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The Journal of Immunology, 2000, 165: 404-410.
Copyright © 2000 by The American Association of Immunologists

Expression of P-Selectin at Low Site Density Promotes Selective Attachment of Eosinophils Over Neutrophils1

Bruce S. Edwards2, Mark S. Curry, Hisashi Tsuji, David Brown, Richard S. Larson and Larry A. Sklar

Departments of Pathology and Cytometry, Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The selective interaction of neutrophils with E-selectin and eosinophils with P-selectin has been previously reported, but the relevance of selectin site density and fluid shear has not been studied in detail. We have developed a new approach to examine these interactions in cell suspensions that integrates an on-line cone-plate viscometer with a flow cytometer. We find that eosinophils and neutrophils both use P-selectin glycoprotein ligand-1 to form stable conjugates with P-selectin Chinese hamster ovary cell transfectants, with a preferential adhesion of eosinophils. Further, the difference in cell adhesion between neutrophils and eosinophils is magnified at P-selectin expression levels below ~20 sites/µm2, a range likely to be relevant to endothelial cell expression levels in conditions associated with eosinophilia. The unique behavior is retained over shear rates ranging from 100 to 1500/s but is magnified at low shear. Results from parallel-plate flow chamber assays suggest that preferential eosinophil adhesion reflects an enhanced efficiency of initial PSGL-1 bond formation with P-selectin rather than a unique ability of eosinophils to mediate rolling interactions of longer duration on low-density P-selectin substrates. These differences may account in part for the increase in eosinophil accumulation in allergic diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Recruitment of leukocytes to an inflammatory site is a regulated process. Leukocytes normally circulate in blood without interacting with other leukocytes or the vascular endothelial cells lining blood vessel walls. However, under inflammatory conditions, they adhere to vascular endothelial cells at sites proximal to tissue damage or inflammation (1, 2, 3, 4).

The recruitment of an eosinophil (Eo)3 to a site of inflammation appears to exhibit many features of a pathway common to all leukocytes. Initial Eo tethering to endothelium is mediated by adhesion to vascular selectins induced on endothelial cell surfaces by inflammatory mediators. Selectin-dependent attachment occurs under fluid shear and results in a slowing down or "rolling" of Eos along the affected vascular segments (5). This event is reversible unless the adhesive activation of the integrins on the Eo follows. In the presence of chemoattractants or cell contact-mediated signals, the integrins become functionally activated and, in some cases, also increase in number. Activated integrins bind cell adhesion molecule counterstructures on endothelial cells, which results in a sustained, strong attachment and cell spreading (5, 6).

There also appear to be unique features of adhesion receptor usage by Eos that could account for the differential patterns of Eo and polymorphonuclear neutrophil (PMN) migration observed in allergic diseases such as asthma. Unlike resting PMNs, Eos express the ß1 integrin very late Ag-4 ({alpha}4ß1) that mediates both rolling and firm adhesion to VCAM-1, a counterstructure whose expression is induced upon endothelial cells by inflammatory cytokines. Consistent with a role in selective recruitment of Eos, VCAM-1 is expressed, albeit weakly, on lung endothelium in clinical asthma (7, 8).

Eos also appear to differ from PMNs in their relative ability to recognize and bind vascular E- and P-selectins. It has been a consistent finding that Eos bind significantly less avidly than PMNs to E-selectin (9, 10, 11). With P-selectin, the relationship has been less clear cut. An increased binding avidity of Eos for P-selectin was implicated as the reason up to 10-fold more Eos than PMNs selectively accumulated on airway endothelium (nasal polyps) in vitro (12). This was consistent with the finding in a murine asthma model that Ag-induced eosinophilia was blocked by preventing associated production of histamine, a selective inducer of endothelial cell P-selectin (13). Recent studies in P-selectin gene knockout mice have also supported P-selectin as an important element in Eos recruitment (14, 15, 16). However, there have been conflicting results in studies of leukocyte adhesion to purified P-selectin immobilized on surfaces. Eos showed only a modestly increased avidity for P-selectin (9, 12) or bound P-selectin apparently less avidly than PMNs (10). These latter results raised questions as to how differential P-selectin recognition could account for selective Eo accumulation.

