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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 512-519, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4806

Platelets Promote Eosinophil Adhesion of Patients with Asthma to Endothelium under Flow Conditions

Laurien H. Ulfman, Dianne P. H. Joosten, Corneli W. van Aalst, Jan-Willem J. Lammers, Ed A. van de Graaf, Leo Koenderman and Jaap Jan Zwaginga

Department of Pulmonary Diseases, University Medical Center, Utrecht; and Central Laboratory for Blood Transfusion, Amsterdam, The Netherlands

Address correspondence to: Laurien H. Ulfman, Heidelberglaan 100 hpn G03 550, 3584 CX, Utrecht, The Netherlands. E-mail: L.Ulfman{at}hli.azu.nl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During the late-phase asthmatic response, eosinophils migrate to the bronchial tissue and cause severe damage. In this study we compared in vivo primed eosinophils from patients with allergic asthma with eosinophils from healthy control subjects in their adhesion behavior to tumor necrosis factor-{alpha}–activated endothelium under flow conditions (0.8 dyn/cm2). More eosinophils from patients with asthma adhered to activated endothelium, compared with cells from healthy control subjects (1,237 ± 126 versus 887 ± 94 cells/mm2, respectively). In the presence of blocking antibodies directed against very late antigen-4 and E-selectin, the residual binding of the cells of individuals with allergic asthma was significantly higher than that of the healthy control subjects (353 ± 64 versus 123 ± 31 cells/mm2, respectively, P < 0.02). In addition, secondary tethering or formation of clusters of the eosinophils of patients with allergic asthma was significantly increased compared with the healthy control subjects (cluster indices 1.8 ± 0.3 versus 0.8 ± 0.2, respectively, P < 0.05). Because patient cells showed an enhanced interaction with platelets during the perfusions, the role of P-selectin on platelets was investigated. Blocking antibodies directed against P-selectin reduced the enhanced binding and clustering of eosinophils of patients with allergic asthma. We conclude that P-selectin–bearing platelets contribute to secondary tethering processes of eosinophils to activated endothelium. Therefore, platelets might play an important role in the chronic inflammatory processes of these patients.

Abbreviations: fluorescence-activated cell sorter, FACS • human serum albumin, HSA • human umbilical vein endothelial cell, HUVEC • intercellular adhesion molecule, ICAM • interleukin, IL • platelet-activating factor, PAF • tumor necrosis factor-{alpha}, TNF-{alpha} • vascular cell adhesion molecule, VCAM • very late antigen, VLA


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Eosinophil accumulation is a hallmark of allergic inflammation, as found in the airway mucosa of patients with asthma. Adhesion of eosinophils to endothelial cells of the blood vessel is one of the initial and crucial steps in the accumulation of eosinophils to these allergic inflammatory sites. As all leukocytes, eosinophils use adhesion molecules in a multistep mechanism to adhere to endothelial cells and migrate to the inflammatory site (reviewed in Ref. 1). First, specific carbohydrate structures interact with selectins expressed by activated endothelium and allow them to slow down from the circulation in a rolling interaction with the substrate. In vitro flow chamber studies have addressed the relative importance for the three selectins, E-, P-, and L-selectin (2–5), in the rolling process of eosinophils to tumor necrosis factor (TNF)-{alpha}–activated endothelium. For eosinophils and monocytes also, the {alpha}4ß1 integrin–vascular cellular adhesion molecule (VCAM)-1 interaction plays a role in the initial adhesion (2). Firm adhesion is subsequently mediated by chemokine-induced activation of ß1- and ß2-integrins (6) that bind to their Ig-superfamily member counterstructures, VCAM-1 and intercellular adhesion molecule (ICAM)-1, respectively, on the endothelium. Finally, the cells spread and migrate to the site of inflammation.

