml of the anti-CD11a-blocking mAb, R3.1, or 20 µg/ml of the anti-CD11b mAb, 60.1. All mAbs were present with the cells during the entire experiment. The percent total interacting polymorphonuclear neutrophil (PMN) refers to the proportion of the cells that were firmly adherent contacting the monolayer. *P < 0.01 compared with control; **P < 0.01 compared with control or with the condition with added anti-CD11a antibody; n = 3, mean ± SEM. Both, a combination of anti-CD11a and anti-CD11b antibodies added. (B) The flow adhesion assays were performed on HUVEC monolayers as described in Materials and Methods. Neutrophils were untreated or preincubated with 10 µg/ml of the anti-CD11a-blocking mAb, R3.1; 20 µg/ml of the anti-CD11b mAb, 60.1; or a combination of these antibodies (Both). All mAbs were present with the cells during the entire experiment. The percent of control adhesion refers neutrophils that had arrested or transmigrated after 9.5 min of flow in the absence of mAb treatment. Data are expressed as the mean ± SEM from at least three separate experiments.

 
Neutrophil rolling behavior on HUVEC before arresting
To assess rolling behavior, the adhesive history of individual neutrophils was evaluated as shown in Figure 1 . Approximately 30% of untreated neutrophils arrested immediately without observable rolling (Table 1 ). Approximately 50% arrested after a rolling distance of 5 µm, and >98% of neutrophils arrested within the field of view. When neutrophils were treated with anti-LFA-1 or anti-Mac-1 mAb alone, 28% and 33% of neutrophils, respectively, arrested immediately, and the rolling distance was not significantly prolonged compared with conditions without blocking antibody (Table 1) . However, when antibodies to CD18, ICAM-1, or LFA-1 and Mac-1 were present, the ability of neutrophils to abruptly arrest was completely blocked. On monolayers pretreated with anti-ICAM-1 F(ab')2, neutrophils rolled somewhat farther before stopping, but 87% of the cells arrested within the field of view. In the presence of anti-CD18 mAb or anti-LFA-1 and Mac-1 mAbs, >90% neutrophils were unable to arrest within the field of view (distance>400 µm). For those neutrophils that could arrest in the presence of anti-CD11a and anti-CD11b mAbs, the median rolling distance before arrest was 101 µm, far longer than with CD18-dependent adhesion mechanisms intact.


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  		Table 1. Rolling Behavior of Isolated Neutrophils on IL-1-Stimulated HUVEC Monolayersa

 
The median rolling velocity of untreated neutrophils on IL-1ß-stimulated HUVEC at 2.0 dynes/cm2 was 2.3 µm/sec (Table 1) . The velocity did not increase significantly when only LFA-1, Mac-1, or ICAM-1 was blocked individually. However, a combination of anti-CD11a and CD11b, or anti-CD18 increased the median rolling velocity significantly. Although anti-ICAM-1 significantly increased the distance rolled, it did not increase the velocity of rolling. This ICAM-1 observation is consistent with studies in vivo [36 ].

Neutrophil migration after firm adhesion to HUVEC
As noted earlier, ~58% of neutrophils associated with the monolayer after 9.5 min of flowing a neutrophil suspension over the endothelial cells had transmigrated. This meant that ~450 neutrophils were beneath each mm2 of endothelial monolayer. The addition of blocking mAbs to only ICAM-1, LFA-1, or Mac-1 did not significantly reduce this number (Fig. 5 ). Blocking LFA-1 and Mac-1 significantly decreased the number of transmigrated neutrophils from 469 ± 29 neutrophils/mm2 in the absence of mAb treatment to 146 ± 31/mm2. Blocking CD18 or CD18 and ICAM-1 similarly decreased the number of transmigrated cells (Fig. 5) . This effect appeared to be a result of reductions in the number of arrested neutrophils, because transmigration appeared to proceed favorably for those neutrophils that were able to firmly adhere under control or antibody-blocking conditions. In each treatment condition, >40 individual neutrophils were followed as described in Figure 1 . Regardless of the blocking antibody combination used, >90% of arrested cells migrated beneath the endothelial monolayer during a 3-min observation time.



