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The Journal of Immunology, 2002, 168: 1380-1388.
Copyright © 2002 by The American Association of Immunologists

TNF-{alpha} Potentiates C5a-Stimulated Eosinophil Adhesion to Human Bronchial Epithelial Cells: A Role for {alpha}5{beta}1 Integrin1

Anne Burke-Gaffney2,*,{dagger}, Kate Blease3,*, Adele Hartnell4,* and Paul G. Hellewell{ddagger}

* Applied Pharmacology and {dagger} Unit of Critical Care, National Heart and Lung Institute Division, Imperial College of Science, Technology and Medicine, London, United Kingdom; and {ddagger} Cardiovascular Research Group, University of Sheffield, Clinical Sciences Center, Northern General Hospital, Sheffield, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cooperative action of inflammatory mediators and adhesion molecules orchestrates eosinophil recruitment during allergic inflammation in the airways. This study investigated the mechanisms involved in increasing eosinophil adhesion to human bronchial epithelial cells (HBEC) following priming and activation of eosinophils with TNF-{alpha} and complement protein C5a, respectively. Under primed conditions, eosinophil adhesion increased 3-fold from basal (16%), and the effect was significantly greater (p < 0.05) than the increase following stimulation with C5a alone (2-fold). Eosinophil contact with HBEC was essential for priming. In contrast to C5a, adhesion of eotaxin-stimulated eosinophils to HBEC was not primed with TNF-{alpha} nor IL-5, a known eosinophil-priming agent. Priming caused activation of {alpha}M{beta}2 integrin; mAb against either the common {beta}2 integrin subunit or its ICAM-1 ligand reduced the primed component of adhesion. Using mAbs against {beta}1 or {alpha}5, but not {alpha}4 integrin subunit, together with anti-{beta}2 integrin mAb, reduced stimulated adhesion to basal levels. Cross-linking {alpha}5{beta}1 integrin increased {alpha}M{beta}2 integrin-dependent adhesion of eosinophils. There are no known adhesion molecule ligands of {alpha}5{beta}1 integrin expressed on HBEC; however, fibronectin, the major matrix protein ligand for {alpha}5{beta}1 integrin, was detected in association with HBEC monolayers. A mAb against fibronectin, in combination with anti-{beta}2 integrin mAb, reduced adhesion to basal levels. In conclusion, {alpha}5{beta}1 integrin may provide a contact-dependent costimulus for eosinophil priming that, together with TNF-{alpha}, potentiated C5a activation of {alpha}M{beta}2 integrin and increased eosinophil adhesion to ICAM-1. Fibronectin, associated with HBEC, may act as a ligand for {alpha}5{beta}1 integrin. Dual regulation of eosinophil priming may prevent inappropriate activation of eosinophils in the circulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Asthma is a major cause of morbidity worldwide, and severity and prevalence are increasing (1). There is now substantial evidence that eosinophils are major effector cells in asthma (2, 3, 4), and an understanding of the mechanisms by which eosinophils accumulate and are activated in airway tissue is fundamental to our understanding of the pathogenesis of this disease. Adhesion molecules play an essential role in eosinophil recruitment (5, 6) and are also considered to play a critical part in eosinophil activation (7, 8, 9). In particular, eosinophil adhesion to human bronchial epithelial cells (HBEC)5 potentiates the release of toxic granule products (7) that can damage the respiratory epithelium, contributing to airway hyperresponsiveness (10).

Despite a number of studies, including our own, that have addressed the subject of eosinophil adhesion to epithelial cells (11, 12, 13, 14, 15), the adhesion pathways governing these interactions remain to be fully elucidated. It is known that eosinophils bind ICAM-1 expressed on HBEC predominantly via the {beta}2 integrin, {alpha}M{beta}2 (CD11b/CD18) (14, 15). Eosinophils also express a number of {beta}1 integrins of which {alpha}4{beta}1 is the best characterized; however, the contribution of this integrin or its adhesion molecule ligand, VCAM-1, to eosinophil-epithelial cell adhesion is debated (13, 14, 15). Eosinophils express {alpha}6{beta}1, and there are conflicting reports of {alpha}2{beta}1 and {alpha}5{beta}1 integrin expression (16, 17, 18). The major ligands for these integrins are matrix proteins; for example, {alpha}5{beta}1 and {alpha}6{beta}1 bind fibronectin and laminin, respectively (19). L-selectin, P-selectin glycoprotein ligand-1, other selectin ligands bearing sialyl-Lewis X, and the integrin {alpha}4{beta}7 (5) are also expressed on eosinophils, but to our knowledge there are no known epithelial ligands for these adhesion molecules.

Many of the features of asthma, including inflammatory cell recruitment and activation, are consistent with the actions of the complement proteins C3a and C5a (20). Despite the well-established association of complement in immune and inflammatory reactions and several studies showing C3a and C5a in bronchoalveolar lavage fluid of asthmatics (21, 22, 23), investigation of the role of complement in asthma has been superseded by research into the cytokine and chemokine networks. A number of recent studies using animal models with genetic alterations in the complement pathways have now established crucial links between complement and allergic inflammation (24, 25, 26) and may provide an impetus for resurgence in the investigation of the role of complement in asthma. Also, despite the relative importance of eosinophil-activating chemokines in asthma and in particular their ability to cause movement of eosinophils from the blood into tissue (27), we showed that a number of these chemokines, eotaxin, RANTES, and macrophage inflammatory protein-1, had little or no effect on eosinophil adhesion to HBEC (14). In contrast, C5a alone of the inflammatory mediators we investigated increased eosinophil adhesion to cytokine-activated and resting HBEC (14).

One way in which eosinophil responses, including adhesion, may be increased during allergic inflammation is by priming the eosinophils. Priming potentiates most eosinophil functions and is defined as exposure of cells to a stimulus that has little or no effect alone but increases the response of a second inflammatory agent (28). Soluble mediators such as the cytokines IL-5 and IL-3 prime eosinophil function, one example being degranulation of C5a-activated eosinophils (29). The synergistic effect between IL-5 and eotaxin to promote eosinophil migration in asthma is also well recognized (30). In addition to soluble mediators, adhesion molecules and in particular leukocyte integrins can also provide priming signals to eosinophils (31, 32).