Studies with animal models have addressed the importance of different adhesion molecules used by eosinophils to migrate to allergic inflammatory sites (reviewed in Ref. 7). A role for P-selectin was found by using P-selectin knockout mice in a ragweed allergen–induced peritonitis. The percentage of rolling eosinophils drastically decreased compared with control mice in the mesentery, as determined by intravital microscopy (8). In this study, eosinophil migration in the peritoneum was not only dependent on P-selectin, but also on ICAM-1 and VCAM-1. Remarkably, in a delayed onset allergic reaction in skin, both E-selectin and P-selectin were needed for eosinophil recruitment (9). Furthermore, {alpha}4- and ß2-integrins were found to play a role in eosinophil migration (10, 11). Also, a role for ICAM-1 was found (i) using ICAM-1 knockout mice in mice models of allergen challenge (12), and (ii) using blocking antibodies in a monkey model of asthma (13). Overall, the in vivo animal studies address the importance of the selectins (E- and P-selectin), the integrins ({alpha}4- and ß2-integrins), and the Ig superfamily members (VCAM-1 and ICAM-1) in homing of eosinophils to allergic inflammatory sites.

Studies showing that endothelial cells in biopsies of patients with allergic asthma after allergen challenge have increased expression of E-selectin, ICAM-1 (14), and VCAM-1 (15) confirm the importance of these molecules in eosinophils adhesion and migration in humans. Static adhesion experiments showed that eosinophils from patients with allergic asthma have increased adhesion to and transmigration through interleukin (IL)-1–stimulated human umbilical vein endothelial cells (HUVECs) (16). Another study showed that eosinophils from patients with asthma had an increased adhesion to VCAM-1 and ICAM-1, compared with eosinophils from healthy control subjects (17). Enhanced physiologic functions of eosinophils of patients with asthma, compared with healthy control subjects, are known to be a result of priming of the cells in vivo (18). Eosinophil priming in vivo can be mimicked in vitro by adding the cytokines IL-3, granulocyte-macrophage colony-stimulating factor, and particularly IL-5 to eosinophils of healthy control subjects. In an in vitro Boyden chamber assay, it was shown that eosinophils from patients with allergic asthma had an enhanced migration toward a platelet-activating factor (PAF) gradient, compared with healthy control subjects. Subsequently, priming of eosinophils from healthy control subjects with IL-3, IL-5, or granulocyte-macrophage colony-stimulating factor induced comparable enhanced migration, as was seen for the eosinophils from the patients (19).

Although it is well established how eosinophils interact with inflamed endothelium under static conditions, few studies have addressed the role of eosinophils from patients with allergic asthma under flow conditions. Sririmarao and coworkers (5) showed that eosinophils of patients with mild allergic asthma use L-selectin and very late antigen (VLA)-4 for rolling interactions with inflamed endothelium in a rabbit model. However, eosinophils from a nonasthmatic control group were not included in the study.

Thus far, it is unclear whether the primed phenotype of eosinophils in the peripheral blood from patients with allergic asthma will influence adhesion to endothelial cells under physiologic flow conditions. Therefore, we investigated whether eosinophils of patients with allergic asthma differ from eosinophils of healthy individuals in rolling and adhesive behavior on TNF-{alpha}-activated endothelial cells in an in vitro flow chamber model. We found an unexpected role for platelets contributing to the adhesion of eosinophils of patients with allergic asthma to inflamed endothelium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
CD16, CD14, and CD3 beads and isolation tools were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Human serum albumin (HSA) was obtained from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (Amsterdam, The Netherlands). Recombinant human TNF-{alpha} was purchased from Boehringer Mannheim (Mannheim, Germany). Incubation buffer contained 20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM KH2 PO4, supplemented with 5 mM glucose, 1.0 mM CaCl2, and 0.5% (wt/vol) HSA. All other materials were reagent grade.

Antibodies
The MAb HP2/1 (anti–VLA-4, CD49 d) was purchased from Immunotech (Marseille, France). Mab BBIG-E4 (5D11, anti–E-selectin, anti CD62e) was purchased from R&D systems (Abingdon, UK). MAb IB4 (anti–ß2-integrin) and WASP12.2 (anti–P-selectin) was isolated from the supernatant of a hybridoma obtained from the American Type Culture Collection (Rockville, MD). The MAbs as mentioned above are functionally blocking antibodies. Control antibody W6/32 (anti–HLA-A,B,C) was isolated from the supernatant of a hybridoma obtained from the American Type Culture Collection. Blocking MAbs were incubated with eosinophils (4 x 106 cells/ml) at 10 µg/ml during 15 min before the experiments. The cell suspensions were diluted once with incubation buffer (final concentration of 5 µg/ml MAb at 2 x 106 cells/ml in incubation buffer), and the coverslips were placed directly in the system. For fluorescence-activated cell sorter (FACS) analysis studies, the following antibodies were used: CD42/PE (clone AN51) and the negative control (mouse IgG1/PE) were purchased from DAKO (Glostrup, Denmark). BBIG-V1 (anti–VCAM-1) was purchased from R&D systems. RR1/1 (anti–ICAM-1) was kindly provided by prof. T. A. Springer (Center for Blood Research, Boston, MA). Goat anti-mouse fluorescein isothiocyanate was purchased from Becton Dickinson (Mountain View, CA).