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  		Figure 5. Transmigrated neutrophils on 4 h IL-1b-stimulated HUVEC monolayers at 2.0 dynes/cm2. The flow adhesion assays were performed on HUVEC monolayers as described in Materials and Methods. Neutrophils were untreated or preincubated with 10 µg/ml of the anti-CD11a-blocking mAb, R3.1; 20 µg/ml of the anti-CD11b mAb, 60.1 F(ab')2; or 20 µg/ml of the anti-CD18 mAb, R15.7. HUVEC monolayers were untreated or pretreated with 100 µg/ml of the anti-ICAM F(ab')2, R6.5. All mAbs were present in the perfusion buffer during the entire experiment. The percent control migration refers to the number of transmigrated neutrophils at the end of 9.5 minutes of flowing neutrophils over the IL-1-stimulated monolayer. Data are expressed as the mean ± SEM from at least three separate experiments. **P < 0.01 compared with untreated, transmigrated neutrophils.

 
The median transmigration time of untreated neutrophils from arrest (i.e., after they were stationary for 10 sec) to complete migration was 83.8 sec. Blocking CD11a or CD11b alone did not change the transmigration time significantly (73.0 sec and 71.9 sec, respectively). Blocking CD18 integrins with anti-CD11a (R3.1) and anti-CD11b (60.1) in combination or with anti-CD18 (R15.7), or blocking ICAM-1 also did not affect the transmigration time significantly (73.3 sec, 69.5 sec, and 93.6 sec, respectively).

The distance that neutrophils moved from the site of arrest to the site of transmigration was also measured. In the absence of blocking mAbs, the mean (SEM) migration distance was calculated to be 5.5 ± 0.7 µm (Fig. 6 ). Regression analysis revealed that this migration distance was independent of the distance each neutrophil rolled before arresting. Although blocking ICAM-1 on the monolayer, blocking CD11a and CD11b on the neutrophil, or blocking CD18 and ICAM-1 in combination reduced the number of cells arresting, there was no effect on the distance that the subset of arrested neutrophils moved before transmigrating (Fig. 6) . In addition, the mean distance (6.5 µm) migrated by cells that abruptly stopped was not significantly different from the total population. These data suggest that flowing or rolling neutrophils arrested close to their site of transmigration, and those cells that arrested in the presence of antibody inhibition of CD18 and ICAM-1 still arrested close to the site of transmigration.



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  		Figure 6. Distance neutrophils moved from the site of arrest to the site of transmigration under flow conditions on 4 h IL-1b-stimulated HUVEC. The flow adhesion assays were performed on HUVEC monolayers as described in Materials and Methods. Neutrophils were untreated or preincubated with 20 µg/ml of the anti-CD18 mAb, R15.7, or with a combination of 10 µg/ml of the anti-CD11a-blocking mAb, R3.1, and 20 µg/ml of the anti-CD11b mAb, 60.1 F(ab')2. HUVEC monolayers were untreated or pretreated with 100 µg/ml of the anti-ICAM F(ab')2, R6.5. All mAbs were present in the perfusion buffer during the entire experiment. Data are expressed as the mean ± SEM from at least 30 neutrophils.

 
Preferential sites of transmigration on HUVEC
Previously, we reported that under static conditions, neutrophil transmigration through IL-1ß-stimulated HUVEC monolayers preferentially (~75%) occurs at tricellular corners, the site where the margins of three endothelial cells meet [22 ]. The remaining 25% transmigrate at the border between two adjacent endothelial cells. By silver staining the HUVEC monolayers after each flow experiment, we were able to determine the location of neutrophils in the process of transmigration at the time the monolayers were fixed. We now report that under flow conditions (2 dynes/cm2), 69.9 ± 1.7% of neutrophils transmigrated at tricellular corners, and the remaining neutrophils transmigrated at the junction of two adjacent endothelial cells.