The aims of the present study were to determine the effect of priming eosinophils on their ability to adhere to HBEC and to assess the contribution of adhesion molecules to priming and adhesion. Eosinophils were primed with the cytokines TNF-{alpha} or IL-5 before stimulation with C5a, or for comparison eotaxin. TNF-{alpha} is increased in the bronchoalveolar lavage fluid of patients with asthma (33) and is known to prime neutrophils (34), but its role as an eosinophil-priming agent is not well established.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture reagents

HBEC prepared from four separate donors and cryopreserved as first passage cultures (Clonetics, San Diego, CA) were purchased from TCS Biologicals (Buckingham, U.K).

Cytokines and other reagents

Human rTNF-{alpha} (sp. act., >1 x 108 U/mg) was obtained from Roche Diagnostics (Lewes, East Sussex, U.K.), and eotaxin was obtained from PeproTech (London, U.K.). C5a and IL-5 were generous gifts from J. J. van Oostrum (Ciba-Geigy, Summit, NJ) and T. Wells (GlaxoWellcome, Geneva, Switzerland), respectively. Percoll was obtained from Pharmacia Biotech (St. Albans, Herts, U.K.); very low endotoxin BSA was obtained from Bayer (Basingstoke, Hants, U.K.); CD16 microbeads were obtained from Miltenyi Biotec (Bisley, Surrey, U.K.); and Calcein-AM was obtained from Cambridge Bioscience (Cambridge, U.K.). One-micron fluorescent microspheres were purchased as a solids-latex (2.5% v/v) stock solution from Park Scientific (Nottingham, U.K.). Heat-inactivated FCS and Dulbecco’s PBS with Ca2+/Mg2+ were from Life Technologies (Paisley, Scotland). All other reagents were from Sigma-Aldrich (St. Louis, MO) or BDH Chemicals (Poole, Dorset, U.K.).

Antibodies

Mouse anti-human {alpha}4 integrin IgG1 (CD49d, clone 2B4) (35) and mouse anti-human ICAM-1 (BBIG-I1) IgG1 whole mAb were gifts from R. Pigott (British Biotech, Oxford, U.K.). F(ab')2 of BBIG-I1 were generated by Cymbus Biotechnology (Chandlers Ford, Hants, U.K.). Mouse IgG1 (MOPC21; whole mAb and F(ab')2) and mouse anti-human {beta}2 integrin mAb (CD18, clone 6.5E) were gifts from M. Robinson (Celltech, Slough, Berkshire, U.K.). Affinity-isolated goat anti-mouse peroxidase conjugate, {gamma}-chain and L chain specific, was obtained from TCS Biologicals. The following whole Abs were from Sigma-Aldrich: mouse IgG2b (MOPC141); mouse anti-human HLA class I Ag IgG2a mAb (clone W6/32); mouse anti-human fibronectin IgG1 (clone FN-15, raised against plasma fibronectin, but reactive with all fibronectin forms). The following were from Serotec (Kidlington, Oxford, U.K.): mouse anti-human {beta}1 integrin IgG1 (CD29, clone 3S3); rabbit anti-mouse IgG-FITC and mouse anti-human {alpha}5 integrin IgG2b (CD49e, clone SAM-1) (36). Mouse anti-human {alpha}5 integrin IgG1 (CD49e, clone A5-PUJ2) (37) was purchased from Upstate Biotechnology (Lake Placid, NY).

Cell culture

HBEC were cultured as we have previously described (14). Cells subcultured onto 1% gelatin-coated 96-well plates and grown to confluence for 5–6 days express low levels of ICAM-1, as determined by ELISA (OD405 of 0.09). Exposure of HBEC to TNF-{alpha} (10 ng/ml) or C5a (10-7 M) for the duration of the adhesion assay (1 h) did not significantly alter ICAM-1 expression.

Isolation of human peripheral blood eosinophils

Eosinophils were isolated from peripheral blood of mildly atopic volunteers with a history of asthma, eczema, or hay fever with symptoms on exposure to common aeroallergens; donors were taking no systemic medication. Blood was taken according to a Royal Brompton Hospital ethics committee-approved protocol. Eosinophils were isolated as described previously by us (14). Briefly, granulocytes were separated from mononuclear cells using discontinuous plasma-Percoll density gradient centrifugation, and eosinophils were purified from granulocytes using anti-CD16 microbeads. Eosinophils (>98% pure) were labeled (30 min, 37°C) with a fluorescent dye, Calcein-AM, and resuspended in Krebs-Ringer phosphate buffer (3.2 mM NaH2PO4, 12.5 mM Na2HPO4, 4.8 mM KCl, 5% glucose, 0.93 mM CaCl2, and 1.2 mM MgSO4) containing 2.5% FCS (14).

Eosinophil adhesion assay

For contact-dependent priming experiments, eosinophils were added with priming agent or buffer to HBEC monolayers for 30 min. For contact-independent priming experiments, eosinophils were primed in suspension (37°C, 30 min) before transfer to HBEC monolayers. Following priming, C5a or eotaxin was added to eosinophils and HBEC for a further 30 min. In selected experiments, TNF-{alpha} and C5a were coincubated (60 min) during eosinophil adhesion to HBEC; also, priming and adhesion were conducted using microtiter plates coated with FCS (10% in PBS; 3 h at 37°C) instead of HBEC. Fluorescence was measured after washing using a Biolite F1 plate reader, and results were expressed as percentage of adhesion, as we have described previously (14). Isotype-matched control mAbs for anti-CAM mAb did not significantly alter eosinophil adhesion under any of the conditions in this study, confirming our previous findings (14). For clarity, control Ab data are omitted from the figures.

ELISA determination of fibronectin and integrin expression on HBEC

To establish association of fibronectin with HBEC monolayers and FCS-coated plates and measure integrin expression, cells were incubated with primary mAbs against fibronectin or {alpha}5 integrin, followed by a secondary peroxidase-linked goat anti-mouse Ab and a peroxidase-sensitive substrate, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid). Chromophore development was determined by measuring OD405 using a Titretec MCC/340 Multiscan microplate reader (ICN Flow Laboratories, Paisley, U.K.) (38).