In the present study, we exposed cell suspensions to fluid shear using a novel on-line cone-plate viscometer integrated with a flow cytometer to detect cell adhesion. This model of cell-cell adhesion reveals a previously unappreciated ability of Eos to selectively recognize and bind to cells expressing very low cell membrane densities of P-selectin. These results suggest that low P-selectin expression may foster selective recruitment of Eos over PMNs to vascular endothelium.


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

Eos were prepared from peripheral blood of healthy volunteers as previously described (17, 18) by separation of granulocytes on Percoll and hypotonic lysis of contaminating erythrocytes. Eos were then purified from the granulocytes by negative selection in the presence of magnetic beads bearing mAbs to CD16 (to remove PMNs) and CD3 (to remove the small number of contaminating T lymphocytes). Such preparations consisted of 95% or more Eos. Because the initial unfractionated granulocyte preparations typically consisted of 95% or more PMNs and only 1–3% Eos, we used them rather than the magnetic bead-coated postfractionation PMNs as the source of PMNs for analysis. The expression by PMNs and Eos of a panel of membrane determinants was assessed by direct immunofluorescence staining with fluorochrome-conjugated mAbs. Expression of each determinant was quantified by comparison of the median fluorescence intensity to a standard curve generated with Quantum Simply Cellular microspheres (Flow Cytometry Standards, San Juan, Puerto Rico) stained in parallel with the same mAb. Levels of PMN Mac-1 (CD11b/CD18) expression (Fig. 1GoA) were similar to what we previously observed in whole-blood preparations of unstimulated PMNs (19), an indication that the preparation procedures did not inadvertently induce cell activation.



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  		FIGURE 1. Characterization of leukocyte preparations and CHO cell P-selectin expression. A, PMNs and Eos were immunofluorescently stained with mAbs to VLA-4 (CD49d), LFA-1 (CD11a), Mac-1 (CD11b), L-selectin (CD62L), PSGL-1 (CD162), Fc{gamma}RIII (CD16), and CD44. Results represent mean ± SD Ab binding sites per cell for eight separate experiments with cells from three different healthy volunteers. B, Representative CHO cell lines expressing a range of transfected human P-selectin levels were immunofluorescently stained with PE-conjugated anti-P-selectin mAbs (CD62P, clone G1). The median number of P-selectin Ab binding sites was determined by comparison to a standard curve generated with Quantum Simply Cellular beads stained with the same Ab. Median P-selectin site density was estimated using these values together with an empirically determined surface area for CHO cells of 897 µm2. The median site density for each cell line is indicated by the solid bar above its respective fluorescence histogram. These values were 11, 36, 99, and 440 sites/µm2 for the four CHO-Psel cell lines. Nonspecific binding of Abs to parental untransfected CHO cells corresponded to a background of 0.7 sites/µm2 (leftmost fluorescence histogram).

 
Chinese hamster ovary (CHO) cells transfected with the gene for human P-selectin (CHO-Psel) have been previously described (20) and were a kind gift from Dr. R. P. McEver (University of Oklahoma). CHO-Psel expressing P-selectin over a 3-log range of site density were generated by flow cytometric sorting of CHO-Psel on the basis of anti-P-selectin immunofluorescence staining intensity. In some experiments, very low but significant levels of membrane P-selectin were also achieved by limited trypsin treatment of CHO-Psel. Incubation of CHO-Psel with 0.25% trypsin (Life Technologies, Grand Island, NY) for 5 min at 37°C typically reduced P-selectin site density 3- to 4-fold without effects on cell viability. P-selectin Ab binding sites were determined by comparison of the median anti-P-selectin fluorescence intensity of immunostained CHO-Psel to standard curves generated with Quantum Simply Cellular microspheres as described above. The mean diameter of CHO cells was empirically determined in a Coulter Counter to be 16.9 µm (Coulter, Palo Alto, CA). The surface area of CHO cells was estimated to be ~900 µm2 using the assumption of the cells as spherical objects of 16.9-µm diameter. This is an obvious oversimplification that does not account for membrane folds and ruffles, but one that is meaningful as an average measure of what leukocytes likely encounter during cell contacts and as a normalization method to permit qualitative comparisons with results obtained in other studies involving different experimental parameters. P-selectin site number determinations were routinely performed at the beginning of each adhesion experiment, and site density was computed as total Ab binding sites/mean cell surface area. Fig. 1GoB illustrates CHO-Psel site densities representative of the experimentally tested range. At high P-selectin surface densities, the number of Abs bound may reflect half the number of P-selectin molecules expressed as these are conditions in which binding of a single Ab to a pair of P-selectin molecules may be favored. At limiting low P-selectin expression, the number of Abs bound may be equal to the number of P-selectins.