Patients
Patients had documented allergy as shown by positive skin-test reactions to several allergens (including inhalation allergens such as house dust mite, pollen, and cat allergens), correlated with raised levels of specific IgE antibodies (Rast > 2). Patients had respiratory complaints and were examined for asthmatic symptoms. For this study patients were included according to the criteria of the American Thoracic Society (ATS, 1986). Briefly, patients were included who showed increased bronchial hyperresponsiveness to histamine and had a reversible lung function (FEV1) > 8% of the predicted value after the use of ß2-agonists. These patients used inhaled bronchodilators when needed (and some were receiving daily therapy with inhaled corticosteroids). The study was approved by the hospital ethics committee, and all patients gave informed consent before entering the study.

Isolation of Eosinophils
Blood was obtained from healthy volunteers anti-coagulated with 0.4% (wt/vol) trisodium citrate (pH 7.4) or by sodium heparin, and from patients with allergic asthma anti-coagulated by sodium heparin. Mixed granulocytes were isolated as described previously (20). Mononuclear cells were removed by centrifugation over isotonic Ficoll (1.077 g/ml). After lysis of the erythrocytes with an isotonic ice-cold NH4Cl solution, the granulocytes were washed and resuspended in isolation buffer. Eosinophils were purified from granulocytes by negative immunomagnetic selection using anti-CD16–conjugated microbeads (MACS; Miltenyi Biotec) (21). To avoid mononuclear cell contamination, anti-CD3– and anti-CD14–conjugated microbeads were also added to the granulocyte suspension. When anti–P-selectin was used in the perfusion experiments, anti–P-selectin antibodies (WASP12.2, 10 µg/ml) were also added to the beads suspension during purification. Purity of eosinophils was always > 95%.

Endothelial Cells
HUVECs were isolated from human umbilical cord veins according to the method of Jaffe and colleagues (22), with some minor modifications (23). The cells were cultured in RPMI 1640 containing 20% (vol/vol) heat-inactivated human serum, 200 µg/ml penicillin/streptomycin (GIBCO Life Technologies, Breda, The Netherlands) and fungizone (GIBCO Life Technologies). Cell monolayers were grown to confluence in 5–7 d. Endothelial cells of the second passage or third passage were used in perfusion assays. HUVEC was activated by TNF-{alpha} (100 U/ml, 5–7 h, 37°C) before the perfusion experiments.

Perfusion Chamber
Perfusions under steady flow were performed in a modified form of transparent parallel plate perfusion chamber as previously described by Van Zanten and coworkers (24). This micro-chamber has a slit height of 0.2 mm and width of 2 mm. The chamber contains a circular plug on which a coverslip (18 mm x 18 mm) with confluent HUVEC was mounted.