Flow cytometric analysis of eosinophil integrin expression

Flow cytometry was used to determine expression of integrins on eosinophils. Eosinophils were resuspended in PBS at 1 x 106 cells/ml. Samples (50 µl) were incubated on ice for 30 min with saturating concentrations (10 µg/ml) of anti-integrin mAbs, washed, and incubated with 100 µl of 1/100 dilution of goat anti-mouse IgG-FITC mAb for 30 min on ice. Nonbinding control mAbs were used to determine nonspecific binding. Samples were resuspended in FACSFlow before analyses using a FACScan flow cytometer (BD Biosciences, Oxford, U.K.) and analyzed using CellQuest software (BD Biosciences). Results were expressed as geometric mean fluorescence intensity (MFI).

Eosinophil adhesion to ACLB

{beta}2 integrin activation was determined by eosinophil binding of albumin-coated latex beads (ACLB) (7) using a modification of a method established with neutrophils (39). Fluorescent latex beads were washed in PBS, resuspended at 2.5% in PBS containing 10 mg/ml human serum albumin, and incubated for 10 min at 25°C. The resultant ACLB were washed again in PBS and resuspended at 0.75% (v/v). Eosinophils (175-µl aliquots at 1 x 106/ml, in PBS) were primed with TNF-{alpha} at 37°C for 15 min in a 96-well plate coated with FCS. Eosinophils were transferred to polypropylene tubes and incubated with ACLB (25 µl of 0.75% solution) and C5a or buffer for a further 15 min. Nonadherent ACLB were removed by washing with PBS, and eosinophils were fixed by addition of 0.5 ml of 0.5% gluteraldehyde; bead binding to the eosinophils was assessed by FACScan analysis, as previously detailed (39).

Cross-linking of {alpha}5{beta}1 molecules by immobilized Abs to culture plates

Immobilization of mAb to culture plates was conducted as previously described (40). Culture plates (96-well plates) were precoated with 100 µl of anti-{alpha}5 (A5-PUJ2), MOPC21 (nonbinding control), and MHC class I (binding control) mAb in phosphate buffer (pH 9) at 25°C. Plates were washed with PBS and incubated with PBS/BSA (1%) for 1 h. Eosinophil adhesion assays were conducted as described above in the presence or absence of an anti-{beta}2 integrin mAb, and adhesion was determined after 1 h at 37°C.

Statistics

Results are expressed as mean ± SEM of n separate experiments. Statistical analysis was conducted using ANOVA, followed by Student-Newman-Keuls multiple comparison post test to compare all values to each other or the Bonferroni post test to make selected comparisons in experiments with anti-adhesion molecule mAb. Instat GraphPad software (GraphPad, San Diego, CA; www.graphpad.com) was used to perform statistical analysis; results were deemed significant if p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF-{alpha} primes eosinophil adhesion to HBEC

To investigate the effect of TNF-{alpha} on C5a-stimulated eosinophil adhesion to HBEC, eosinophils and TNF-{alpha} were added to resting HBEC monolayers for 30 min; C5a was added for a further 30 min and adhesion was measured. TNF-{alpha} (10 ng/ml; 6 x 10-10 M) alone did not increase eosinophil adhesion from basal levels of 16.2 ± 2.3%, whereas stimulation with C5a (10-7 M) alone significantly (p < 0.01) increased adhesion 2-fold (Fig. 1Go). Preincubating eosinophils with TNF-{alpha} before C5a addition increased adhesion 3-fold over basal, and the effect was significantly (p < 0.05) greater than with C5a alone (Fig. 1Go). In contrast, coincubation of eosinophils with TNF-{alpha} and C5a for 60 min did not increase adhesion to HBEC above that seen with C5a alone (data not shown), suggesting that TNF-{alpha} may act in a priming capacity to increase eosinophil adhesion. Priming eosinophils with a lower (1 ng/ml) or higher (100 ng/ml) concentration of TNF-{alpha} also increased adhesion 3-fold, but 0.1 ng/ml TNF-{alpha} had no effect. Eosinophil adhesion was also increased (2.5-fold, n = 3) following priming with TNF-{alpha} (10 ng/ml) and stimulation of eosinophils with a lower concentration of C5a (10-8 M). Coincubation of HBEC and eosinophils with TNF-{alpha} (10 ng/ml) for 15 or 30 min resulted in similar increases in adhesion of C5a-stimulated eosinophils, i.e., 51.4 ± 3.9 and 53.8 ± 1% (n = 3), respectively. In contrast, preincubation with TNF-{alpha} for 5 min was not sufficient to prime C5a-stimulated eosinophil adhesion (30.4 ± 4.2%). In subsequent experiments, eosinophils were preincubated with 10 ng/ml TNF-{alpha} for 30 min before stimulation with C5a at 10-7 M, also for 30 min.



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  		FIGURE 1. Effects of priming eosinophils with TNF-{alpha} on adhesion to HBEC. Eosinophils were incubated with TNF-{alpha} (10 ng/ml) or buffer for 30 min on HBEC monolayers. C5a (10-7 M) was added for 30 min, and adhesion was measured. Results are expressed as percentage of adhesion ± SEM; n = 5. *, p < 0.01; **, p < 0.001. Asterisks denote significant differences compared with basal adhesion (buffer alone). #, p < 0.01, a significant difference compared with effects of C5a alone.

 
Contact between eosinophils and HBEC is required for priming

Several approaches were taken to determine the contribution of contact between the eosinophils and HBEC to priming of eosinophil adhesion. First, priming under adherent and nonadherent conditions was compared. Eosinophils were preincubated for 30 min with TNF-{alpha} under nonadherent conditions and then added to HBEC for a further 30 min in the presence of C5a. Contact-independent priming of eosinophils did not increase adhesion above the effect of C5a alone (Table IGo); eosinophils used in these experiments primed normally under contact-dependent conditions (data not shown). Therefore, contact was essential for TNF-{alpha} priming of eosinophil adhesion to HBEC.