We compared the observed behavior of arrested neutrophils with a stochastic model that assigned random sites of arrest on endothelial cells under flow. Previously, we determined that the average perimeter of an endothelial cell under the culture conditions used in these studies was 142 µm and that each endothelial cell formed an average of 5.3 tricellular corners with adjacent cells in the monolayer under the culture conditions used in the current studies [22 ]. For our model of predicted transmigration distance, we approximated each endothelial cell as a pentagon with a perimeter of 142 µm, with the center of a tricellular corner at each vertex (Fig. 7 ). Each pentagon was assigned a different shape to reflect the heterogeneity in the observed shapes of endothelial cells in a HUVEC monolayer. The center of each neutrophil was represented by a point. To determine the predicted average distance from the point of arrest to the site of transmigration, 150 points (neutrophils) were randomly generated within the pentagons (endothelial cells), and the distance from each point to the nearest vertex (i.e., tricellular corner) and the minimum distance to the nearest margin were determined. The assumption that neutrophils migrated in a straight line was the most conservative comparison with our empirical data. We calculated the average weighted distances to reflect the distribution of transmigration at tricellular corners and between two endothelial cells (i.e., 70% x distance to nearest tricellular corner + 30% x distance to nearest margin). The frequency histograms of the distances generated from the model suggested that randomly arrested neutrophils would migrate significantly farther than the actual transmigration distances observed in our experiments (Fig. 7B) . In most instances, the body of the neutrophil appeared to remain at the site of initial arrest, simply extending a pseudopod to the point of eventual transmigration. As noted above, >90% of the arrested cells transmigrated, and neutrophils were rarely seen migrating over the apical surface of the endothelial cells. The rare neutrophils that migrated across the apical surface of the endothelial monolayer often did not locate a site for transmigration during the observation period.



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  		Figure 7. Comparison of actual and predicted transmigration distances. (A) The distance that individual neutrophils moved from their site of arrest to their site of transmigration was predicted as described in Materials and Methods. Endothelial cells were approximated as pentagons of perimeter 142 µm, with each vertex representing the center of a tricellular corner. (a) Neutrophils were modeled as points randomly distributed in a region of three adjacent endothelial cells (pentagons). (b) (1) The distance to the nearest tricellular corner was the distance from each point to the nearest vertex. (2) The distance to the nearest cell margin was the minimum distance from each point to the margin between two adjacent endothelial cells (pentagons). (B) Transmigration distance is the distance moved from the site of arrest to the site of transmigration (see Fig. 1 ). The histograms are frequency distributions, expressed as percentage of the total number of neutrophils observed. For the actual migration distance, the flow adhesion assays were performed with neutrophils on IL-1-stimulated HUVEC monolayers in the absence of mAbs as described in Materials and Methods. Data are expressed as a frequency histogram of at least 40 neutrophils. The predicted transmigration distances were calculated as described in Results and represent data from 150 neutrophils randomly arrested on a region of three adjacent endothelial cells. The histogram shows the weighted distribution of transmigration distance to cell margins and tricellular corners. The median actual distance migrated was 5.5 µm, and the median predicted distance was 9.4 µm, P < 0.001. (C) The predicted transmigration distance was calculated by changing the model in only one aspect. The sites of random arrest were confined to endothelial borders of an assigned width of 2 µm. These new predicted distances are plotted along with the same actual values shown in (B) for comparison. This model appears to be consistent with the empirical data.

 
Based on these data, we next modified the stochastic model by assuming that stationary adhesion occurred on endothelial cell borders with an assigned width of 2 µm. Migration distances to borders and tricellular corners were determined and plotted in Figure 7C . In contrast to the model assuming random adhesion over the endothelial cell body, this model is consistent with the observed migration distances, and in experiments under static conditions, neutrophil adhesion to IL-1-stimulated endothelial monolayers was on the cell border >85% of the time (Fig. 8 ). We have previously shown that adhesion to borders occurs under flow conditions [37 ].