Eosinophil Perfusion and Evaluation
Eosinophil adhesion under flow conditions was performed as described before (2, 6). In short, eosinophils were aspirated from a reservoir through the perfusion chamber. Eosinophil perfusions were performed as individual runs under shear conditions by 37°C. During the perfusion, the flow chamber was mounted on a microscope stage (DM RXE; Leica, Weitzlar, Germany) which was equipped with a B/W CCD-video-camera (Sanyo, Osaka, Japan), coupled to a VHS video recorder. Perfusion experiments were recorded on video tape. Video images were evaluated for the number of adherent cells and cluster indices using dedicated routines made in the image-analysis software Optimas 6.1 (Media Cybernetics systems, Silver Springs, MD). The eosinophils that were in contact with the surface appeared as bright white-centered cells after proper adjustment of the microscope during recording. The adhering cells on the HUVEC were detected by the image analyzer. The eosinophil suspension was perfused during 5 min at shear stress 0.8 dyn/cm2. The number of surface-adherent eosinophils and cluster index (25) were measured after 5 min perfusion at a minimum of 25 randomized high power fields (total surface of at least 1 mm2). For each adherent cell in these 25 fields the number of cells in the surrounding area of ~ 1,750 µm2 was measured. In the case of a random distribution, the expected number of cells inside this area was calculated based upon the mean number of surface-adherent cells per mm2. The cluster index was set to be the difference between the measured and the expected number of cells inside an arbitrary area around the cell. In equation: Cluster index per cell = |m - (X · a/A - 1)|, in which m is the measured number of cells in the rectangle area, X is the total number of cells in the image, A is the size of the total image, and a is the size of a rectangular cell-surrounding area. Percentage of rolling was measured by the capture of a sequence of 50 frames representing an adjustable time interval ({Delta}t, with a minimal interval of 80 ms). At each frame, the position of every cell was detected, and for all subsequent frames the distance moved by each cell and the number of images in which a cell appears in focus was measured. The velocity of a cell (v) in µm/s was calculated from the equation: v = L/{delta}t(x - 1), in which L is the covered distance (µm), {delta}t is the time interval between images (s), and x is the number of images in which a cell appears. The cutoff value to distinguish between rolling and static adherent cells was set at 1 µm/s.

Flow Cytometry
HUVEC. Confluent HUVE cells were incubated in the presence or absence of TNF-{alpha} for 7 h. Cells were incubated with a HEPES buffer containing 5 mM EDTA (15 min, 4°C) to allow them to detach from the surface. Primary antibodies (WASP12.2; anti–P-selectin, R1/1; anti–ICAM-1, D6; negative control, BBIG-V1; anti-VCAM-1 and 5D11; anti–E-selectin) were incubated with the cells for 30 min (4°C). Cells were washed twice and then incubated with a secondary antibody (goat anti-mouse–fluorescein isothiocyanate). Cells were washed twice and fluorescence of 5,000 cells was measured on the FACS.

Eosinophil/platelet complexes. Whole blood was incubated with primary directly labeled antibodies against CD42 or a negative control. Erythrocytes were lysed in isotonic ice-cold NH4Cl solution for 15 min. Cell sample was washed and immediately measured on FACS. The granulocytes were gated according their forward and sideward scatter. From this granulocyte population, the eosinophils were gated on basis of their high scatter (26). The percentage of CD42-positive events in the eosinophil gate was calculated.

Statistical Analysis
Results are expressed as dot plots with mean values. Statistical analysis of the data was performed using the Student's t test and the Pearson correlation test. P values < 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased Adhesion of Eosinophils of Patients with Allergic Asthma Compared with Healthy Control Subjects
To investigate whether eosinophils from patients with allergic asthma showed increased adhesion to activated HUVEC compared with cells from healthy individuals, cells were perfused over activated HUVEC at shear stress 0.8 dyn/cm2 for 5 min in an in vitro flow chamber.

Figure 1A shows that 1240 ± 130 patient cells adhered to the surface, compared with 890 ± 90 control cells that did not reach significance (P = 0.059). The adhesion molecules responsible for capturing eosinophils from healthy individuals from the flow stream are {alpha}4-integrins on the eosinophils and E-selectin on TNF-{alpha}–activated endothelium, as determined by a study using blocking Moabs (2). To investigate whether these adhesion molecules mediated the increased adhesion of patient cells, blocking Moabs against {alpha}4 integrins and E-selectin were used. This resulted in 353 ± 64 cells/mm2 that remained adherent (Figure 1B). There was a significant correlation (Pearson correlation test, P = 0.023) between the amount of total adhesion of patients (Figure 1A, right panel) and the number of adherent cells of patients when {alpha}4- integrins and E-selectin were blocked (Figure 1B, right panel). Blocking both {alpha}4 integrins on the healthy control cells and E-selectin on the endothelium resulted in 123 ± 31 adherent cells/mm2 (Figure 1B). The latter was comparable with previous data (2) and differed significantly from the residual binding of patient cells (P < 0.02). These results show that in addition to {alpha}4 integrins and E-selectin, additional adhesive interactions mediate the increased adhesion of eosinophils of patients with allergic asthma to activated endothelial cells.



