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(Journal of Leukocyte Biology. 2002;72:702-710.)
© 2002 by Society for Leukocyte Biology

Increased PSGL-1 expression on granulocytes from allergic-asthmatic subjects results in enhanced leukocyte recruitment under flow conditions

Bao Dang*, Shahina Wiehler{dagger} and Kamala D. Patel*,{dagger}

Departments of
* Physiology and Biophysics and
{dagger} Biochemistry and Molecular Biology, Immunology Research Group, University of Calgary, Calgary, Alberta, Canada

Correspondence: Dr. K. D. Patel, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr., N.W., Calgary, Alberta, Canada T2N 4N1. E-mail: kpatel{at}ucalgary.ca


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ABSTRACT
 
Allergic asthma is increasing in incidence and severity in many industrial countries. Leukocyte recruitment into the airways of affected individuals contributes to the severity of the disease. In this study, whole blood from normal, allergic, asthmatic, or allergic-asthmatic subjects was perfused over immobilized adhesion molecules using an in vitro flow chamber system to determine if there were differences in leukocyte recruitment in these patient populations. Leukocytes from allergic-asthmatic subjects showed a threefold increase in recruitment on P-selectin as compared with normal controls. In both patient populations, the accumulated cells were exclusively neutrophils and eosinophils. Increased granulocyte recruitment was specific for P-selectin, as neither purified E-selectin nor vascular cell adhesion molecule-1 (VCAM-1) supported enhanced leukocyte recruitment from allergic-asthmatics. Leukocyte accumulation on P-selectin was completely blocked by an anti-P-selectin or anti-P-selectin glycoprotein ligand-1 (PSGL-1) monoclonal antibody. Flow cytometry revealed that neutrophils and eosinophils from allergic-asthmatic subjects had increased expression of PSGL-1, whereas expression of another adhesion molecule, L-selectin, was unchanged. PSGL-1 expression on peripheral blood mononuclear cells of allergic-asthmatic patients was unaffected. The increased PSGL-1 expression on granulocytes from allergic-asthmatic patients also led to enhanced leukocyte recruitment on interleukin-4-stimulated human umbilical vein endothelial cells, which express P-selectin and VCAM-1. Thus, increased PSGL-1 expression on granulocytes from allergic-asthmatic subjects resulted in increased leukocyte recruitment on P-selectin under flow conditions.

Key Words: selectins • rolling • endothelial cells • adhesion


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INTRODUCTION
 
Asthma is an inflammatory disease characterized by leukocyte infiltration, mucus hypersecretion, epithelial cell shedding, and bronchoconstriction, resulting in airway hyper-responsiveness [1 , 2 ]. Leukocytes that have infiltrated into the bronchial mucosa are thought to participate in the acute and chronic aspects of the disease [1 , 2 ]. Traditionally, investigators have focused on eosinophils and lymphocytes in asthma. Infiltrating lymphocytes release cytokines such as interleukin (IL)-4 and IL-5, which contribute to the chronic inflammation observed in asthma [3 ]. Eosinophils are primarily implicated in the tissue damage characteristic of asthma as a result of the release of cationic proteins, such as major basic protein and eosinophil cationic protein [4 , 5 ]. The potential role of neutrophils in this disease has largely been ignored. However, recent evidence suggests that neutrophils may indeed participate in asthma, particularly in more severe forms of the disease [6 , 7 ]. Thus, understanding the mechanisms that regulate the infiltration of all these leukocyte subclasses is critical.

Leukocyte trafficking proceeds in a multistep manner that is initiated by leukocyte tethering and rolling along the endothelium. These rolling interactions occur in post-capillary venules under hydrodynamic, shear conditions and require specialized adhesive mechanisms [8 , 9 ]. Leukocyte tethering and rolling are mediated predominately by the selectin family of adhesion proteins [8 ], as has been demonstrated in vivo and in vitro [10 11 12 ]. The selectins mediate tethering and rolling by interacting with sialylated, fucosylated, and sometimes sulfated carbohydrate structures present on ligands, such as P-selectin glycoprotein ligand-1 (PSGL-1), CD34, and GlyCAM-1 [13 , 14 ]. The {alpha}4-integrins ({alpha}4ß1 and {alpha}4ß7) and {alpha}Dß2 can also mediate tethering and rolling by interacting with their respective immunoglobulin (Ig) superfamily ligands, such as vascular cell adhesion molecule-1 (VCAM-1) [15 , 16 ] and mucosal addressin cell adhesion molecule-1 [17 , 18 ].

Adhesion molecule expression on endothelial cells and circulating leukocytes is regulated. Under baseline conditions, endothelial cells do not support tethering or rolling of leukocytes. In response to inflammatory stimuli such as cytokines or histamine, endothelial cells up-regulate an array of adhesion molecules, promoting leukocyte rolling and adhesion [8 , 9 ]. Circulating leukocytes can also undergo functional changes during inflammatory states. Ibbotson et al. [19 ] recently demonstrated that circulating neutrophils from septic patients have increased expression of {alpha}4ß1, which enables them to tether and adhere to VCAM-1 [19 ]. Other studies have shown elevated levels of CD11a/CD18 (lymphocyte function-associated antigen-1, {alpha}Lß2) but not CD11b/CD18 [macrophage antigen-1 (MAC-1), {alpha}Mß2] or {alpha}4ß1 on peripheral blood eosinophils from allergic-asthmatic patients [20 ]. In rheumatoid arthritis, there is increased MAC-1 expression on eosinophils and neutrophils from the peripheral blood [21 ]. These are a few examples of data showing that modified adhesion molecule expression on endothelial cells and circulating leukocytes during specific disease states can contribute to increased tissue recruitment.

In this study, we determined whether leukocytes from normal and allergic-asthmatic donors demonstrated functional differences in their leukocyte trafficking. We used an in vitro flow chamber system in which whole blood was perfused over immobilized adhesion molecules and/or IL-4-stimulated human umbilical vein endothelial cells (HUVEC) under flow conditions [22 , 23 ]. Following perfusion, the number of accumulated cells was quantified, then the plate was disassembled, and the accumulated cells were identified. This system has the advantage of observing interactions of all leukocytes in the context of the whole blood with a minimum degree of manipulation. We found that granulocytes but not peripheral blood mononuclear cells (PBMCs) from allergic-asthmatic subjects demonstrated enhanced rolling on P-selectin but not E-selectin or VCAM-1. Flow cytometry revealed that PSGL-1 expression was increased on granulocytes from allergic-asthmatic subjects. Consistent with this, leukocyte rolling could be blocked with antibodies directed against P-selectin or PSGL-1, suggesting that the enhanced rolling of granulocytes from allergic-asthmatic patients was a result of interactions between P-selectin and its ligand PSGL-1. These data suggest that up-regulation of PSGL-1 on granulocytes could serve to increase leukocyte trafficking in allergic asthma.