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  		Figure 8. Site of neutrophil adhesion on activated endothelial monolayers. HUVEC or CJVEC (DEC) monolayers were grown to confluence and then stimulated with IL-1 (10 U/ml) or endotoxin (10 ng/ml), respectively, for 3 h. Isolated human or canine neutrophils were allowed to adhere for 4 min, and then the monolayers were rinsed gently, fixed, and stained with silver nitrate to define the interendothelial junctions. Using light microscopy, the percent of neutrophil overlying borders or overlying the body of the endothelial cells (Body) was determined. The mean ± SEM for five separate experiments is plotted.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The experimental model used in this report revealed features of the interactions of neutrophils with endothelial cells under conditions of flow that provide potentially important insights. First, LFA-1 or Mac-1 may support the arrest of tethered neutrophils at venular shear rates, a result that would not have been predicted from earlier in vitro studies of adhesion under static conditions. Second, on IL-1-stimulated endothelial cells, the efficiency of conversion of rolling adhesion to firm adhesion can be very high for neutrophils. Third, the efficiency of transmigration is very high for arrested neutrophils and may not require CD18 integrins. Fourth, neutrophils arrest very close to the site on the endothelial monolayer where transendothelial migration will occur; and fifth, this transmigration occurs preferentially at tricellular corners in the endothelial monolayer.

The relative contributions of LFA-1 and Mac-1
Although the contribution of CD18 integrins to the arrest of rolling neutrophils has been established in many laboratories using cell culture models and intravital techniques, the relative contributions of LFA-1 and Mac-1 remain uncertain. The in vitro model used in the present studies reveals that either of these ß2 integrins can stop rolling neutrophils, a conclusion supported by the finding that neither anti-CD11a nor anti-CD11b alone affected any reduction in the number of arrested cells, whether in the percent of leukocytes that abruptly arrested, the rolling velocity, or the total that were firmly adherent at any of the observation times. The mAbs used in these studies have been shown to block the functions of LFA-1 and Mac-1 [5 ], and when used in combination in the current experiments, these mAbs were as inhibitory as anti-CD18.

As shown in Figure 4 , we found a significant distinction between adhesion under static conditions and under flow. Blocking LFA-1 was without effect on the arrest of neutrophils moving in response to fluid shear, but blocking LFA-1 produced almost 50% inhibition of adhesion to the HUVEC monolayers when neutrophils contacted the endothelial cells in the absence of shear. Thus, shear stress appears to increase the contribution of Mac-1 to neutrophil/endothelial cell adhesion. The limited involvement of Mac-1 in static adhesion to cytokine-stimulated endothelium has been previously reported [18 ], but the potentiating effect of shear is undefined.

One possible distinction between adhesion under static and flow conditions is that selectins may interact with the neutrophils differently in these two conditions. L-selectin has been shown to exhibit a shear threshold (i.e., an inability to function as an adhesion molecule until the wall shear stress applied to the neutrophil reaches a threshold of >0.5 dynes/cm2 [38 , 39 ]). Isolated E- and P-selectin also exhibit similar shear thresholds at site densities of 200/µm2 or less [39 ]. It appears that shear-induced torque is needed to maintain selectin-dependent adhesion (rolling) [39 ], because gravitational force is sufficient to detach cells from L-, E-, and P-selectin binding. The shear threshold of selectins is unexplained in molecular terms but functionally could mean different forms of selectin interaction under static and flow conditions. Of potential relevance to this discussion is the fact that L-selectin can function as a signaling molecule on neutrophils [40 , 41 ], triggering, among other things, an increase in the adhesive function of Mac-1 [42 ]. L-selectin ligation can also significantly potentiate the effect of chemotactic stimulation on Mac-1 function [24 ], and current evidence indicates that chemotactic stimulation [via IL-8 and platelet-activating factor (PAF)] occurs when neutrophils contact cytokine-stimulated endothelial cells under static or flow conditions [43 44 45 ]. In recent unpublished studies, we have shown that E-selectin binding to neutrophils under shear, but not static conditions, can also activate Mac-1 adhesion (Simon et al., unpublished results), and Evangelista et al. [46 ] have provided evidence that P-selectin interacting with its ligand PSGL-1 can trigger enhanced Mac-1 adhesion. The E- and P-selectin effects on Mac-1 adhesion were sensitive to tyrosine kinase inhibitors. Thus, the functional significance of selectin-dependent capture under flow may extend beyond simply the support of rolling adhesion to include potentiation of chemotactic activation of Mac-1 adhesion.