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  		Table I. Contact-independent priming with TNF-{alpha} on eosinophil adhesion to HBEC

 
Second, an alternative contact-dependent priming method was used, i.e., contact with FCS-coated microtiter plates; under these conditions, TNF-{alpha} had no effect on basal adhesion (5.3 ± 2.6%) but significantly increased C5a-stimulated adhesion from 44.3 ± 7.1 to 64.5 ± 5.3% (n = 3; p < 0.05). These results further confirm the necessity of contact for eosinophil priming with TNF-{alpha} and by extrapolation suggest that soluble mediators released from HBEC are unlikely to be required for priming eosinophil adhesion to HBEC.

Third, contact between eosinophils and HBEC was established before addition of TNF-{alpha}. In these experiments, priming was established as before (i.e., coincubation of eosinophils with HBEC and TNF-{alpha} for 30 min) and also by preincubating eosinophils with HBEC for 15 min before the addition of TNF-{alpha}, which was then added for a further 15 min. Because exposing eosinophils to HBEC and TNF-{alpha} for 15 or 30 min gave a similar priming effect, it was assumed that any observed increase in adhesion would be the result of the prior contact between eosinophils and HBEC before addition of TNF-{alpha}. Indeed, as shown in Fig. 2Go, adhesion of eosinophils preincubated with HBEC before the addition of TNF-{alpha} was significantly greater (p < 0.01) than adhesion of eosinophils and TNF-{alpha} added simultaneously, supporting the importance of contact for priming.



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  		FIGURE 2. Effect of preincubating eosinophils with HBEC, before addition of TNF-{alpha}, on eosinophil adhesion. Eosinophils were coincubated with HBEC at time (t) = 0. TNF-{alpha} (10 ng/ml) or buffer was added either at t = 0 or at t = 15. C5a (10-7 M) was added at time t = 30, and adhesion was measured at t = 60. Results are expressed as percentage of adhesion, and data are mean ± SEM from three experiments. *, p < 0.001; asterisk denotes a significant difference compared with eosinophils exposed to buffer alone under the corresponding conditions. #, p < 0.001; symbol denotes a significant difference compared with eosinophils exposed to C5a under the corresponding conditions. $, p < 0.01; symbol denotes a significant difference between bars joined by a line.

 
Lack of effect of eotaxin on eosinophil adhesion to HBEC

When eosinophils were stimulated with eotaxin (100 ng/ml; 1.2 x 10-8 M) instead of C5a at a concentration we showed previously increased eosinophil adhesion, substantially, to cytokine-activated endothelial cells (41), neither TNF-{alpha} nor IL-5 primed eosinophil adhesion to HBEC (Fig. 3Go). Eotaxin used in these experiments caused a small increase in adhesion of eosinophils to cytokine-activated HBEC (data not shown), supporting our previous findings (14). In parallel experiments, TNF-{alpha} and IL-5 (10-9 M) primed adhesion of C5a-stimulated eosinophils, with IL-5 increasing adhesion 2.5-fold (Fig. 3Go). These results suggest that C5a may play a more significant role than eotaxin in facilitating eosinophil adhesion to the airway epithelium.



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  		FIGURE 3. Effects of priming eosinophils with TNF-{alpha} or IL-5 on eotaxin-stimulated eosinophil adhesion to HBEC. Eosinophils were incubated with TNF-{alpha} (10 ng/ml; 6 x 10-10 M), IL-5 (10-9 M), or buffer for 30 min in the presence of HBEC monolayers. C5a (10-7 M) or eotaxin (100 ng/ml; 1.2 x 10-8 M) was added for 30 min, and adhesion was measured. Results are expressed as percentage of adhesion ± SEM and n = 3–5. *, p < 0.05; **, p < 0.01; asterisks denote significant differences compared with basal adhesion (buffer alone). #, p < 0.05; ##, p < 0.01; symbol denotes significant differences compared with effects of C5a alone.

 
{beta}2 integrins and ICAM-1 contribute to adhesion of primed eosinophils

Having established that TNF-{alpha} caused contact-dependent priming of eosinophil adhesion to HBEC, we investigated the adhesion pathways involved. We confirmed our previous finding (14) that mAbs against the {beta}2 integrin subunit and its ICAM-1 ligand do not inhibit basal or C5a-stimulated eosinophil adhesion to resting HBEC (Fig. 4Go). In contrast, both mAb reduced primed adhesion to levels seen with C5a alone (Fig. 4Go), but a combination of these mAbs did not reduce adhesion further (data not shown). These results suggest that a {beta}2 integrin/ICAM-1-dependent pathway mediates the primed component of the adhesion response. F(ab')2 of anti-ICAM-1 were used in this study because the whole mAb caused aggregation of eosinophils, as observed microscopically, dramatically increasing adhesion. In contrast, whole mAb and F(ab')2 of anti-{beta}2 integrin mAb gave similar inhibitory results (data not shown).



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  		FIGURE 4. Effects of Abs against the {beta}2 integrin chain and ICAM-1 on eosinophil adhesion to HBEC. Eosinophils were coincubated with HBEC in the presence of buffer, TNF-{alpha} (10 ng/ml), and also 10 µg/ml F(ab')2 anti-ICAM-1 (BBIG-I1) and anti-{beta}2 integrin (6.5E; 10 µg/ml). Buffer or C5a (10-7 M) was then added for a further 30 min, and adhesion was measured at 60 min. Results are expressed as percent adhesion ± SEM, n = 3. *, p < 0.001; asterisk denotes a significant decrease compared with TNF-{alpha}/C5a-treated eosinophils without anti-{beta}2 and anti-ICAM-1 mAbs. Isotype-matched control F(ab')2 and whole mAb (MOPC21) did not significantly alter eosinophil adhesion (data not shown).