Cell suspension adhesion assays

Eos and PMNs were labeled with green fluorescent fluo3 (7.5 µM, 15 min 37°C), and CHO-Psel were labeled with red fluorescent hydroethidine (250 µM, 15 min, 37°C). In some experiments, PMNs were labeled with red fluorescent Fura Red (7.5 µM, 15 min, 37°C). Labeled cells were washed, resuspended in HHB buffer (110 mM NaCl, 10 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 30 mM HEPES, 10 mM glucose, rendered nonpyrogenic by affinity chromatography over Polymixin B, pH 7.4), and stored on ice until used in assays. In initial experiments, cell adhesion was assessed under conditions of fluid shear generated by a magnetic stir bar. Cell suspensions containing 105 leukocytes plus 105 CHO-Psel in 0.5 ml HHB were stirred in a 12 x 75-mm polystyrene tube with a 2 x 5-mm magnetic stir bar (400 rpm, 37°C). After 5 min of continuous stirring, cells were sampled and analyzed in the flow cytometer to determine numbers of nonadherent singlet leukocytes (green/nonred fluorescent), nonadherent singlet CHO-Psel (red/nongreen fluorescent), and cell conjugates containing leukocytes adherent to CHO-Psel (red/green cofluorescent). The percentage of adherent leukocytes was calculated on the basis of the decrease in singlet leukocyte numbers: 100 x (I - O)/I in which I was the original number of input leukocytes and O was the number of leukocytes observed in the singlet population at the time of sampling.

To provide an adhesion environment in which fluid shear forces were uniform and subject to precise control, cell suspension adhesion assays were also performed in a cone-plate viscometer (CPV) (Brookfield DV-III, Stoughton, MA). The CPV consists of a stationary plate beneath a rotating cone designed to apply a uniform shear rate to all parts of a sample placed between the opposing cone and plate surfaces (21). The shear rate is independent of distance from the cone center and equal to the cone angular velocity divided by the tangent of the cone angle (22). Cones with angles of 0.8°, 1.565°, and 3° were used. Cell suspensions consisted of 2.5 x 104 Fluo 3-labeled PMNs or Eos plus 105 hydroethidine-labeled CHO-Psel in 0.5 ml HHB, which were combined and placed in the CPV just before application of shear. In one set of experiments, Fluo3-labeled Eos and Fura Red-labeled PMNs (2 x 104 of each) were combined together with 1.2 x 105 unlabeled CHO-Psel. The increase in the ratio of CHO-Psel to leukocytes to >=3:1 in CPV experiments was to ensure that the CHO cells were present in excess and thus not limiting to the adhesion interaction. Samples were aspirated through a port that penetrated the plate at the mid-point of cone rotation.