The use of anti-ICAM-1 antibody in these studies significantly diminished the ability of the neutrophils to stop rolling. However, the effect is minimal compared with that of anti-CD18 or combined anti-CD11a and -CD11b. Although ICAM-1 has been shown to be a ligand for LFA-1 and Mac-1 on neutrophils [27 ], the binding of Mac-1 to ICAM-1 is relatively weak [4 ], and Mac-1 apparently recognizes another uncharacterized ligand on endothelial cells [14 , 47 ]. Because our data indicate that Mac-1 or LFA-1 can support stationary adhesion of neutrophils under flow, it is likely that Mac-1 is primarily interacting with a ligand other than ICAM-1 under these flow conditions on stimulated HUVEC. This point is supported by the finding that combined use of anti-CD11b and anti-ICAM-1 antibodies resulted in reductions in firm adhesion that were as marked as combinations of anti-CD11b and anti-CD11a antibodies.

In contrast to the important role played by the CD18 integrins in arrest of rolling cells, the role for these integrins in transmigration was not distinguishable from their role in stable adhesion. When individual arrested neutrophils were continuously observed, >90% transmigrated regardless of antibody treatment. It appeared that if a neutrophil could arrest in the presence of anti-CD18 antibody, it could transmigrate. Our analysis raises the possibility that CD18 functions are limited to adhering the leukocyte to endothelial cells, and that the process of transendothelial migration involves other mechanisms. CD18-independent mechanisms of emigration have been demonstrated in vivo [48 ]. Jung et al. [49 ] found that transendothelial migration was highly efficient for arrested leukocytes in TNF{alpha}-stimulated cremasteric vessels in CD18-deficient mice. It appears that reducing adhesion by blocking or removing CD18 integrins indirectly reduces transmigration, and it may be that CD18 integrins are functional in bringing the neutrophil to the site of transmigration but not contributing to the interaction of neutrophils with the endothelial membranes in the interendothelial cleft. Anti-CD18 would thus reduce transmigration under flow (the present study) or under static conditions (numerous earlier studies [5 , 50 ]). The observation that anti-CD18 reduced the number of transmigrated neutrophils to <20% of the control level indicates the major importance of these integrins in promoting stationary adhesion under flow.

Efficiency of neutrophil arrest and transmigration
We found that under a shear stress of 2.0 dynes/cm2, neutrophils are able to interact with cytokine-stimulated endothelial cells at significant levels. The accumulation of neutrophils was linear over time (Fig. 2) . However, recent studies of neutrophil recruitment on purified E- and P-selectin monolayers have reported an exponential accumulation of neutrophils [33 , 34 ]. These and other studies have proposed that bound neutrophils are able to support the capture (tethering) of neutrophils from the free stream, and thus amplify neutrophil recruitment [33 34 35 , 51 ]. Our results suggest that the recruitment of neutrophils from the free stream by bound neutrophils did not significantly increase cell capture on cytokine-stimulated endothelial monolayers in our experimental model. The topography of the monolayer of endothelial cells may be such that bound neutrophils are not as accessible to those in the free stream as neutrophils bound to a monolayer of purified E- or P-selectin on a planar surface. Neutrophils are able to transmigrate under HUVEC monolayers within ~90 sec, a timeframe that may be too rapid to enable many neutrophils to be recruited before transmigration.

In experiments where the behavior of individual neutrophils was followed from the time of the initial observed contact, a remarkably high percentage of cells arrested and transmigrated. The observed efficiency was consistently >90% for both events, and the median rolling distance (5 µm) was less than the diameter of an individual neutrophil. If data collection were to be limited simply to categorizing the behavior of all cells in a field at a given time in the flow experiment, it would be impossible to realize this high efficiency. Such data collection has characterized many previous studies in vivo and in vitro [52 ], leaving the question of the efficiency with which rolling cells are captured by activated endothelium unresolved.