 
An increase in {beta}2 integrin-mediated adhesion may result from an increase in integrin expression and/or integrin activation. Having established that priming eosinophils with TNF-{alpha} is dependent on contact, experiments were carried to measure {beta}2 integrin expression and activation on eosinophils primed during contact with FCS-coated microtiter plates (see Materials and Methods for justification). In these experiments, TNF-{alpha} altered neither basal nor C5a-induced expression of {alpha}cm or {beta}2 integrin subunits (data not shown), suggesting that primed eosinophil adhesion to HBEC was unlikely to be the result of changes in levels of expression of {alpha}m{beta}2 integrin. When ACLB were used to assess {alpha}m{beta}2 activation, contact-dependent priming of eosinophils with TNF-{alpha}, followed by stimulation with C5a significantly (p < 0.05) increased ACLB binding compared with binding to nonprimed, C5a-stimulated eosinophils (Fig. 5Go); binding was abolished with anti-{alpha}M and {beta}2 integrin mAbs (data not shown). It is also of note that TNF-{alpha} alone did not significantly alter ACLB binding, suggesting that the priming effect of TNF-{alpha} on C5a activation of {alpha}m{beta}2 integrin is likely to be at an intracellular signaling level rather than directly on the adhesion molecule. The lack of effect of TNF-{alpha} on {alpha}m{beta}2 integrin activation is concordant with the lack of effect of TNF-{alpha} on eosinophil adhesion to HBEC.



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  		FIGURE 5. Effects of TNF-{alpha} priming on eosinophil adhesion to ACLB. Eosinophils were primed with TNF-{alpha} (10 ng/ml) on a 10% FCS-coated plate for 15 min at 37°C and then transferred to a tube containing ACLB (25 µl) and mixed for 15 min, in the presence of C5a (10-7 M). The reaction was terminated with 0.5 ml of 0.5% gluteraldehyde. Samples were analyzed by flow cytometry, and the percentage of eosinophils with ACLB attached was calculated. a, A representative flow cytometry histogram of unstimulated eosinophils binding ACLB. b, A histogram of TNF-{alpha}/C5a-treated eosinophils binding ACLB. The x-axis shows the MFI and the y-axis shows the relative cell number. The lowest fluorescent peak (far left) represents eosinophils with no attached ACLB, then with increasing binding; single, double, and triple bead binding can be distinguished. The percentage of eosinophils with attached ACLB under priming conditions was calculated by gating out the far left peak determined from the control sample and shows a 30% increase in this example. c, A bar graph of mean data from four experiments showing eosinophil binding to ACLB in the presence of buffer (control), TNF-{alpha}, C5a, and C5a following TNF-{alpha} priming. *, p < 0.05; **, p < 0.001; asterisks denote significant increases compared with unstimulated eosinophils binding ACLB. #, p < 0.05, significant increase compared with C5a-stimulated eosinophils binding ACLB.

 
As a control to confirm the requirement for contact for priming, eosinophils were preincubated with TNF-{alpha} in suspension. In these experiments, binding of C5a-stimulated eosinophils to ACLB was similar, with or without pretreatment with TNF-{alpha} (55.1 ± 2% and 59.9 ± 1.4%; n = 4), suggesting contact was required for priming of {alpha}m{beta}2 activation. Together these results suggest that contact-dependent activation of {alpha}m{beta}2 integrin may account for increased adhesion of C5a-stimulated eosinophils to HBEC following TNF-{alpha} priming.

{alpha}5{beta}1 integrin is involved in adhesion of TNF-{alpha}-primed eosinophils

In addition to {beta}2 integrins, eosinophils also express a number of {beta}1 integrins. Having established that inhibition of {beta}2 integrins alone abolished adhesion of TNF-{alpha}-primed, C5a-stimulated eosinophil adhesion to HBEC, we assessed the effects of a mAb against the {beta}1 integrin subunit alone and in combination with anti-{beta}2 integrin mAb. Again, C5a-stimulated eosinophil adhesion was unaffected by anti-{beta}2 integrin mAb, and the anti-{beta}1 integrin mAb was also ineffective; however, in combination, these mAb reduced adhesion to basal levels (Fig. 6Goa), suggesting that involvement of either of these adhesion molecules may only be apparent when both are blocked. The primed component of the adhesion response was reduced to levels seen with C5a alone using anti-{beta}1 or anti-{beta}2 integrin mAbs, and a combination of these mAbs reduced primed adhesion to basal levels (Fig. 6Goa). As {beta}2 integrins contributed to the primed component of the adhesion response (Fig. 4Go), {beta}1 integrin(s) may provide the initial contact-dependent priming signal, contribute to adhesion of C5a-stimulated and primed eosinophils to HBEC, or may be involved in both processes.



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  		FIGURE 6. Effects of Abs against {beta}1 integrin chain (a), {alpha}4 integrin (b), {alpha}5 integrin chain (c), and fibronectin (d), alone and in combination with anti-{beta}2 integrin Ab, on eosinophil adhesion to HBEC. Eosinophils were coincubated with HBEC in the presence of buffer or TNF-{alpha} (10 ng/ml) for 30 min, C5a (10-7 M) was added for a further 30 min, and adhesion was measured at 60 min. Abs against {beta}1, {beta}2, {alpha}4, or {alpha}5 integrins (3S3, 6.5E, 2B4, A5-PUJ2) or fibronectin (FN-15) at concentrations of 10 µg/ml each were present for the duration of the assay. Results are expressed as percentage of adhesion ± SEM; n = 3–4. *, p < 0.01; **, p < 0.001; asterisks denote significant decreases compared with eosinophils in the absence of inhibitory mAb. Appropriate isotype-matched control Abs had no significant effect on eosinophil adhesion, but data are not shown for clarity of the figure.

 
To determine the {beta}1 integrin(s) involved, we used mAbs against two of the {alpha}-chains associated with the {beta}1 integrin subunit, specifically {alpha}4 and {alpha}5. The most well-established {beta}1 integrin on eosinophils is {alpha}4{beta}1; however, in the present study, anti-{alpha}4 mAb (2B4), at a concentration that we previously showed inhibited eosinophil adhesion to cytokine-activated endothelial cells (41), had no effect on C5a-stimulated or primed eosinophil adhesion to HBEC (Fig. 6Gob). Furthermore, anti-{alpha}4 mAb did not alter the responses seen with the anti-{beta}2 integrin mAb (Fig. 6Gob). There are conflicting reports of {alpha}5{beta}1 integrin expression on eosinophils (16, 17, 18). In the present study, we showed that an anti-{alpha}5 integrin mAb, A5-PUJ2, attenuated primed adhesion and in combination with anti-{beta}2 integrin mAb reduced C5a-stimulated and also primed adhesion, to basal levels (Fig. 6Goc), suggesting a functional role for {alpha}5 in eosinophil adhesion to epithelial cells.