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MATERIALS AND METHODS
 
Proteins and antibodies
Anti-PSGL-1 antibodies (PL1 and PL2), anti-P-selectin antibody (G1), and soluble recombinant P-selectin protein (sPS) were kindly provided by Dr. Rodger McEver (University of Oklahoma, Oklahoma City). Phycoerythrin (PE)-conjugated anti-PSGL-1 (KPL-1) was from BD Biosciences (San Diego, CA). Anti-CD18 monoclonal antibody (mAb; 7E4), fluorescein isothiocyanate (FITC)-conjugated anti-CD11b (VIM12), FITC-conjugated anti-L-selectin (FMC46), and FITC- and PE-conjugated isotype control antibodies were purchased from Serotec (Oxford, England). Membrane P-selectin (mPS) was purified from human platelets as described [24 ]. Soluble recombinant human VCAM-1, soluble recombinant human E-selectin, and human IL-4 were purchased from R&D Systems (Minneapolis, MN).

Subjects
The subjects used in this study were classified as normal, allergic, asthmatic, or allergic-asthmatic in collaboration with Dr. Robert Cowie, Dr. Stephen Field, and Margot Underwood at the Calgary Asthma Clinic (Alberta, Canada). A questionnaire was administered to all participants to identify a history of allergic disease and/or asthma. Thirteen normal subjects (age 36.8±8.8; one male), nine allergic subjects with no evidence of asthma (age 41.1±9.3; two males), seven asthmatic subjects with no evidence of allergy (age 52.4 ± 12.2; no males), and 13 allergic-asthmatic subjects (age 33.9±8.8; five males) were used. All subjects were nonsmokers or ex-smokers that had discontinued smoking at least 1 year before inclusion in the study. All asthmatic subjects met the criteria for asthma as defined by the American Thoracic Society [25 ], as demonstrated by spirometry, with an increased forced expiratory volume in 1 s by 12% after inhalation of short-acting ß-agonists or hyper-responsiveness following bronchoprovocation using methacholine (PC20 of <8 mg/ml). All asthmatic subjects were using ß2-agonists on an as-needed basis only and/or were using <1000 µg/d inhaled glucocorticoids. All other medications, including antihistamines, were discontinued at least 1 week before the study. Allergy was determined by positive reaction (wheal of >3 mm) using skin-prick tests for several common allergens including dust mite, grass and tree pollens, and cat dander. The Research Ethics Board at the University of Calgary (Alberta, Canada) granted approval for this study, and informed consent was obtained from all subjects.

Cell preparation and isolation
Blood from normal and allergic-asthmatic donors was drawn into heparinized syringes and used immediately in rolling experiments as previously described [22 , 23 ]. White blood cell (WBC) counts and leukocyte differentials were performed on all patients (Table 1 ). First passage HUVEC were isolated as described [26 ].


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  		Table 1. White Blood Cell Counts and Leukocyte Differentials from Patient Populationsa

Preparation of rolling substrates
Adhesion molecules were immobilized on culture dishes as previously described [27 ]. Briefly, P-selectin (mPS or sPS), E-selectin, or VCAM-1 was diluted to the specified concentrations in Hanks’ balanced saline solution (HBSS) and was absorbed onto 35-mm tissue culture plates (Corning, Corning, NY) for at least 2 h at 37°C. The plates were then washed four times with HBSS and blocked overnight with HBSS containing 1% human serum albumin at 4°C. Plates were brought to 37°C prior to assembly of the flow chamber.

Several forms of P-selectin can be used to create substrates for leukocyte binding, including sPS and mPS, purified from human platelets. mPS still contains the transmembrane domain of the protein, whereas sPS is a recombinant form of P-selectin in which the transmembrane domain has been deleted [28 ]. Although sPS and mPS bind leukocytes, we have previously reported that equivalent coating concentrations of sPS and mPS do not result in equivalent functional expression of P-selectin [27 ]. In particular, mPS is required to immobilize proteins at high-site densities on culture dishes [27 ]. Thus, to examine leukocyte binding at high-site densities, we chose to use mPS.

Adhesion under flow conditions
Leukocyte interactions under flow conditions were determined as described [22 , 23 ]. Briefly, freshly drawn, heparinized whole blood was perfused through the flow chamber at the specified shear rate for 4 min, followed by perfusion of HBSS as described. Accumulated cells were visualized at 100x and 200x magnifications using bright field optics and recorded onto videotape via a CCD camera (Hitachi Denshi, Ltd., Tokyo, Japan) attached to an inverted microscope (Zeiss Canada, Don Mills, Ontario, Canada) as described. Accumulated leukocytes were quantified by recording multiple fields and counting the cells present in the visual field. P-selectin and E-selectin surfaces supported leukocyte rolling only, whereas VCAM-1 and IL-4-stimulated HUVEC supported rolling and firm adhesion as previously reported. Rolling velocities were calculated capturing images 10 s apart and then measuring the distance traveled by leukocytes during the 10 s. In some experiments, whole blood was preincubated for 10 min with anti-PSGL-1 antibodies (10 µg/ml) prior to perfusion through the chamber. Alternatively, culture dishes were pretreated for 10 min with an anti-P-selectin antibody (10 µg/ml) prior to assembly of the flow chamber. Antibodies were maintained in the perfusate. Leukocytes bound to the plate were identified by disassembling the flow chamber and staining the accumulated cells with Wright-Giemsa. At least 200 cells were identified and counted. The percentage of each leukocyte subclass bound on the plate was divided by the total percentage of each leukocyte subclass in the whole blood differential to determine the recruitment factor (R-factor). A R-factor of 1 would indicate no selective recruitment.