Rainger et al. [17 , 53 ] followed in vitro the behavior of individual neutrophils on endothelial monolayers that had been stimulated with hypoxia/reoxygenation or exposure to hydrogen peroxide. These stimuli provoke mobilization of P-selectin to the apical surface of endothelial cells from Weibel-Palade bodies [54 , 55 ]. Although many rolling neutrophils were observed, arrest was undetected. Stationary adhesion could be induced, however, by pretreating the stimulated endothelium with IL-8 or PAF and washing away the unbound chemotactic factor. A significant percentage ( 30%) of rolling cells then converted to stable adhesion under flow. The rate of this conversion was remarkably fast, consistently occurring within less than a second.

Given the current assumption that IL-1- or TNF-stimulated endothelial cells present IL-8 and PAF [44 , 56 ] as stimuli that activate CD18 integrin-dependent stable adhesion, and given the observations of Rainger et al.[53 ] that conversion occurred in a median rolling duration of 720 msec, it is possible that these chemotactic substances contribute to the high-efficiency transition of neutrophils from flowing or rolling to arrested cells. In our experiments, the median time for this transition was ~2 sec. Our current model differs from the experiments of Rainger et al. in that the primary selectins supporting rolling on cytokine-stimulated endothelial monolayers are L- and E-selectin [15 , 43 ], and the wall shear stress used was higher. Under similar conditions and site densities, neutrophils appear to roll more slowly on E-selectin than P-selectin [57 ]. Moreover, the rolling velocities of neutrophils at a wall shear stress of 2 dynes/cm2 on P-selectin-expressing monolayers (10–15 µm/s) [53 , 58 ] may be somewhat higher than on IL-1- or TNF-stimulated HUVEC monolayers [3 , 15 ]. Rolling velocity is affected by the level of cytokine stimulation of endothelial cells [59 ] and the site density of selectins [60 ].

Under control conditions, nearly 30% of the interacting neutrophils were able to arrest immediately after capture at 2.0 dynes/cm2 without observable rolling. It appears that neutrophil rolling is not essential for arrest. However, it is possible that rolling was more rapid than the minimum time period for detection in our analysis system, ~30 msec. Lawrence and Springer [61 ] estimated that a rolling neutrophil would contact an E-selectin-containing lipid bilayer for only 0.1–1 sec before arrest. They reasoned that adhesion strengthening must occur within this time period. For the neutrophils that appeared to abruptly arrest in our system, adhesion strengthening might have occurred within 30 msec. Another possibility is that some neutrophils became stimulated while flowing across the activated monolayer before capture and arrest. The complete inability to abruptly arrest when CD18 or ICAM-1 was blocked confirms the importance of CD18 and ICAM-1 in mediating rapid adhesion strengthening and arrest.

The arrest of rolling leukocytes in vivo has been evaluated in many studies by continuous, direct, microscopic observation of a single vessel segment [36 ]. The rolling flux appears very high compared with the number of leukocytes that stop within the single field of view, leaving the impression that the efficiency of conversion from rolling to arrest is quite low. Lack of information, though, on the type and fate of each passing cell makes it impossible to directly answer the question of efficiency. What seems likely at the present time is that the rate of conversion to stable adhesion after endothelial contact is much higher in the in vitro models than in vivo. Hydrodynamic and technical factors probably account for some of this apparent difference, as well as some possible activation of leukocytes, during the isolation for in vitro studies, but direct evidence about mechanisms is not available.