To acquire and deliver the samples from the CPV to the flow cytometer, an automated device was used that involved the integrated actions of a syringe, a reciprocating two-position eight-port valve, and a pressure-driven fluid line. Fourteen 35-µl aliquots of suspension were aspirated from the CPV over a 2-min period as the cone was continuously rotating. From each aliquot, a 5-µl sample was collected in one of the valve sample loops, injected into the pressurized fluid line by switching the valve, and delivered to the flow cytometer by the moving fluid for analysis of cell adhesion. This sampling device is described in more detail in a separate publication (23). There was an interval of 6–8 s between initial sample aspiration and analysis. For each condition of shear, a suspension of leukocytes plus control parental untransfected CHO cells was analyzed to determine the cumulative total of input leukocytes recovered from all 14 aspirated samples (I). This was compared with the observed cumulative number of nonadherent leukocytes recovered in the singlet populations when leukocytes were subjected to comparable shear in the presence of CHO-Psel (O). The percentage of adherent leukocytes was then calculated as 100 x (I - O)/I. In an alternate approach, Fura Red-labeled PMNs and Fluo3-labeled Eos were combined together in suspension with unlabeled CHO-Psel. Singlet Eos and PMNs exhibited relatively low and comparable forward light scatter intensity signals, which shifted to the distinctively higher light scatter intensity profile characteristic of CHO cells when the leukocytes adhered to CHO-Psel. In these experiments, the number of input Eos and PMNs (I) was determined as the cumulative total of PMNs and Eos detected in the singlet leukocyte forward light scatter gate when CHO-Psel were pretreated with anti-P-selectin function-blocking mAbs. This was compared with the observed (O) cumulative number of nonadherent Eos and PMNs recovered in the singlet population when sheared with CHO-Psel in the absence of blocking mAbs. The percentage of adherent Eos and PMNs was then calculated as 100 x (I - O)/I as above. Function-blocking mAbs G1, directed against P-selectin, and PL-1 (Fab) directed against P-selectin glycoprotein ligand-1 (PSGL-1) were gifts from Dr. R. P. McEver.

The singlet-depletion method used for adhesion analysis assumes that a decrease in cell numbers in the singlet leukocyte population strictly reflects the formation of heterologous doublets and higher order clusters between leukocytes and CHO-Psel. The validity of this assumption is supported by our observation that the loss of leukocyte singlet numbers in the presence of CHO-Psel is completely reversed in the presence of P-selectin blocking Abs (see Fig. 3GoA below). Because only the CHO cells express P-selectin, only heterologous adhesion should be affected and the total observed decrease in singlet leukocyte numbers must be attributable to this cause. Thus, singlet depletion due to homotypic leukocyte-leukocyte adhesion or leukocyte death were not of detectable consequence in our adhesion measurements. We have found that direct enumeration of heterologous conjugates (i.e., two-color events) consistently underestimates the number of adherent leukocytes. This is due in part to the presence of higher order heterologous clusters and the difficulty under our analysis conditions of accurately quantifying the number of leukocytes present in such heterologous clusters.



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  		FIGURE 3. Analysis of leukocyte adhesion to CHO-Psel under conditions of uniform fluid shear in the CPV. Eos or PMNs were mixed with CHO-Psel, exposed to continuous shear for 5 min at 37°C, then injected into the flow cytometer for analysis. The percent of adherent leukocytes was determined by comparison to parallel assays in which untransfected parental CHO cells were used or EDTA was added to disrupt P-selectin-dependent adhesion. A, Leukocytes and CHO-Psel expressing 9 sites/µm2 P-selectin were sheared at 225/s in the presence or absence of 20 µg/ml G1 anti-P-selectin Abs. B, Leukocytes were sheared at 114/s with CHO-Psel expressing 15 sites/µm2 P-selectin in the presence or absence of 20 µg/ml of PL-1 anti-PSGL-1 Ab Fab. C, Adhesion assays were performed at shear rates of 114/s (open symbols) and 225/s (filled symbols) using Eos (triangles) or PMNs (square) and CHO-Psel expressing indicated levels of P-selectin. All illustrated results are representative of two or more separate experiments.