An alternative name for {alpha}5{beta}1 integrin is the fibronectin receptor; as the name suggests, the matrix protein fibronectin is a major ligand for this integrin. It has been reported that fibronectin can be detected on the upper surface of cell monolayers in culture. In these studies, ELISA determination showed association of fibronectin (OD405, 0.39) with HBEC monolayers (and also FCS-coated plates; OD405, 0.14), but not eosinophils, as assessed by flow cytometry (data not shown). Anti-fibronectin mAb alone and in combination with anti-{beta}2 integrin mAb gave a similar profile of inhibition to anti-{beta}1/anti-{beta}2 integrin mAbs on eosinophil-HBEC adhesion (Fig. 6God). As with the anti-{beta}2 integrin mAb, the anti-fibronectin mAb did not cause eosinophil aggregation, as observed microscopically. As fibronectin was detected on HBEC, but not eosinophils, we conclude that inhibition of adhesion results from blocking HBEC-associated fibronectin. We suggest that fibronectin may act as a ligand for eosinophil {alpha}5{beta}1 integrin in these studies.

Detection of {alpha}5 integrin on eosinophils and HBEC

Unlike fibronectin, {alpha}5{beta}1 integrin is expressed on epithelial cells (19), in addition to the likelihood that it is expressed on eosinophils. Therefore, it was necessary to establish whether A5-PUJ2 used in the adhesion studies blocked {alpha}5 integrin on eosinophils and/or epithelial cells. We investigated the capacity of A5-PUJ2 and in comparison another anti-{alpha}5 integrin mAb SAM-1, to bind to HBEC and eosinophils. A5-PUJ2 showed significant (p < 0.001) binding to eosinophils (MFI 494 ± 39, n = 4) compared with an isotype-matched control mAb (MFI 9.7 ± 1, n = 4; Fig. 7Goa), whereas there was no significant binding of SAM-1 to eosinophils (MFI 3.2 ± 0.1; n = 4) compared with a control mAb (MFI 4 ± 0.2; n = 4, Fig. 7Gob). In contrast, SAM-1 bound to HBEC, as detected by ELISA (OD 0.28, n = 4), but A5-PUJ2 did not bind under the conditions used in this study. These results suggest that A5-PUJ2 blocks eosinophil and not epithelial cell {alpha}5 integrin in eosinophil-HBEC adhesion assays.



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  		FIGURE 7. Determination of {alpha}5 integrin expression on eosinophils. The expression of {alpha}5 integrin was determined with mAb clones SAM-1 (IgG2b) and A5-PUJ2 (IgG1) in comparison with appropriate isotype-matched control mAbs, MOPC141 (IgG2b) and MOPC21 (IgG1). Representative flow cytometry histograms for {alpha}5 integrin expression on eosinophils using A5-PUJ2 (a) and SAM-1 (b) are depicted; the x-axis shows the MFI and the y-axis shows the relative cell number. The filled and open histograms represent {alpha}5 integrin and control mAb binding, respectively; these overlap in b.

 
Cross-linking {alpha}5{beta}1 increases {beta}2 integrin-dependent adhesion

Finally, it was necessary to establish that activation of {alpha}5{beta}1 integrin activation could, in fact, cause {beta}2 integrin-dependent adhesion. A well-established method of integrin activation is to cross-link the integrin by incubating the integrin-expressing cells with mAb against the integrin immobilized to cell culture plates. Thus, in these experiments, eosinophils were incubated with anti-{alpha}5 mAb (A5-PUJ2) immobilized on culture plates also coated with BSA to provide a {beta}2 integrin ligand for adhesion. Adhesion increased from basal levels of 8.4 ± 2.3 to 27.6 ± 2.9%, and the increase was abolished using anti-{beta}2 integrin mAb (Fig. 8Go). In control experiments, adhesion to plates coated with a nonbinding (MOPC21) or binding (MHC I) isotype-matched control mAbs was not increased, suggesting that nonspecific activation of eosinophils was unlikely to account for increased adhesion (Fig. 8Go). These results confirm that activation of {alpha}5{beta}1 integrin is capable of triggering {beta}2 integrin-dependent adhesion.



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  		FIGURE 8. Effect of cross-linking {alpha}5 integrin on eosinophil adhesion to BSA-coated plates. Ninety-six-well plates were coated for 2 h with 5 µg/ml anti-{alpha}5 (A5-PUJ2) or nonbinding (MOPC21) or binding (anti-MHC class I) control mAbs. Plates were washed and incubated for 1.5 h with BSA (1%) to block nonspecific binding of eosinophils to plastic and also to facilitate {beta}2 integrin-dependent adhesion. Eosinophils were added with or without anti-{beta}2 integrin (6.5E; 10 µg/ml) for 1 h at 37°C, and adhesion was measured. Results are expressed as percentage of adhesion ± SEM; n = 4. *, p < 0.01; asterisk denotes a significant increase compared with adhesion without cross-linking of {alpha}5 integrin. #, p < 0.01; symbol denotes a significant inhibition compared with adhesion in the absence of anti-{beta}2 integrin mAb.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Eosinophil adhesion is considered to play an essential role in the accumulation and activation of eosinophils in the airways during allergic inflammation (6). In this study, we showed that TNF-{alpha} potentiated adhesion of C5a-stimulated eosinophils to HBEC, and that preincubation or priming with TNF-{alpha}, rather than coincubation of eosinophils with TNF-{alpha} and C5a, was necessary to enhance adhesion. The magnitude of primed adhesion response was dependent on the concentration of TNF-{alpha} and C5a and also on the duration of priming. Adhesion was also increased when eosinophils were primed with IL-5 before C5a stimulation. In contrast, a priming effect of TNF-{alpha} or IL-5 on adhesion was not observed if eotaxin, rather than C5a, was used to stimulate eosinophils. These results support a potential role of complement proteins in the pathogenesis of asthma.