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  		Figure 1. Adhesion of eosinophils of control donors versus patients with allergic asthma on TNF-{alpha}–activated HUVEC under flow conditions. Cells were perfused during 5 min at shear rate 0.8 dyn/cm2, and then the amount of cells and the cluster index was determined. (A) The amount of adherent cells of control subjects (887 ± 94) versus patients (1237 ± 126). (B) Eosinophils were incubated with anti–{alpha}4-integrin Moab and HUVEC was incubated with anti–E-selectin Moab; number of adherent cells/mm2 is depicted. *P < 0.02.

 
Platelet-Derived P-Selectin and Activated ß2-Integrins Mediate Residual Binding of Eosinophils from Patients with Allergic Asthma
Remarkably, the residual adhesion of patient cells in the presence of blocking Moabs against {alpha}4-integrins and E-selectin was characterized by clusters in marked contrast to healthy control cells (Figures 2A and 2B, respectively). Indeed, when this clustering was expressed as index for nonrandom adhesion (see MATERIALS AND METHODS), patient cells showed a significant increase in the cluster index as compared with control subjects (1.8 ± 0.3 versus 0.8 ± 0.2, respectively, P < 0.05; Figure 2C). Furthermore, it was clear that patient cells interacted with platelets forming clear eosinophil–platelet rosettes on the endothelial surface (Figure 3A). The eosinophil–platelet interactions participated in the formation of flow orientated strings of clustered cells, also known as secondary tethering. This was not seen with control cells (Figure 3B). Also, many platelets showed transient interactions with adherent eosinophils, suggesting that the interaction between eosinophils and platelets was short-term with a low affinity. This is typical for the molecules within the selectin family (Figure 3C). Because the platelet–eosinophil interactions occurred in the in vitro flow chamber, we tested the hypothesis if this interaction was already present in whole blood. For this purpose, total leukocytes were stained for a platelet marker (CD42). The percentage of CD42-positive eosinophils was counted by FACS analyses. For both groups, the percentage of platelet positive eosinophils was low and similar (7.4 ± 4.5% and 7.1 ± 2.4% for healthy control subjects and patients, respectively).



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  		Figure 2. Cluster index of eosinophils of patients with allergic asthma versus control donors on TNF-{alpha}–activated HUVEC under flow conditions. When the adhesion molecules {alpha}4-integrins on eosinophils and E-selectin on the endothelium were blocked, cells of patients but not of healthy control subjects clustered on the endothelium. (A) A nonrandom, clustered distribution of eosinophils of patients with allergic asthma. (B) A random distribution of eosinophils of control subjects. (C) Quantified cluster indices of control subjects versus patients with asthma. Unpaired Student's t test, *P < 0.05.

 


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  		Figure 3. (A) Eosinophils of patients with allergic asthma, and (B) of healthy control subjects, were perfused on activated HUVEC cells. Platelets are present on the eosinophils of patients but not of healthy control subjects. Platelets are indicated by arrows. (C) Transient platelet–eosinophil interaction is shown. Platelet is positioned in the circle. In time, the platelet tethers to the already bound eosinophils.