Flow cytometry
Blood from allergic-asthmatic and control donors was collected. Flow cytometry was performed on the whole blood. Blood (100 µl) was mixed with 100 µl phosphate-buffered saline (PBS), and L-selectin-FITC, PSGL-1-PE, CD11b-FITC, or directly conjugated isotype control antibodies were added. CD16-TriColor was used in all cases to distinguish between eosinophils and neutrophils. After 20 min at room temperature, CalLyse (Caltag, distributed by Cedar Lane, Hornby, Ontario) was used according to the manufacturer’s instructions to fix the WBC and lyse the red blood cells. The cells were then spun and resuspended in PBS for fluorescence-activated cell sorter (FACS) analysis. Granulocytes, lymphocytes, and monocytes were identified by forward- and side-scatter properties. Eosinophils were differentiated from neutrophils based on staining with anti-CD16-TriColor. Data were acquired using a Becton Dickinson FACScan flow cytometer, and data were analyzed using FlowJo software (Mountain View, CA).

Statistics
All experiments were performed at least five times using a minimum of three independent donors from the specified patient groups. The data were analyzed using a Student’s t-test or using a Mann-Whitney U-test. P values < 0.05 were considered significant.


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RESULTS
 
Leukocytes from allergic-asthmatic subjects demonstrate enhanced rolling on P-selectin
In this study, we used a whole blood recruitment assay to determine if peripheral blood leukocytes from allergic-asthmatic patients demonstrated differences in the ability to bind to vascular adhesion molecules. Blood was obtained from normal, allergic, asthmatic, or allergic-asthmatic subjects, and WBC counts and leukocyte differentials were performed (Table 1) . Blood was then perfused over immobilized P-selectin. We found that leukocytes from all patients interacted with P-selectin (Fig. 1A ); however, there was a threefold increase in the total number of leukocytes from allergic-asthmatic subjects accumulating on P-selectin (Fig. 1A) . Subjects with allergies or asthma alone showed no difference in leukocyte recruitment compared with normal subjects; thus, only normal and allergic-asthmatic subjects were compared in subsequent studies. We next examined the effect of shear stress and P-selectin site density on leukocyte recruitment. Elevated leukocyte recruitment from allergic-asthmatic subjects was observed at all of the shear rates examined (Fig. 1B) and was more evident at higher concentrations of P-selectin (Fig. 1C) . Thus, leukocytes from allergic-asthmatics showed enhanced binding to P-selectin under flow conditions.



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  		Figure 1. Leukocytes from allergic-asthmatic donors demonstrate enhanced rolling on P-selectin. (A) Blood was collected from normal, allergic, asthmatic, or allergic-asthmatic subjects and perfused through the flow chamber at a shear rate of 200 s-1 over P-selectin (0.6 µg/ml sPS) immobilized on 35-mm dishes as described in Materials and Methods. (B) Alternatively, blood from normal or allergic-asthmatic subjects was perfused over (B) 0.6 µg/ml sPS at the specified shear rates or (C) mPS at the specified concentrations. In all cases, blood was perfused for 4 min, after which time the blood was chased with HBSS and the leukocytes were visualized and counted. The data represent the mean ± SEM. *P < 0.01; ns = not significant.

Leukocytes from allergic-asthmatic subjects roll slower on P-selectin than leukocytes from normals
Increased binding is often associated with a change in the rolling velocity, which is indicative of increased adhesive strength; thus, we next examined the rolling velocity of leukocytes from normal and allergic-asthmatic patients on P-selectin. We found that cells from normal subjects rolled as a single population with an average velocity of 32.9 ± 2.23 µm/s (n=167; Fig. 2 ). When rolling velocities of leukocytes from allergic-asthmatic subjects were examined, at least two populations were observed (Fig. 2) : one with a mean velocity of 1.82 ± 0.56 µm/s (n=56) and the other with a mean velocity of 46.82 ± 2.49 µm/s (n=117). The slow-rolling population was nearly stationary at 200 s-1; however, the cells did not flatten or change shape, suggesting that they had not been activated. In addition, an anti-ß2-integrin mAb did not alter the rolling profile of these cells, and an anti-P-selectin mAb could detach the bound cells (data not shown). These data suggest that the leukocytes were rolling very slowly on P-selectin, and their integrins had not been activated.



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  		Figure 2. Leukocytes from allergic-asthmatics roll slower on P-selectin than those from normal subjects. An intermediate concentration of mPS (2.5 µg/ml) was immobilized on 35-mm plates, and whole blood from normal or allergic-asthmatic subjects was perfused through the flow chamber at a shear rate of 200 s-1. After 4 min of perfusion, blood was chased with HBSS, and the interacting cells were visualized. Leukocyte-rolling velocities were determined by measuring the distance traveled by each leukocyte during a 10-s interval. The data represent the velocities of >150 cells from four experiments with independent donors.

Only granulocytes from allergic-asthmatic subjects demonstrate enhanced accumulation on P-selectin under flow conditions
Reinhardt and Kubes [22 ] have previously shown that P-selectin supports the binding of predominately granulocytes (neutrophils and eosinophils) from normal subjects. Our data also showed that neutrophils and eosinophils were the primary cells recruited on P-selectin from all of the patient populations examined (Fig. 3 and data not shown). To examine the degree of selective recruitment, the percentage of each cell type bound to the plate was divided by the percentage found in the peripheral blood to give a R-factor. A R-factor of 1 indicates that there is no selectivity for that particular cell type, and a R-factor greater than 1 indicates selectivity for a particular cell type. Eosinophils and neutrophils showed an increased R-factor in both patient populations, indicating that there is selective recruitment of these cell types on P-selectin (Fig. 3D) . Eosinophils showed a greater R-factor than neutrophils at virtually all of the concentrations examined (Fig. 3D) . In addition, when the ratio of eosinophils to neutrophils was directly compared, we found a ratio of 1:25 in the peripheral blood but a ratio of 1:11.5 on immobilized P-selectin. This was observed with normal and allergic-asthmatic patients. Thus, eosinophils are selectively recruited on surfaces expressing P-selectin, but this selectivity is not based on patient disease status.



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  		Figure 3. Granulocytes are preferentially recruited on P-selectin. Whole blood from normal or allergic-asthmatic subjects was perfused over the specified concentration of immobilized mPS or sPS at a shear rate of 200 s-1. After 4 min, the blood was chased with HBSS, and the accumulated cells were counted as descried in Materials and Methods. (A–C) The percentage of each leukocyte subclass bound on the plate was determined. (D) The percentage of each leukocyte subclass bound on the plate was divided by the total percentage of each leukocyte subclass in the whole blood differential to determine the R-factor. A R-factor of 1 would indicate no selective recruitment. The data represent the mean ± SEM.