Using several different approaches, we demonstrated the significance of surface contact of eosinophils, during exposure to TNF-{alpha}, for enhancing the adhesion response. We showed that adhesion was enhanced when eosinophils were preincubated with TNF-{alpha} in contact with HBEC, but not in suspension. Eosinophil contact with FCS-coated plates also enhanced adhesion, suggesting that release of soluble mediators from HBEC was not required for priming. We also showed that establishing eosinophil-HBEC contact before addition of TNF-{alpha} promoted a greater increase in adhesion than when eosinophils and TNF-{alpha} were coincubated with HBEC, further highlighting the importance of contact to the priming process.

We established that {beta}1 and {beta}2 integrin adhesion pathways contributed to primed adhesion. Specifically, we suggested that {beta}1 integrins were involved in the contact-dependent priming step and also contributed to adhesion of primed eosinophils to HBEC. Of the {beta}1 integrins investigated, we identified that {alpha}5{beta}1 integrin was expressed on eosinophils and could provide a contact-dependent priming signal. We also suggested that fibronectin associated with HBEC monolayers may act as a possible ligand for {alpha}5{beta}1 integrin. We showed that the manifestation of the contact-dependent priming event was activation of {alpha}M{beta}2 integrin, leading to increased eosinophil adhesion to HBEC via ICAM-1. Finally, we confirmed that activation of {alpha}5{beta}1 integrin could trigger {beta}2 integrin-dependent eosinophil adhesion.

The importance of eotaxin together with IL-5 in promoting the initial movement of eosinophils into airway tissue in asthma is not in dispute (30); however, our findings suggest that eotaxin may be ineffective at a later stage in the process, i.e., adhesion of eosinophils to airway epithelium. We showed previously that eotaxin increased eosinophil adhesion to human lung microvascular endothelial cells via activation of {beta}2 and {alpha}4{beta}1 integrins and adhesion to ICAM-1 and VCAM-1, respectively (41). Resting HBEC used in the present study expressed no VCAM-1 and only low levels of ICAM-1 (OD405 0.09 ± 0.02, n = 8); this may account for the lack of effect of eotaxin on eosinophil adhesion to HBEC. Furthermore, de-adhesive properties of eotaxin on {alpha}4{beta}1 integrin-mediated eosinophil adhesion have been reported (42), and these may also contribute to an apparent lack of effect of eotaxin on eosinophil adhesion to HBEC. A functional consequence of the difference between eotaxin and C5a on adhesion is seen in the disparity between effects on eosinophil degranulation. Specifically, adhesion-dependent processes are a prerequisite for eotaxin-induced release of granule products (43, 44, 45), while adhesion potentiates, but is not essential for, C5a-induced degranulation (7, 8, 45). We speculate that C5a may be more effective than eotaxin at releasing tissue-damaging granule products from eosinophils in the vicinity of the epithelium in asthma.

C5a-stimulated eosinophil adhesion to resting HBEC involved {beta}2 and {beta}1 integrins, as shown by us in the present study and others previously (15). Our results also show that the involvement in adhesion of one integrin may only be revealed when the second is inhibited. A similar cooperation between integrins contributed to eosinophil adhesion to endothelial cells (41, 46). The importance of integrin cooperation to eosinophil adhesion is particularly apparent in the present study. We showed that an anti-{beta}2 integrin mAb together with a mAb against either the {alpha}- or {beta}-chain of {alpha}5{beta}1 abolished adhesion to HBEC of TNF-{alpha}-primed/C5a-stimulated eosinophils. Together with our findings that contact-dependent priming of eosinophils with TNF-{alpha} activated {alpha}M{beta}2 integrin and cross-linking {alpha}5{beta}1 integrin caused an {alpha}m{beta}2 integrin-dependent increase in eosinophil adhesion, we suggest that activation of {alpha}5{beta}1 integrin during the priming step contributes to subsequent activation of {alpha}M{beta}2 integrin and adhesion to ICAM-1. It is likely that the role of {alpha}5{beta}1 integrin is not only confined to priming eosinophils but also contributes to the adhesion process. We speculate that {alpha}5{beta}1 integrin provides a priming effect at an early time point, but at later times during the adhesion assay this integrin may also serve as an adhesion ligand.

Given the propensity for signaling or cross-talk between integrins (47, 48, 49), it is not surprising that activation of one influences the activity of another. Studies with monocytes and neutrophils originally identified cross-talk between {alpha}5{beta}1 and {alpha}M{beta}2 as an important pathway for activation of these cells under inflammatory conditions (40, 50). Our study now suggests that cross-talk from {alpha}5{beta}1 to {alpha}M{beta}2 integrin may also contribute to activation of eosinophils in allergic inflammation. In our study, it is unlikely that activation of {alpha}M{beta}2 integrin occurs first, leading to {alpha}5{beta}1 integrin activation, because this would increase adhesion following priming alone, and this was not observed. Cooperative effects, similar to those of TNF-{alpha} and {alpha}5{beta}1 integrin, have been described for {alpha}5{beta}1 integrin with insulin on adhesion to fibronectin of Chinese hamster ovary cells and with IL-11 for CD34+ hemopoietic stem/progenitor cell adhesion (51, 52). Requirement for a soluble (TNF-{alpha}) and an adhesion-dependent ({alpha}5{beta}1 integrin) signal for eosinophil priming may prevent inappropriate activation of eosinophils in the circulation while enhancing adhesion in the airways. Evidence from studies with neutrophils suggests that leukocytes often require stimulation via adhesion molecules and soluble mediators, providing a dual control mechanism to regulate leukocyte activation (53, 54).