 
Parallel-plate flow chamber assay

Physiologic flow conditions were produced in vitro using a flow chamber with parallel-plate geometry (Glycotech, Rockville, MD). The parallel-plate flow chamber used in this study has been described in detail (24). Briefly, the chamber produces a well-defined laminar flow over cell monolayers grown on coverslips. A suspension of Eos or PMNs (2 x 106 ml) in RPMI 1640 media, 1% human serum albumin was perfused through the chamber at a defined flow rate. Defined levels of shear are applied to CHO cell monolayers expressing P-selectin by drawing perfusion media through the parallel-plate chamber via a syringe pump (Harvard Apparatus, Natick, MA). The entire time period of leukocyte perfusion was videotaped under phase-contrast microscopy (Olympus, New Hyde Park, NY). All experiments were recorded with a Vicon VC240 CCD video camera and Toshiba KV-7168A recorder (ADI, Albuquerque, NM). Videotaped data frames were digitized for data analysis. Two quantities were measured in the analysis: the rate of initial attachment and the total number of rolling cells. The total number of rolling cells was determined by analysis of videotape recordings of 8–10 random fields surveyed over a 5-min interval (x20 phase-contrast objective). The rate of initial attachment was determined by counting the number of leukocytes that attached for at least 1 s during the first 3 min of observation in one field (x20 phase-contrast objective) per experiment.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
To assess leukocyte recognition of P-selectin, CHO-Psel expressing different levels of P-selectin (20–2400 sites/µm2) were combined in cell suspensions with Eos or PMNs and exposed to magnetic stir-bar-induced fluid shear. Preliminary experiments indicated that leukocytes were adherent to CHO-Psel but not to untransfected control CHO cells and that a steady-state plateau level of adherent leukocytes was attained within 5 min of continuous stirring at 37°C. Thus, although P-selectin is generally considered to be a receptor that mediates rapidly reversible adhesive interactions associated with cell rolling, these results indicated that it was also capable of mediating prolonged cell-cell adhesion in stirred cell suspensions. In all subsequent experiments, cell mixtures were routinely exposed to fluid shear for 5 min before the cell adhesion assessment was made.

In four separate experiments involving four different healthy blood donors, we observed a statistically significant log linear relationship to exist between P-selectin site density and the percentage of leukocytes adherent to CHO-Psel (Fig. 2Go, p = 0.01 and 0.003 for linear fit of PMN and Eo data, respectively). A greater percentage of Eos than PMNs bound to CHO-Psel in each paired comparison. Moreover, as P-selectin site density decreased, the ratio of adherent Eos to adherent PMNs increased. At the lowest tested level of P-selectin (20 sites/µm2), ~40% of Eos were adherent as compared with 10% of PMNs, a 4:1 ratio (Fig. 2Go). These initial results suggested that low levels of P-selectin expression might favor selective adhesion of Eos.



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  		FIGURE 2. Effect of P-selectin site density on adhesion of PMNs and Eos to CHO-Psel in stirred cell suspensions. Leukocytes and CHO cells were stirred together for 5 min at 37°C with a magnetic stir bar (400 rpm), then analyzed in the flow cytometer to determine the percent of adherent leukocytes as described in Materials and Methods. Regression lines computed for each leukocyte population were: % adherent Eos, 18.1 + 14.4 x log P-selectin site density (p = 0.003); and % adherent PMNs, -22.1 + 21.9 x log P-selectin site density (p = 0.01).

 
The fluid shear field around a rotating stir bar is nonuniform and poorly defined. To assess the role of shear in P-selectin recognition, we adapted the adhesion assay to an alternate cell suspension system in which fluid shear forces could be more precisely defined and controlled. This consisted of a CPV interfaced with the flow cytometer via a recently developed coupling valve technology (23). The design of the CPV enables uniform shear to be applied to the entire cell suspension (21). After exposure of cell suspensions to continuous shear in the CPV for 5 min at 37°C, the cells were delivered directly to the flow cytometer under computer automated control so that the analysis of cell adhesion was completed within 8 s of cell removal from the CPV. Adhesion of both PMNs and Eos to untransfected control CHO cells was negligible (data not shown), and adhesion to P-selectin expressing CHO cells was abrogated in the presence of Abs to either CHO cell P-selectin (G1, Fig. 3GoA) or leukocyte PSGL-1 (PL-1, Fig. 3GoB). At physiological shear rates (114–225/s), PMN adhesion to CHO-Psel precipitously declined as P-selectin site densities decreased from ~20 to less than 10 sites/µm2 (Fig. 3GoC). By contrast, Eos exhibited only a moderate reduction in adhesion, confirming the results observed in the stir-bar-driven adhesion experiments. The most pronounced difference between PMNs and Eos occurred at the lower tested conditions of shear rate and P-selectin site density in which <5% of PMNs were adherent as compared with >50% of Eos (Fig. 3GoC).