 
Platelets, when activated, express P-selectin. This adhesion molecule has been shown to mediate initial tethering of leukocytes to platelets that are present and activated at injured vessel wall models (27, 28). Moreover, binding of platelets to monocytes in circulation has been shown to mediate rolling on activated HUVEC (29). The influence of platelet-decorated leukocytes (in this case eosinophils) on adhesion in the context of clustering behavior has not been studied before. This secondary tethering process results in cells that cluster on the endothelial surface. To investigate if increased adhesion of eosinophils from patients is due to P-selectin that is expressed by adherent platelets, cells were isolated and perfused in the presence of a blocking anti–P-selectin monoclonal antibody (WASP12.2). Figure 4A depicts the total amount of adherent eosinophils from patients and control individuals treated with anti–P-selectin Moabs. Where the anti–P-selectin treatment did not significantly effect eosinophil adhesion from healthy control subjects (in the absence or presence of anti–P-selectin treatment the total adhesion was 887 ± 94 and 665 ± 121, respectively), the treatment did inhibit the adhesion of patient eosinophils by 50% from 1,237 ± 126 to 627 ± 166; the latter value being comparable to adhesion of control eosinophils. Additionally, when combining anti–P-selectin Moabs, anti–{alpha}4-integrin Moabs, and anti–E-selectin Moabs, the number of adherent cells/mm2 of control subjects and patients became comparable (94 ± 26 and 89 ± 44, respectively; Figure 4B). The mix of anti–{alpha}4-integrin Moab, anti–P-selectin Moab, and anti–E-selectin Moab (Figure 4C) in perfusions with patient cells resulted in a cluster index of 0.24 ± 0.07, which was significantly different from 1.8 ± 0.3 when anti–P-selectin Moab was omitted from the Moab mix (Figure 2C). However, the additional presence of anti–P-selectin Moab, besides blocking {alpha}4-integrins and E-selectin, in healthy individuals did not result in a significant change in the cluster index (0.58 ± 0.25 versus 0.78 ± 0.2). To show that P-selectin was absent on 7 h TNF-{alpha}–activated HUVEC, we performed flow cytometry experiments. Figure 5 shows that P-selectin was not present on 7 h TNF-{alpha}–activated HUVEC, whereas E-selectin, ICAM-1, and VCAM-1 were. Together, these results show that P-selectin–expressing platelets on eosinophils of patients are responsible for this phenomenon. Second, upon inhibition of platelet function by blocking anti–P-selectin antibodies, the phenotype of increased adhesion and cluster index was abrogated.




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  		Figure 4. Adhesion behavior of eosinophils of control subjects versus patients with allergic asthma. Data of Figures 1 and 2C are incorporated in this figure for easy comparison. (A) The amount of adherent eosinophils/mm2 in the presence or absence of P-selectin and in the presence of a control Moab W6/32. (B) The number of adherent cells/mm2 in the absence or presence of anti–P-selectin antibodies when E-selectin on the endothelium and {alpha}4-integrins in the eosinophil suspension were blocked. (C) The cluster index.

 


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  		Figure 5. Flow cytometry profiles of unstimulated (A) and 7 h TNF-{alpha}–stimulated HUVEC (B) are shown. HUVECs were stained for P-selectin (grey line), ICAM-1 (dotted line), VCAM-1 (dashed-dotted line) and E-selectin (dashed line). The negative control (D6) is shown by the black line.

 
In the presence of the mix of blocking Moabs against {alpha}4-integrins, E-selectin and P-selectin eosinophils still adhered to activated HUVEC (94 ± 26 cells/mm2 from healthy control subjects and 89 ± 44 cells/mm2 of the patients; Figure 4B). When, in addition, ß2-integrins were inhibited, both for cells of patients and for those of healthy control subjects, the number of adherent cells decreased to 22 ± 8 cells/mm2 of healthy contol subjects and 12 ± 2 cells/mm2 of patients (Figure 6, panel with P-selectin treatment).



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  		Figure 6. Adhesion behavior of eosinophils of control subjects versus patients with allergic asthma when E-selectin on the endothelium and {alpha}4- and ß2-integrins are blocked in the eosinophil suspension in the presence or absence of anti–P-selectin Moabs. Number of adherent cells/mm2 is depicted.