Leukocytes from allergic-asthmatic donors do not show enhanced accumulation on E-selectin or VCAM-1
As E-selectin and VCAM-1 are also expressed on activated endothelial cells and support leukocyte tethering and rolling [17 , 29 ], we determined if leukocytes from allergic-asthmatics would demonstrate enhanced accumulation on these other adhesion molecules. Whole blood from normal or allergic-asthmatic subjects was perfused over immobilized E-selectin or VCAM-1, and leukocyte interactions were observed. We found that there was no difference in leukocyte recruitment between these patient populations on either of these proteins (Fig. 4A and 4C ). We also examined the leukocyte subclasses recruited on these surfaces. E-selectin bound exclusively neutrophils, whereas VCAM-1 bound predominately eosinophils and lymphocytes (Fig. 4B and 4D) . In both cases, the leukocyte subclasess recruited were unaffected by the nature of the donor population (Fig. 4B and 4D) . These data suggest that enhanced adhesion to P-selectin was specific and did not represent a global increase in granulocyte adhesiveness.



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  		Figure 4. Leukocytes from allergic-asthmatic donors do not show enhanced accumulation on E-selectin or VCAM-1. (A and B) E-selectin or (C and D) VCAM-1 was immobilized on 35-mm plates at the specified concentrations. (A and C) Whole blood from normal or allergic-asthmatic donors was perfused through the flow chamber at a shear rate of 200 s-1. After 4 min, the blood was chased with HBSS, and the accumulated cells were visualized as described in Materials and Methods. (B and D) Plates were removed from the flow chamber, and the leukocytes were identified as described in Materials and Methods. The data represent the mean ± SEM.

All leukocyte recruitment is mediated by PSGL-1
Enhanced accumulation of allergic-asthmatic leukocytes on P-selectin could be a result of several factors, including increased PSGL-1 expression, altered PSGL-1 function, or the expression of a novel P-selectin ligand. We addressed the role of PSGL-1 in granulocyte rolling and accumulation on P-selectin by using neutralizing antibodies. We used anti-P-selectin or anti-PSGL-1 mAb to block leukocyte accumulation on immobilized P-selectin. As expected, both antibodies blocked accumulation of normal leukocytes on immobilized P-selectin (data not shown). Neutralizing antibodies directed against P-selectin or PSGL-1 were also able to completely block leukocyte accumulation on P-selectin when allergic-asthmatic subjects were used (Fig. 5 ). These data suggest that interactions between leukocytes from allergic-asthmatic subjects and P-selectin were mediated by PSGL-1 under flow conditions.



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  		Figure 5. Leukocytes from allergic-asthmatics interact with P-selectin via PSGL-1. Whole blood from allergic-asthmatic subjects was perfused over immobilized P-selectin at a shear rate of 200 s-1. In some experiments, blood was preincubated for 10 min with 10 µg/ml blocking (PL1) or nonblocking (PL2) antibody directed against PSGL-1. Alternatively, plates were incubated for 10 min with 10 µg/ml blocking P-selectin antibody (G1) prior to assembly of the flow chamber. Antibodies were maintained in the perfusate. After 4 min, blood was chased with HBSS, and the accumulated cells were counted as described in Materials and Methods. The data represent the mean ± SEM. *P < 0.01; ns = not significant.

PSGL-1 expression is increased on granulocytes from allergic-asthmatic subjects
We next examined PSGL-1 expression on leukocytes from normal and allergic-asthmatic subjects using whole blood flow cytometry. Eosinophils have been previously reported to express higher levels of PSGL-1 than neutrophils [30 ]. We found this to be the case in normal and allergic-asthmatic patients (Table 2 ). In addition, there was a significant increase in PSGL-1 expression on neutrophils and eosinophils from allergic-asthmatic subjects as compared with normal controls (Table 2) . In contrast, there was no difference in the PSGL-1 expression on lymphocytes or monocytes. Neutrophils and eosinophils from allergic-asthmatic subjects expressed a single population of PSGL-1-positive cells (data not shown). In parallel studies, there was no difference in the expression of L-selectin or CD11b (Table 2 and data not shown) on any of the leukocyte populations, indicating that the difference in PSGL-1 expression was not a result of leukocyte activation.


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  		Table 2. PSGL-1 Expression but Not L-Selectin Expression Is Increased on Granulocytes from Allergic-Asthmatic Subjectsa

Granulocytes from allergic-asthmatic subjects demonstrate enhanced accumulation on IL-4-stimulated HUVEC as compared with normal controls
IL-4 is a T helper cell type 2 (Th2) cytokine frequently used to model allergic inflammation in vitro. Endothelial cells stimulated with IL-4 express P-selectin and VCAM-1 and support the adhesion of eosinophils under flow conditions [5 , 31 ]. In addition, these endothelial cells support the selective recruitment of eosinophils from the blood using a whole blood recruitment assay [23 ]. Using whole blood from normal or allergic-asthmatic subjects, we determined if there were differences in total leukocyte recruitment or in the recruitment of particular subclasses of leukocytes on IL-4-stimulated HUVEC. We found nearly a twofold increase in total leukocyte accumulation on IL-4-stimulated endothelial cells when blood from allergic-asthmatic subjects was used as compared with blood from normal subjects (Fig. 6A ). Although there was an increase in the total number of cells accumulated on IL-4-stimulated HUVEC, the percentage of rolling cells was unchanged between these patient populations (normal 27.8±4.3% vs. allergy+asthma 24.4±9.45%; n=5, P=0.753). The increase in accumulated cells was a result of a significant increase in the total number of eosinophils and neutrophils recruited on IL-4-stimulated HUVEC (Fig. 6B) . In contrast, the donor status had no effect on the number of PBMCs bound to IL-4-stimulated HUVEC. This was consistent with the flow cytometry data showing increased PSGL-1 expression on granulocytes but not PBMCs. Eosinophils were selectively recruited from normal and allergic-asthmatic patients, with a R-factor of approximately 4.0 (Fig. 6C) . In contrast, the R-factor for neutrophils from normal patients was 0.63 ± 0.11, but this value increased to 1.62 ± 0.19 when recruitment from allergic-asthmatic patients was examined (Fig. 6C) . Previously, we showed that all neutrophil recruitment in this model could be blocked with an anti-P-selectin or an anti-PSGL-1 mAb [23 ]. Consistent with this, an anti-PSGL-1 mAb completely blocked neutrophil recruitment from allergic-asthmatic patients (data not shown) on IL-4-stimulated HUVEC.