In subsequent experiments, PMNs and Eos were mixed with CHO-Psel at shear rates ranging from 114/s to 1500/s. The effect of shear at low P-selectin site density was confirmed in repeated experiments with leukocytes from four different healthy donors in which CHO P-selectin site density was 9 ± 2 sites/µm2. The ratio of adherent Eos to adherent PMNs was ~9:1 at the lowest shear rate of 114/s, then decreased to a plateau range of between 2:1 and 3:1 as shear rate increased to 225/s and 1500/s (Fig. 4GoA). This was in distinct contrast to the adhesion pattern observed when CHO-Psel expressing ~7-fold higher levels of P-selectin (75 ± 16 sites/µm2) were tested in similar experiments (Fig. 4GoB). Although Eos again consistently exhibited a larger adherent cell fraction than PMNs at all levels of shear, the differences were of much smaller magnitude, and the influence of shear rate on adhesion was less pronounced.



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  		FIGURE 4. Effects of fluid shear upon leukocyte recognition of P-selectin. Adhesion assays in A and B were performed in the CPV at the indicated levels of shear as in Fig. 3Go. A, P-selectin expressed on CHO cells at 9 ± 2 sites/µm2. The percentage adherent Eos was significantly greater than the percentage adherent PMNs at each shear rate (p < 0.05 for each, Student’s t test). B, P-selectin expressed on CHO cells at 75 ± 16 sites/µm2. Represented in A and B are pooled results of four separate experiments with leukocytes from four different healthy volunteers. C, Cell suspensions containing a 1:1 mixture of PMNs and Eos, together with CHO-Psel (14 ± 7 P-selectin sites/µm2), were sheared 5 min at 180/s. Results represent the percentage adherent cells in each leukocyte population as compared with parallel suspensions sheared in the presence of anti-P-selectin function blocking mAbs. The percentage adherent Eos was significantly greater than the percentage adherent PMNs (p < 0.02 for three separate experiments with leukocytes from three different donors).

 
To determine whether preferential adhesion of Eos to CHO-Psel occurred in the presence of PMNs, cell suspensions containing both Eos and PMNs together with CHO-Psel (P-selectin, 14 ± 7 sites/µm2) were sheared 5 min at 180/s and analyzed. In three separate experiments, the ratio of adherent Eos to adherent PMNs averaged 3.2:1 and ranged from 2.7:1 to 4.1:1 (Fig. 4GoC). Thus, Eos were enriched several fold from leukocyte mixtures by low P-selectin on CHO cells.

These experiments represent, to our knowledge, the first studies of how defined shear conditions affect P-selectin-dependent adhesion of leukocytes in free-cell suspensions. In previous work, such adhesive interactions have been exclusively characterized between flowing leukocytes and immobilized P-selectin or planar monolayers of cells expressing P-selectin (9, 10, 12, 25, 26). The collisions of free-flowing cells with immobilized cell monolayers in these latter experimental systems differ in certain physical aspects from collisions between cells in free suspension, and these differences are likely to have important consequences for adhesion receptor operation. Mathematical models comparing these two different classes of intercellular collision suggest that when cells collide in free suspension the average initial contact angle between cells is greater (i.e., closer to head-on) and the average receptor-independent duration of cell contact is ~26 times longer (27, 28). Both of these factors are likely to foster increased numbers of receptor-ligand bonds; the first because of the broadened contact area between opposing cell surface membranes promoted by increased compressive forces of collision, the second because of the extended time frame in which adhesion receptors are allowed to interact. Thus, the relatively stable associations between leukocytes and CHO-Psel observed in our cell suspension assays probably reflected the formation of a multiplicity of PSGL-1/P-selectin bonds between individual pairs of colliding cells. To further refine the physiological implications of our sheared cell suspension adhesion data, we next compared the effect of P-selectin site density on initial attachment and rolling of PMNs and Eos on CHO cell monolayers in a conventional parallel-plate flow chamber assay. This assay simulates interactions observed in vivo in which circulating leukocytes are captured to vessel walls and roll along the vascular endothelium.