Neutrophils arrest very near the site of transendothelial migration
Neutrophils move less than the diameter of the leukocyte from the point of arrest to the site of transendothelial migration. This appears to be true whether neutrophils abruptly stop from the flow stream or roll for relatively long distances, because no correlation was found between the distance rolled and the distance migrated after arrest. The efficiency of transendothelial migration in our in vitro model is very high, exceeding 90%, and distinctly higher than the ~60% seen under static conditions when cells are allowed to simply settle onto monolayers making random contact with endothelial cells [22 ]. The site of arrest under flow appears to be spatially and functionally associated with the site of transmigration. The distance that an arrested neutrophil would migrate before migration was consistent with a model in which they adhered preferentially at endothelial borders. Such a condition would likely require the colocalization of adhesion molecules and chemotactic stimuli, but little is known about the topography of IL-8 and PAF presentation, and ICAM-1 and E-selectin appear to be distributed uniformly over the surface of cytokine-stimulated endothelial cells. We have recently obtained evidence that P-selectin is concentrated at endothelial cell junctions following histamine stimulation [37 ], but there is little evidence that this adhesion molecule participates in rolling or stationary adhesion on HUVEC monolayers stimulated for 4 h with IL-1. ICAM-2 appears to be concentrated at the junctions of unstimulated endothelial cells (unpublished results). There is evidence that Mac-1 and LFA-1 can interact with this molecule [7 , 62 ], and Issekutz et al. [50 ] have shown that ICAM-2 can participate in transmigration of neutrophils under static experimental conditions. Another feature of the monolayer that may contribute to the stationary adhesion is the surface contour of the monolayer. Davies and colleagues [63 , 64 ] using atomic force microscopy have mapped the 3-D shape of the luminal endothelial surface and found it to be an important determinant of spatial distribution of stress gradients from the elevated central regions of endothelial cells to the lower junctional areas. Such gradients could favor stationary adhesion at the cellular regions of lower shear stress (i.e., at cell junctions).

The high percentage of neutrophils migrating at tricellular corners where three endothelial cells meet is consistent with observations under static conditions. Burns et al. [22 ] have shown that this region is the site of some discontinuity in the junctional complexes between endothelial cells (e.g., occludin) and have argued that this discontinuity obviates the need for disrupting intercellular adhesion of endothelial cells. Walker et al. [65 ] and Barry et al. [66 ] have provided ultrastructural and immunohistochemical evidence that junctional structures are discontinuous at endothelial tricellular corners in vivo. The static experiments allowed neutrophils to settle at unit gravity onto monolayers of cytokine-stimulated HUVEC, and the location of transmigrating neutrophils was determined. It is assumed that there was random contact of settling cells with endothelial cells and that migrating neutrophils would seek the sites for transmigration. Under flow conditions, neutrophils stopped rolling within a few µm from the site of transmigration and because for ~70% of the cells, this site was at the tricellular corner, there seems to be a localized endothelial determinant that promotes stationary adhesion at distinct sites. It remains to be determined if neutrophil-activating factors and/or ligands for CD18 integrins are spatially organized to target neutrophil arrest near tricellular corners. The possible significance of this site may lie in the observation that intercellular junctions are inherently discontinuous at tricellular corners, and leukocyte migration at this point may minimize the disruption of integrity of the endothelial monolayer.


   ACKNOWLEDGEMENTS
 
This work was supported in part by the following grants: A.R.B. is the recipient of a Chao Fellowship and grants from the Methodist Hospital Foundation and from the American Lung Association (RG-068-N). L.V.M. is supported by grants HL18670, NS 23327, and Robert A. Welch Foundation Grant C-938. C.W.S. is supported by grants AI19031, HL42550, and ES06091. We are grateful to Dr. C.V. Ananth for assistance with the statistics and Drs. Robert Rothlein, James Ruschie, and Timothy Springer for generously providing monoclonal antibodies. We also wish to thank Drs. Yasunori Abe and Sriram Neelamegham for many useful discussions, Lisa Thurmon for technical assistance, and Michelle Swarthout for her secretarial assistance. We are grateful to the delivery nurses and technicians at Columbia Women’s Hospital of Texas and Ben Taub General Hospital for providing us with human umbilical cords.

Received October 30, 1999; revised January 18, 2000; accepted January 20, 2000.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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