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  		Figure 6. Leukocytes from allergic-asthmatics demonstrate enhanced accumulation on IL-4-stimulated HUVEC as compared with normal subjects. Confluent monolayers of HUVEC were stimulated for 24 h with M199/A alone (Control) or M199/A containing 20 ng/ml IL-4 (IL-4). The monolayers were washed, the flow chamber was assembled, and whole blood from normal or allergic-asthmatic patients was perfused through the chamber at a shear rate of 200 s-1. (A) After 4 min, the blood was chased with HBSS, and the accumulated cells were visualized and counted as described in Materials and Methods. (B) The plate was then removed from the chamber, and the accumulated leukocytes were identified as described in Materials and Methods. (C) The percentage of each leukocyte subclass bound on the plate was divided by the total percentage of each leukocyte subclass in the whole blood differential to determine the R-factor. The data represent the mean ± SEM. *P < 0.05; ns = not significant.


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DISCUSSION
 
The general mechanisms that govern leukocyte recruitment into the tissue during inflammation have been carefully dissected over the last decade, and roles for leukocyte and endothelial cell-specific adhesion molecules have been proposed. Selectins have clearly emerged as the primary mediators of tethering and rolling, the first steps in the leukocyte recruitment cascade [32 ]. {alpha}4-integrins and VCAM-1 can also mediate these early steps in leukocyte recruitment but under more restricted conditions [17 , 18 ]. Chemokines and integrins then take over, leading to activation, firm adhesion, and transmigration of leukocytes into the tissue. With many of the players identified, the focus is now on understanding how these mediators are altered during specific disease states and how these changes affect disease pathogenesis.

In this study, we examined the effects of allergies and asthma on leukocyte rolling and accumulation under flow conditions using a whole blood recruitment assay system. We have previously used this system with blood from normal patients to show that eosinophils are selectively recruited on IL-4- but not tumor necrosis factor-stimulated HUVEC [23 ]. In this study, granulocytes from allergic-asthmatic patients showed increased recruitment on immobilized P-selectin and on P-selectin expressed by IL-4-activated endothelial cells (Fig. 6) . The primary ligand for P-selectin is PSGL-1. Using flow cytometry, we found that eosinophils and neutrophils from allergic-asthmatic patients expressed more PSGL-1 than granulocytes from normal patients (Table 2) . Blocking PSGL-1 abolished neutrophil and eosinophil binding to immobilized P-selectin (Fig. 5) , suggesting that the enhanced recruitment of granulocytes from allergic-asthmatics is due to, at least in part, increased PSGL-1 expression.

Selectin interactions with their ligands are not only dependent on total protein expression but also on post-translational modifications [14 ]. For example, tyrosine sulfation, {alpha}1,3 fucosylation, {alpha}2,3 sialylation, and core 2 branched O-glycosylation are required for PSGL-1 to bind to P-selectin [14 , 33 34 35 ]. Changes in these post-translational modifications alter the adhesive interactions between P-selectin and PSGL-1 under static and flow conditions [35 ]. Although we did not directly examine the post-translational modifications of PSGL-1 in this study, our data do suggest that functional differences exist between PSGL-1 expressed on granulocytes from normal versus allergic-asthmatic patients. When we examined leukocyte rolling velocities, we found that granulocytes from normal patients rolled as a single population, whereas granulocytes from allergic-asthmatic patients rolled as a much more heterogeneous population (Fig. 2) , indicating a high degree of variability in terms of adhesive strength. This was not a result of heterogeneous expression of PSGL-1 itself, as FACS analysis showed that PSGL-1 was expressed on allergic-asthmatic granulocytes as a single peak, similar to control granulocytes (data not shown). Thus, in addition to increased protein expression, there may also be functional differences in PSGL-1 expressed on granulocytes from allergic-asthmatic patients.

Additional support for functional changes in PSGL-1 on granulocytes from allergic-asthmatic patients comes from our data showing increased granulocyte recruitment on P-selectin, but not on immobilized E-selectin (Fig. 4) . PSGL-1 can bind to all of the selectins under appropriate conditions in vitro and in vivo [33 , 36 37 38 39 ]; however, several studies have shown differential requirements for the biosynthesis of P-selectin and E-selectin ligands. For instance, core 2 ß-1, 6-N-glucosaminyltransferase (C2GnT) is essential for the formation of P-selectin ligands on Th1 cells [40 , 41 ] and to a lesser extent, neutrophils [41 ], whereas this enzyme is not essential for the formation of E-selectin ligands in these cell types [40 , 41 ]. Our study showed that despite a global increase in PSGL-1 expression on granulocytes from allergic-asthmatic patients, there was no increase in binding to E-selectin, a finding consistent with changes in post-translational modifications of PSGL-1.

A potential mechanism for altering PSGL-1 expression and/or function on granulocytes from allergic-asthmatics is through the action of cytokines. There is a wealth of data showing that cytokines influence selectin ligand biosynthesis. Wagers et al. [42 ] have clearly shown that naïve CD4+ T cells lack {alpha}1,3 fucosyltransferase-VII (FT-VII), an enzyme essential for forming selectin ligands [42 ]. As a result, these cells fail to bind vascular selectins. Cytokine-induced maturation of these T cells increases FT-VII expression, which then confers selectin binding [42 ]. Antigen-specific activation of naïve T cells in the presence of IL-12 increases FT-VII and C2GnT mRNA, resulting in the generation of ligands for P- and E-selectin [40 ]. Natural killer cell differentiation also results in the expression of a novel, L-selectin-binding epitope on PSGL-1, which acts independently of tyrosine sulfation [37 ].