 
When P-selectin was not inhibited but {alpha}4-integrins and E-selectin were inhibited, we showed that 123 ± 31 cells/mm2 of healthy control subjects and 353 ± 64 cells/mm2 of patients remained adhered (Figure 1B). The question of whether this residual binding is mediated by ß2-integrins was addressed. Figure 6 shows that in the presence of blocking Moabs against ß2–integrins, in addition to blocking Moabs against {alpha}4-integrins and E-selectin the residual binding of both healthy control subjects and patients decreased to 36 ± 16 and 75 ± 27 cells/mm2, respectively. Thus, to reduce virtually all adhesion in cells of patients we need blocking of at least E-selectin, {alpha}4-integrins, and ß2-integrins. However, anti–P-selectin Moabs can further decrease the number of adherent cells of patients on the endothelium. Most likely, P-selectin mediates tethering processes of eosinophil/platelet conjugates of patients to the endothelium and ß2-integrins mediate firm adhesion.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we show that eosinophils of patients with allergic asthma have an augmented adhesion to TNF-{alpha}–activated HUVEC under flow conditions, compared with eosinophils from healthy control subjects. Three observations suggest that platelets that bound to eosinophils of patients with asthma caused the increase in adhesion as compared with eosinophils of healthy control subjects: (i) platelets interacted with eosinophils of patients but not of healthy individuals in the in vitro flow chamber on a HUVEC surface; (ii) increased adhesion of patient eosinophils to activated HUVEC was prevented by treatment with anti–P-selectin Moabs; and (iii) the formation of cell clusters of patient eosinophils on activated HUVEC was blocked by anti–P-selectin treatment. In Figure 7, a model is depicted that illustrates the consequences of the presence of platelets on eosinophils of patients with allergic asthma but not of healthy control subjects in the in vitro flow chamber.



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  		Figure 7. Model for platelet-mediated secondary clustering of eosinophils of patients with allergic asthma. (A) Eosinophils of healthy control subjects do not bind platelets. Adhesion to the endothelium is mediated via primary tethering processes. (B) Eosinophils of patients with allergic asthma bind platelets. Platelet-derived P-selectin–dependent secondary tethering processes increase the number of cells that are recruited to the surface.

 
It is known that eosinophils can bind P-selectin very well, especially under flow conditions. Recently, Woltmann and colleagues showed that eosinophils, but not neutrophils, rolled on P-selectin–expressing HUVEC cells (30). In that study, the asthma-related Th2 cytokines IL-13 and IL-4 induced the P-selectin on the endothelium. We stimulated endothelial cells with TNF-{alpha}, which is also a cytokine made by inflammatory cells in allergic sites. Our TNF-{alpha}–activated HUVEC (passage 3) did not express P-selectin (Figure 5). Moreover, we have never observed P-selectin–mediated rolling of eosinophils on TNF-{alpha}–activated HUVEC (2). Therefore, the effect of anti–P-selectin in our studies is most likely caused by platelet-derived P-selectin and not by endothelial-derived P-selectin.

For leukocytes other than eosinophils, an important role for platelets has already been established. Neutrophils and monocytes are known to bind platelets on damaged vessel wall models in a P-selectin–dependent manner. Moreover, circulating complexes of leukocytes with platelets on their membrane have been implicated in the pathogenesis of atherosclerosis (27, 28; for review see Ref. 31). However, platelets are also regarded to play an important role in allergic asthmatic diseases (reviewed in Ref. 32). Wardlaw and coworkers showed that eosinophils of patients with allergic asthma were positive for ß3-integrins. However, these were not ß3-integrins expressed on the eosinophils but on platelets that bound very tightly to the eosinophils. Electron microscopy demonstrated this tight interaction (33). Also, platelets have been shown to be in contact with the vasculature of the bronchi of patients with asthma, but not those of healthy control subjects (34). Another study showed that inhalation of PAF-acether in a baboon model of asthma increased the amount of platelets in the pulmonary vasculature in contrast to the periphery. This increase in platelets preceded the increase in eosinophils (35). Furthermore, in a rabbit model of allergen-induced late asthmatic airway obstruction, Coyle and colleagues showed that platelet depletion by injecting anti-platelet serum reduced the eosinophil infiltration in the bronchoalveolar lavage 24 h after allergen challenge and bronchohyperresponsiveness (36). Also, the release of platelet-derived secretion products in individuals with allergic asthma has been reported (37). In this respect, although not consistently, platelet factor 4 (PF4) has been detected in the plasma of patients with allergic asthma, but not in the plasma of healthy control subjects. For secreting PF4, platelets have to be activated by stimuli like collagen, thrombin, and adenosine diphosphate. Thus, a correlation between activated platelets and eosinophil infiltration exists. In this study, we additionally suggest that P-selectin–bearing platelets associated with eosinophils could be a mechanism for an increased recruitment of these cells on the stimulated vessel wall.