Leukocytes were perfused over monolayers of CHO cells expressing P-selectin over a range of 7–140 sites/µm2. The rate of fluid flow was adjusted to achieve a wall shear rate of ~120/s. When P-selectin was expressed at relatively high site densities (48–140 sites/µm2), the frequency of rolling Eos was consistently greater than that of PMNs (Fig. 5GoA). By contrast, both leukocyte types exhibited similar very low frequencies of rolling cells on CHO cell monolayers expressing seven P-selectin sites/µm2. All observed leukocyte rolling was P-selectin dependent as there was no detectable rolling on untransfected or mock-transfected control CHO cell monolayers (data not shown). It has been previously shown that when P-selectin site density falls below ~15 sites/µm2, PMNs are transiently captured or tethered to the substrate but often fail to subsequently roll (29, 30). These attachment events are thought, on the basis of binding kinetics data, to reflect single P-selectin/PSGL-1 bond interactions (29, 30). Therefore, we reevaluated PMN and Eo interactions with low-density P-selectin substrates to determine whether differences in initial attachment could be discerned. When leukocytes were perfused over CHO-Psel monolayers expressing 8–12 P-selectin sites/µm2, the rate of Eo initial attachment was significantly higher than that of PMNs (Fig. 5GoB).



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  		FIGURE 5. Analysis of Eo and PMN adhesive interactions with P-selectin in parallel-plate assays. PMNs ({blacksquare}) and Eos ({square}) were perfused over monolayers of CHO cells expressing the indicated levels of P-selectin at a wall shear rate of ~120/s. A, Number of rolling leukocytes per mm2. Represented are pooled results from three separate experiments. Rolling Eos were significantly greater than rolling PMNs at 48, 87, and 142 P-selectin sites/µm2 (p < 0.05 for each, Student’s t test). B, Number of leukocytes forming initial attachments to the P-selectin substrate per mm2 per min. Results at eight P-selectin sites/µm2 represent the mean and SD of two separate experiments with leukocytes from two different donors. Results at 12 P-selectin sites/µm2 represent the mean and SD of triplicate determinations made on the same day with leukocytes from a single donor. The initial attachment rate of Eos was significantly greater than that of PMNs (p < 0.05 for each P-selectin condition, Student’s t test). There was no detectable rolling or attachment of PMNs or Eos in parallel control experiments in which monolayers of untransfected or mock-transfected CHO cells were used (data not shown).

 
These results confirmed and extended previous findings showing important differences between Eos and PMNs in adhesive interactions with P-selectin under shear flow. A novel finding in our sheared cell suspension assays was the importance of low P-selectin site density as a major determinant of selective Eo adhesion. The most pronounced selectivity was observed at P-selectin site densities of <20 sites/µm2. This corresponded to conditions in parallel-plate assays in which Eos formed initial attachments to the P-selectin CHO cell substrate more efficiently than PMNs even though they, like the PMNs, only rolled for a brief time period (1–3 s) before detaching. We conclude from this that the selectivity of Eo adhesion to P-selectin most likely reflects the greater efficiency with which Eos form initial attachments to P-selectin under shear flow. Eos have been shown to use P-selectin in concert with VCAM-1 for firm adhesion to IL-4-activated HUVEC monolayers under physiological shear, even though they do not roll but immediately arrest upon initial contact (31). Under such conditions, endothelial cell P-selectin has been reported to be at most only slightly elevated (<10 sites/µm2) (32). This suggests that limited tether bond formation between Eo PSGL-1 and P-selectin such as detected in the present studies is sufficient to facilitate an adhesion receptor cascade capable of recruiting Eos from the circulation.