In allergic-asthmatics, there is increased expression of cytokines such as IL-4, IL-5, and IL-9. When added to eosinophils from normal donors, some of these cytokines can induce functional changes that mimic what is observed in eosinophils from allergic-asthmatic patients. For example, in vitro treatment with IL-5 primes eosinophils for increased metabolic activity, oxygen consumption, and superoxide anion production [4 ]. In addition, IL-5 induces a hypodense phenotype in eosinophils from normal donors, which is similar to that found in eosinophils obtained from allergic-asthmatic donors. Finally, Moser et al. [43 ] found that eosinophils from normal donors do not transmigrate across endothelial cells unless they are primed with IL-3 or granulocyte macrophage-colony stimulating factor, whereas, eosinophils from allergic-asthmatic donors bind and transmigrate without any previous stimulus. In addition, granulocytes from allergic-asthmatic patients show altered expression and/or function of several surface proteins. Eosinophils isolated from allergic-asthmatics demonstrate increased static adhesion to intercellular adhesion molecule-1 and VCAM-1 as compared with eosinophils from normal donors [44 ]. This increase in adhesiveness is not associated with an increase in receptor density. Instead, the authors suggest that there is functional up-regulation of these adhesion molecules [44 ]. Neutrophils from allergic-asthmatic patients also demonstrate functional changes in surface markers. For example, neutrophils from allergic-asthmatics can express and/or up-regulate receptors for IL-9, IL-4, and IgE [45 46 47 ]. Our data showed that neutrophils and eosinophils from allergic-asthmatic subjects had increased expression of PSGL-1, which led to functional changes in granulocyte recruitment. Studies are currently underway to examine the role of cytokines in inducing changes in the expression and function of PSGL-1 on granulocytes from allergic-asthmatic patients.

Alteration in the expression or post-translational modification of PSGL-1 has been shown to impact leukocyte trafficking in several models. Th1 cells generated from PSGL-1-deficient mice demonstrate decreased recruitment into sites of inflammation in the skin [38 ]. Similarly, Th1 cells expressing PSGL-1, but not C2GnT, also demonstrate impaired trafficking to sites of inflammation [41 ]. These data show that changes in the amount or the function of PSGL-1 alters leukocyte trafficking to inflammatory sites. P-selectin and PSGL-1 also participate in leukocyte recruitment in models of allergic asthma [48 , 49 ]. P-selectin-deficient mice show a decrease in airway hyper-responsiveness and leukocyte recruitment in an ovalbumin model of allergic asthma [48 ]. Examination of the bronchoaveolar lavage (BAL) fluid from these mice shows that recruitment of all leukocyte subclasses is impaired [48 ], suggesting that P-selectin is not acting selectively on any particular leukocyte population. In another study using the same model, the administration of a soluble form of PSGL-1 dramatically reduces eosinophil and lymphocyte infiltration into the airways and peribronchial regions [50 ]. These data show that PSGL-1 and P-selectin can participate in leukocyte recruitment to sites of allergic inflammation in vivo, suggesting that increased expression of PSGL-1 on granulocytes from allergic-asthmatic patients may result in increased recruitment to sites of allergic inflammation in human disease.

Much of the literature has focused on the selective recruitment of eosinophils during asthma; however, it is clear that there is increased recruitment of neutrophils as well, although the role of these cells in the pathogenesis of asthma has yet to be clearly defined [6 , 7 , 51 ]. BAL fluid from allergic-asthmatic children shows increased neutrophil infiltration and activation that is particularly evident in more severe forms of the disease [52 ]. As described earlier, neutrophils from allergic-asthmatic patients also express receptors, such as the IL-9R, IL-4R, and high-affinity Ig ER, which could facilitate their activation during allergic disease [45 46 47 ]. In this study, we found that eosinophils and neutrophils from allergic-asthmatic patients expressed increased amounts of PSGL-1 on their surface, resulting in a threefold increase in recruitment to P-selectin under flow conditions. We propose that increased PSGL-1 expression on granulocytes from allergic-asthmatics acts to increase the number of granulocytes capable of tethering and rolling on inflamed endothelium. Once bound, additional mediators can act to selectively recruit eosinophils or neutrophils into specific sites of inflammation.


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ACKNOWLEDGEMENTS
 
This work is supported by grants from the Alberta Lung Association and the Canadian Institutes for Health Research (MT-14180). K. D. P. is a Canada Research Chair and an Alberta Heritage Foundation for Medical Research Senior Scholar. B. D. was supported by a studentship from the Alberta Heritage Foundation for Medical Research. We thank Evelyn Lailey for her excellent technical assistance; Dr. Robert Cowie, Dr. Stephen Field, and Margo Underwood at the Calgary Asthma Clinic for their assistance with obtaining and characterizing the subjects used in this study; Unit 51 at the Foothills Hospital in Calgary, Alberta, for their assistance in obtaining umbilical cords; and Dr. Paul Kubes and Susan Cuvelier for critical reading of this manuscript.

Received December 27, 2001; revised May 31, 2002; accepted June 4, 2002.