Under physiologic flow conditions, anti–P-selectin Moabs reduced both the total adhesion and the cluster index of eosinophils to activated endothelial cells. Cluster formation occurs in several ways: (i) already adherent eosinophil–platelet complexes serve as secondary tethering platforms recruiting fast-flowing cells; (ii) free flowing eosinophil–platelet complexes get recruited to eosinophils already adhered to the endothelial surface. Even transient interactions of free-flowing platelets with eosinophils will facilitate both these mechanisms and increase the eosinophil recruitment to the surface. We conclude that P-selectin at least mediates the interactions between eosinophils and platelets, because anti–P-selectin Moabs not only blocked the enhanced adhesion but also the clustering of the cells. Indeed, it has been shown by FACS analyses that activated platelets bind to eosinophils in a P-selectin–dependent manner (38). In this study, isolated eosinophils of healthy subjects were incubated with 10 times as many platelets, and 13% of the eosinophils bound unactivated platelets as determined by FACS analyses. When platelets were activated with thrombin, the percentage of platelet-positive eosinophils increased to 90%. In agreement with this, we found eosinophil–platelet complexes in whole blood. However, no difference was found in the percentage of eosinophils from healthy control subjects and patients that bound platelets (7.4 and 7.1%, respectively). Thus, both preparations showed low amounts of bound platelets in the flow cytometer. This indicates that relatively few platelets can bind with high affinity to eosinophils under conditions of high shear (as found in the sample line of the flow cytometer). However, we observed in our in vitro flow chamber experiments transient and probably low-affinity interactions between platelets and eosinophils (Figure 3C). We hypothesize that these low-affinity interactions between platelets and eosinophils cannot be measured in the FACS. Therefore, new tools should be developed to study low-affinity interactions between platelets and leukocytes in vivo.

Besides the difference in platelet binding, the ß2-integrins expressed by eosinophils in the patient group seemed slightly activated. Whereas in the case of inhibited E-selectin and {alpha}4-integrins, patient adhesion and clustering is increased, additional inhibition of ß2-integrins reduced both patient and control adhesion values to ~ 5% of the control situation. The presence or absence of anti–P-selectin Moabs (Figure 6) under the latter condition makes no additional difference. One might state that the activated ß2-integrins on eosinophils of patients with allergic asthma allows the increase in P-selectin clustering to become clear. In agreement with this, a study performed under static conditions showed that eosinophils from patients who were symptomatic (with a peak expiratory flow rate variability of > 10%) had an increased adhesion to the ß2-integrin ligand ICAM-1 compared with nonsymptomatic patients (with a peak expiratory flow variability of < 10%) (17). The same group showed that eosinophils from patients allergic to birch pollen were primed in the adhesion to VCAM-1 and ICAM-1 during birch pollen season, but not before this season (39). This suggests that only during pollen season, when these patients have complaints, conditions for increased adhesion are present. Similarly, all patients in our study had complaints at the moment of drawing blood. Thus, it seems that eosinophils of symptomatic patients with allergic asthma have increased adhesion to endothelial adhesion molecules.

The transient platelet binding to eosinophils of patients with allergic asthma might be due to (i) platelet activation, or (ii) the primed phenotype of eosinophils (18). Many effector functions of eosinophils like respiratory burst (40), production of lipid mediators, degranulation, and migration (16) are sensitive for priming. The priming agents PAF and IL-5 can increase the affinity of ß2-integrins on eosinophils. This might subsequently lead to the increased binding to platelets. We found a minor role for activated ß2-integrins in this study that could be due to priming of the eosinophils in vivo.

In summary, platelets are prone to bind eosinophils of patients with allergic asthma, a phenomenon that might influence secondary tethering and thereby eosinophil adhesion and migration to inflammatory sites. These processes may contribute to the increased eosinophils found in lung tissue in patients with allergic asthma.


   Acknowledgments
 
This study was supported by the Netherlands Asthma Foundation, project number 96.49. The authors thank Dr. J. A. M. van der Linden for developing the image analyses programs in Optimas 6.1, and Glenda Heynen for growing the HUVEC cells.

Received in original form December 27, 2001

Received in final form October 3, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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