It is noteworthy that the cell suspension adhesion assay used in this study provided a uniquely sensitive method by which to discern the differences between PMN and Eo attachment to P-selectin. The present results suggest it to be a productive methodology for modeling important physiologically relevant features of initial receptor bond formation under shear flow. Perhaps the most important aspect of the suspension assay in this regard is that it permits long cell-cell contact times at moderate shear that could only be observed under near static conditions in a parallel-plate assay. Thus, the suspension assay complements the more conventional parallel-plate assay and highlights features of adhesion receptor interactions that might not be immediately obvious from the latter assay. Moreover, the suspension assay methodology requires minimal numbers of leukocytes (~25,000 per assay) so that low frequency leukocyte populations such as Eos may be routinely prepared from relatively small amounts of blood.

An interesting observation was the decline of PMN but not Eo adhesion to near baseline levels that occurred in suspension assays when the shear rate was decreased from 225 to 114/s (Fig. 4GoA). This suggested that between these two shear levels was a shear threshold below which stable PMN adhesion to CHO-Psel was less efficiently established. This was somewhat higher than the shear threshold of ~70/s reported for rolling of HL-60 cells on ~50 sites/µm2 P-selectin in parallel-plate assays (33). This discrepancy may have reflected the reported variation of shear threshold with P-selectin site density (33), our use of PMNs rather than HL-60 cells, and possibly a difference in the cumulative number of PSGL-1/P-selectin bonds required for formation of stable conjugates in the suspension assay. In any event, these results suggest that Eos and PMNs may exhibit differences in shear thresholds for P-selectin adhesion deserving of further investigation.

An additional important finding in our parallel-plate flow chamber studies was that Eos showed increased rolling with increasing P-selectin site density, whereas PMN rolling showed a plateau (Fig. 5GoA). This suggests that not only do Eos bind more efficiently than PMNs to P-selectin when it is present at very low site density, but they are also able to more efficiently use higher levels of P-selectin for rolling. Because anti-PSGL-1 function blocking mAbs completely blocked adhesion of both PMNs and Eos to P-selectin (Fig. 3Go, A and B), it seems unlikely that other potential leukocyte P-selectin counterligands (34, 35, 36, 37) could have been significantly involved. Therefore, differences between Eos and PMNs in P-selectin recognition may reflect qualitative or quantitative differences in PSGL-1 expression. Eos express nearly 2-fold higher levels of PSGL-1 than PMNs (Ref. 12 , Fig. 1GoA). The molecular structure of PSGL-1 also reportedly differs in Eos vs PMNs (12, 38). It is also possible that differences in PSGL-1 organization on microvilli may be involved. Further studies will be required to distinguish among these possibilities. It is interesting that despite their increased initial attachment rate at low P-selectin site densities, Eos were no more able than PMNs to subsequently mediate rolling interactions of >1- to 3-s duration. This suggests that Eos and PMNs share a common minimum P-selectin site density requirement for more sustained rolling interactions that is independent of the efficiency of the initial PSGL-1/P-selectin tether bond formation.


   Footnotes
 
1 This work was supported in part by American Lung Association Grant ARC 4-00982-4100, National Institutes of Health Grants RR14175 and HL56384, American Heart Association Grant 990318Z, and the State of New Mexico Cigarette Tax. Back

2 Address correspondence and reprint request to Dr. Bruce S. Edwards, Cytometry, CRF Room 217, University of New Mexico Health Sciences Center, 2325 Camino de Salud, Albuquerque, NM 87131. Back

3 Abbreviations used in this paper: Eo, eosinophil; CHO, Chinese hamster ovary; CHO-Psel, CHO cells expressing transfected human P-selectin; CPV, cone-plate viscometer; PMN, polymorphonuclear neutrophil; PSGL, P-selectin glycoprotein ligand-1. Back

Received for publication December 6, 1999. Accepted for publication April 12, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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