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REFERENCES
 
    1
  1. Fahy, J. V., Corry, D. B., Boushey, H. A. (2000) Airway inflammation and remodeling in asthma Curr. Opin. Pulm. Med. 6,15-20[Medline]
    2. 2
  2. Elias, J. A., Zhu, Z., Chupp, G., Homer, R. J. (1999) Airway remodeling in asthma J. Clin. Investig. 104,1001-1006[Medline]
    3. 3
  3. Erb, K. J., Le Gros, G. (1996) The role of Th2 type CD4+ T cells and Th2 type CD8+ T cells in asthma Immunol. Cell Biol. 74,206-208[Medline]
    4. 4
  4. Wardlaw, A. J., Moqbel, R., Kay, A. B. (1995) Eosinophils: biology and role in disease Adv. Immunol. 60,151-266[Medline]
    5. 5
  5. Wardlaw, A. J. (1999) Molecular basis for selective eosinophil trafficking in asthma: a multistep paradigm J. Allergy Clin. Immunol. 104,917-926[Medline]
    6. 6
  6. Jatakanon, A., Uasuf, C., Maziak, W., Lim, S., Chung, K. F., Barnes, P. J. (1999) Neutrophilic inflammation in severe persistent asthma Am. J. Respir. Crit. Care Med. 160,1532-1539[Abstract/Free Full Text]
    7. 7
  7. Wenzel, S. E., Schwartz, L. B., Langmack, E. L., Halliday, J. L., Trudeau, J. B., Gibbs, R. L., Chu, H. W. (1999) Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics Am. J. Respir. Crit. Care Med. 160,1001-1008[Abstract/Free Full Text]
    8. 8
  8. Springer, T. A. (1995) Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration Annu. Rev. Physiol. 57,827-872[Medline]
    9. 9
  9. Ley, K. (1996) Molecular mechanisms of leukocyte recruitment in the inflammatory process Cardiovasc. Res. 32,733-742[Medline]
    10. 10
  10. Lawrence, M. B., Springer, T. A. (1991) Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins Cell 65,859-873[Medline]
    11. 11
  11. Kanwar, S., Johnston, B., Kubes, P. (1995) Leukotriene C4/D4 induces P-selectin and sialyl Lewis(x)-dependent alterations in leukocyte kinetics in vivo Circ. Res. 77,879-887[Abstract/Free Full Text]
    12. 12
  12. Von Andrian, U. H., Hansell, P., Chambers, J. D., Berger, E. M., Torres Filho, I., Butcher, E. C., Arfors, K. E. (1992) L-selectin function is required for beta 2-integrin-mediated neutrophil adhesion at physiological shear rates in vivo Am. J. Physiol. 263,H1034-H1044[Abstract/Free Full Text]
    13. 13
  13. McEver, R. P. (1997) Selectin-carbohydrate interactions during inflammation and metastasis Glycoconj. J. 14,585-591[Medline]
    14. 14
  14. Varki, A. (1997) Selectin ligands: will the real ones please stand up? J. Clin. Investig. 99,158-162[Medline]
    15. 15
  15. Van der Vieren, M., Le Trong, H., Wood, C. L., Moore, P. F., St. John, T., Staunton, D. E., Gallatin, W. M. (1995) A novel leukointegrin, alpha d beta 2, binds preferentially to ICAM-3 Immunity 3,683-690[Medline]
    16. 16
  16. Grayson, M. H., Van der Vieren, M., Sterbinsky, S. A., Michael Gallatin, W., Hoffman, P. A., Staunton, D. E., Bochner, B. S. (1998) alphadbeta2 Integrin is expressed on human eosinophils and functions as an alternative ligand for vascular cell adhesion molecule 1 (VCAM-1) J. Exp. Med. 188,2187-2191[Abstract/Free Full Text]
    17. 17
  17. Alon, R., Kassner, P. D., Carr, M. W., Finger, E. B., Hemler, M. E., Springer, T. A. (1995) The integrin VLA-4 supports tethering and rolling in flow on VCAM-1 J. Cell Biol. 128,1243-1253[Abstract/Free Full Text]
    18. 18
  18. Berlin, C., Bargatze, R. F., Campbell, J. J., von Andrian, U. H., Szabo, M. C., Hasslen, S. R., Nelson, R. D., Berg, E. L., Erlandsen, S. L., Butcher, E. C. (1995) alpha 4 Integrins mediate lymphocyte attachment and rolling under physiologic flow Cell 80,413-422[Medline]
    19. 19
  19. Ibbotson, G. C., Doig, C., Kaur, J., Gill, V., Ostrovsky, L., Fairhead, T., Kubes, P. (2001) Functional alpha4-integrin: a newly identified pathway of neutrophil recruitment in critically ill septic patients Nat. Med. 7,465-470[Medline]
    20. 20
  20. Lantero, S., Alessandri, G., Spallarossa, D., Scarso, L., Rossi, G. A. (2000) Stimulation of eosinophil IgE low-affinity receptor leads to increased adhesion molecule expression and cell migration Eur. Respir. J. 16,940-946[Abstract]
    21. 21
  21. Torsteinsdottir, I., Arvidson, N. G., Hallgren, R., Hakansson, L. (1999) Enhanced expression of integrins and CD66b on peripheral blood neutrophils and eosinophils in patients with rheumatoid arthritis, and the effect of glucocorticoids Scand. J. Immunol. 50,433-439[Medline]
    22. 22
  22. Reinhardt, P. H., Kubes, P. (1998) Differential leukocyte recruitment from whole blood via endothelial adhesion molecules under shear conditions Blood 92,4691-4699[Abstract/Free Full Text]
    23. 23
  23. Patel, K. D. (1999) Mechanisms of selective leukocyte recruitment from whole blood on cytokine-activated endothelial cells under flow conditions J. Immunol. 162,6209-6216[Abstract/Free Full Text]
    24. 24
  24. Moore, K. L. (1994) Purification of P-selectin (CD62P) from human platelets J. Tissue Culture Methods 16,255-259
    25. 25
  25. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, November 1986 Am. Rev. Respir. Dis. 1987;136,225-244[Medline]
    26. 26
  26. Zimmerman, G. A., McIntyre, T. M., Prescott, S. M. (1985) Thrombin stimulates the adherence of neutrophils to human endothelial cells in vitro J. Clin. Investig. 76,2235-2246
    27. 27
  27. Patel, K. D., McEver, R. P. (1997) Comparison of tethering and rolling of eosinophils and neutrophils through selectins and P-selectin glycoprotein ligand-1 J. Immunol. 159,4555-4565[Abstract]
    28. 28
  28. Ushiyama, S., Laue, T. M., Moore, K. L., Erickson, H. P., McEver, R. P. (1993) Structural and functional characterization of monomeric soluble P-selectin and comparison with membrane P-selectin J. Biol. Chem. 268,15229-15237[Abstract/Free Full Text]
    29. 29
  29. Lawrence, M. B., Springer, T. A. (1993) Neutrophils roll on E-selectin J. Immunol. 151,6338-6346[Abstract]
    30. 30
  30. Edwards, B. S., Curry, M. S., Tsuji, H., Brown, D., Larson, R. S., Sklar, L. A. (2000) Expression of P-selectin at low site density promotes selective attachment of eosinophils over neutrophils J. Immunol. 165,404-410[Abstract/Free Full Text]
    31. 31
  31. Patel, K. D. (1998) Eosinophil tethering to interleukin-4-activated endothelial cells requires both P-selectin and vascular cell adhesion molecule-1 Blood 92,3904-3911[Abstract/Free Full Text]
    32. 32
  32. Tedder, T. F., Steeber, D. A., Chen, A., Engel, P. (1995) The selectins: vascular adhesion molecules FASEB J. 9,866-873[Abstract]
    33. 33
  33. Snapp, K. R., Ding, H., Atkins, K., Warnke, R., Luscinskas, F. W., Kansas, G. S. (1998) A novel P-selectin glycoprotein ligand-1 monoclonal antibody recognizes an epitope within the tyrosine sulfate motif of human PSGL-1 and blocks recognition of both P- and L-selectin Blood 91,154-164[Abstract/Free Full Text]
    34. 34
  34. Liu, W., Ramachandran, V., Kang, J., Kishimoto, T. K., Cummings, R. D., McEver, R. P. (1998) Identification of N-terminal residues on P-selectin glycoprotein ligand-1 required for binding to P-selectin J. Biol. Chem. 273,7078-7087[Abstract/Free Full Text]
    35. 35
  35. Jain, R. K., Piskorz, C. F., Huang, B. G., Locke, R. D., Han, H. L., Koenig, A., Varki, A., Matta, K. L. (1998) Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2-3Galbeta1-3GalNAc sequences Glycobiology 8,707-717[Abstract/Free Full Text]
    36. 36
  36. Li, F., Wilkins, P. P., Crawley, S., Weinstein, J., Cummings, R. D., McEver, R. P. (1996) Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin J. Biol. Chem. 271,3255-3264[Abstract/Free Full Text]
    37. 37
  37. Andre, P., Spertini, O., Guia, S., Rihet, P., Dignat-George, F., Brailly, H., Sampol, J., Anderson, P. J., Vivier, E. (2000) Modification of P-selectin glycoprotein ligand-1 with a natural killer cell-restricted sulfated lactosamine creates an alternate ligand for L-selectin Proc. Natl. Acad. Sci. USA 97,3400-3405[Abstract/Free Full Text]
    38. 38
  38. Hirata, T., Merrill-Skoloff, G., Aab, M., Yang, J., Furie, B. C., Furie, B. (2000) P-selectin glycoprotein ligand 1 (PSGL-1) is a physiological ligand for E-selectin in mediating T helper 1 lymphocyte migration J. Exp. Med. 192,1669-1676[Abstract/Free Full Text]
    39. 39
  39. Norman, K. E., Katopodis, A. G., Thoma, G., Kolbinger, F., Hicks, A. E., Cotter, M. J., Pockley, A. G., Hellewell, P. G. (2000) P-selectin glycoprotein ligand-1 supports rolling on E- and P-selectin in vivo Blood 96,3585-3591[Abstract/Free Full Text]
    40. 40
  40. Lim, Y. C., Xie, H., Come, C. E., Alexander, S. I., Grusby, M. J., Lichtman, A. H., Luscinskas, F. W. (2001) IL-12, STAT4-dependent up-regulation of CD4(+) T cell core 2 beta-1,6-n-acetylglucosaminyltransferase, an enzyme essential for biosynthesis of P-selectin ligands J. Immunol. 167,4476-4484[Abstract/Free Full Text]
    41. 41
  41. Snapp, K. R., Heitzig, C. E., Ellies, L. G., Marth, J. D., Kansas, G. S. (2001) Differential requirements for the O-linked branching enzyme core 2 beta1-6-N-glucosaminyltransferase in biosynthesis of ligands for E-selectin and P-selectin Blood 97,3806-3811[Abstract/Free Full Text]
    42. 42
  42. Wagers, A. J., Waters, C. M., Stoolman, L. M., Kansas, G. S. (1998) Interleukin 12 and interleukin 4 control T cell adhesion to endothelial selectins through opposite effects on alpha1, 3-fucosyltransferase VII gene expression J. Exp. Med. 188,2225-2231[Abstract/Free Full Text]
    43. 43
  43. Moser, R., Fehr, J., Bruijnzeel, P. L. (1992) IL-4 controls the selective endothelium-driven transmigration of eosinophils from allergic individuals J. Immunol. 149,1432-1438[Abstract]
    44. 44
  44. Hakansson, L., Bjornsson, E., Janson, C., Schmekel, B. (1995) Increased adhesion to vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 of eosinophils from patients with asthma J. Allergy Clin. Immunol. 96,941-950[Medline]
    45. 45
  45. Abdelilah, S., Latifa, K., Esra, N., Cameron, L., Bouchaib, L., Nicolaides, N., Levitt, R., Hamid, Q. (2001) Functional expression of IL-9 receptor by human neutrophils from asthmatic donors: role in IL-8 release J. Immunol. 166,2768-2774[Abstract/Free Full Text]
    46. 46
  46. Girard, D., Paquin, R., Beaulieu, A. D. (1997) Responsiveness of human neutrophils to interleukin-4: induction of cytoskeletal rearrangements, de novo protein synthesis and delay of apoptosis Biochem. J. 325,147-153
    47. 47
  47. Gounni, A. S., Lamkhioued, B., Koussih, L., Ra, C., Renzi, P. M., Hamid, Q. (2001) Human neutrophils express the high-affinity receptor for immunoglobulin E (Fc epsilon RI): role in asthma FASEB J. 15,940-949[Abstract/Free Full Text]
    48. 48
  48. De Sanctis, G. T., Wolyniec, W. W., Green, F. H., Qin, S., Jiao, A., Finn, P. W., Noonan, T., Joetham, A. A., Gelfand, E., Doerschuk, C. M., Drazen, J. M. (1997) Reduction of allergic airway responses in P-selectin-deficient mice J. Appl. Physiol. 83,681-687[Abstract/Free Full Text]
    49. 49
  49. Strauss, E. C., Larson, K. A., Brenneise, I., Foster, C. S., Larsen, G. R., Lee, N. A., Lee, J. J. (1999) Soluble P-selectin glycoprotein ligand 1 inhibits ocular inflammation in a murine model of allergy Investig. Ophthalmol. Vis. Sci. 40,1336-1342[Abstract/Free Full Text]
    50. 50
  50. Borchers, M. T., Crosby, J., Farmer, S., Sypek, J., Ansay, T., Lee, N. A., Lee, J. J. (2001) Blockade of CD49d inhibits allergic airway pathologies independent of effects on leukocyte recruitment Am. J. Physiol. Lung Cell Mol. Physiol. 280,L813-L821[Abstract/Free Full Text]
    51. 51
  51. Tonnel, A. B., Gosset, P., Tillie-Leblond, I. (2001) Characteristics of the inflammatory response in bronchial lavage fluids from patients with status asthmaticus Int. Arch. Allergy Immunol. 124,267-271[Medline]
    52. 52
  52. Barbato, A., Panizzolo, C., Gheno, M., Sainati, L., Favero, E., Faggian, D., Giusti, F., Pesscolderungg, L., La Rosa, M. (2001) Bronchoalveolar lavage in asthmatic children: evidence of neutrophil activation in mild-to-moderate persistent asthma Pediatr. Allergy Immunol. 12,73-77[Medline]



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