The signaling mechanisms responsible for integrin cross-talk and cooperative effects with other cell stimuli are a rapidly expanding area of research; however, the similarity and intimate nature of these signals make it difficult to dissect the precise contribution of each. It was not the aim of this study to determine the signaling mechanisms responsible for priming of eosinophil adhesion, but from what is known of the second messenger pathways involved in C5a, TNF-{alpha}, and {alpha}5{beta}1 integrin signaling in eosinophils and other cells it is possible to speculate. With others, we have shown that wortmannin, a selective inhibitor of phosphatidylinositol (PI) 3-kinase, reduced {beta}2 integrin-dependent eosinophil adhesion (55) and also inhibited, in part, adhesion of TNF-{alpha}-primed/C5a-stimulated eosinophils to HBEC (our unpublished observation). Eosinophils from allergic asthmatics are known to have elevated basal levels of PI 3-kinase that is enhanced further by TNF-{alpha} (56). Ligation of {alpha}5{beta}1 integrin enhanced protein kinase B activity, a downstream component of PI 3-kinase, and dramatically enhanced the ability of growth factor to stimulate this pathway in intestinal epithelial cells (57). In summary, we suggest that {alpha}5{beta}1 integrin, TNF-{alpha}, and C5a may converge on a similar signaling pathway, perhaps with PI 3-kinase as a common element, and enhance adhesion in this way.

Studies investigating expression of {alpha}5{beta}1 integrin on eosinophils, including our own, have produced conflicting results that may, in part, be explained by differences in mAbs and eosinophil isolation procedures (16, 17, 18). Using a negative selection technique to isolate eosinophils and two different anti-{alpha}5{beta}1 mAbs, we showed that while A5-PUJ2 gave positive staining of eosinophils, SAM-1 showed negative staining. Of the previous three studies, two also used a negative selection method to purify eosinophils (16, 18). Supporting our findings, Weber and colleagues (18) showed positive staining with A5-PUJ2, and Georas et al. (16) showed negative staining with SAM-1. In contrast, a third study used fMLP-induced changes in specific gravity to purify eosinophils and showed positive staining with SAM-1 (17). It was claimed that the concentration of fMLP used for the isolation procedure did not activate eosinophils (58); however, this does not exclude the possibility that subtle changes in cell phenotype, such as clustering of {alpha}5{beta}1 integrin on the cell surface, may occur that would facilitate Ab binding. An example of this is the binding of mAb to CD147 molecules on PHA-activated T cells (59). In this case, binding was due to bivalent binding of relatively low-affinity mAbs to clustered or more densely expressed CD147 molecules on the cell surface rather than to recognition of a true activation-dependent neoepitope (59). We and others also showed functionality of {alpha}5{beta}1 integrin expression on eosinophils. Specifically, we demonstrated a 50% inhibition of TNF-{alpha}-primed/C5a-stimulated eosinophil adhesion to HBEC, while others showed 20 (A5-PUJ2) and 36% (SAM-1) reduction in eosinophil migration through endothelial cells and adhesion to fibronectin, respectively (17, 18).

As A5-PUJ2 did not bind to HBEC monolayers, we suggest that the adhesion-blocking properties of this Ab are due to its capacity to block {alpha}5 integrin expressed on eosinophils. Epithelial cells do express {alpha}5 integrin, and, in fact, we showed that SAM-1 bound to HBEC in this study. One explanation for binding of SAM-1 to HBEC, but not A5-PUJ2 and the reverse for eosinophils, may be that epitopes on {alpha}5 integrin vary between cells. This phenomenon was described for L-selectin on eosinophils and neutrophils (60) and may also apply to other adhesion molecules. Alternatively, we speculate that A5-PUJ2 may recognize an epitope that is blocked by cellular fibronectin produced by HBEC, whereas the SAM-1 epitope may be removed from the ligand-binding site and may thus be available for mAb binding during an ELISA.

Fibronectin, a matrix protein ligand for {alpha}5{beta}1 integrin, was detected on HBEC, and an anti-fibronectin mAb reduced eosinophil adhesion in a manner similar to anti-{beta}1 integrin mAb. This suggests that fibronectin, a matrix protein implicated in the pathogenesis of asthma (61, 62, 63), may act as an HBEC-associated ligand for eosinophil {alpha}5{beta}1 integrin. In contrast, we could not find a role for {alpha}4{beta}1 integrin in adhesion of primed eosinophils to HBEC using a mAb (2B4) that we and others have previously shown reduces eosinophil adhesion to fibronectin and VCAM-1 (35, 41, 64). Different regions of fibronectin bind to {alpha}5{beta}1 and {alpha}4{beta}1 integrins; {alpha}5{beta}1 binds to the middle portion of the fibronectin polypeptide containing the amino acid sequence Arg-Gly-Asp (RGD), whereas {alpha}4{beta}1 binds to a spliced variant containing the connecting segment region (CS-1) (65, 66). One explanation for the lack of effect of anti-{alpha}4 mAb compared with an inhibitory effect with anti-{alpha}5 mAb in our study may be that the CS-1-containing variant of fibronectin is not produced by HBEC, or may not be presented in a form that {alpha}4{beta}1 integrin recognizes. Another explanation may be the following: studies investigating the contribution of {alpha}4{beta}1 and {alpha}5{beta}1 integrins to eosinophil adhesion to purified fibronectin suggested that {alpha}4{beta}1 integrin contributed significantly to adhesion of unactivated eosinophils (67), whereas {alpha}5{beta}1 integrin only contributed to adhesion of activated eosinophils (17). In our study, a role for {alpha}5{beta}1 integrin in eosinophil adhesion to HBEC is only apparent when eosinophils are activated with C5a or TNF-{alpha} and C5a. Adhesion of unactivated eosinophils to HBEC was not inhibited with an anti-{alpha}5{beta}1 integrin mAb, suggesting that this integrin is unlikely to mediate adhesion of unactivated eosinophils to HBEC. For similar reasons, adhesion of TNF-{alpha}/C5a-activated eosinophils to FCS, known to contain fibronectin with both CS-1 and RGD binding sites (68), may also involve {alpha}5{beta}1 integrin.

In summary, our results emphasize the potential importance of C5a as a mediator that promotes eosinophil adhesion to airway epithelial cells. We showed that TNF-{alpha} primed eosinophil adhesion, as does IL-5, and demonstrated that {alpha}5{beta}1 integrin expressed on eosinophils plays an essential role in increasing eosinophil adhesion to epithelial cells under inflammatory conditions. A recent preliminary in vivo study showed that anti-{alpha}5 mAb (5H10-27) inhibited eosinophil accumulation in the lungs of allergen-challenged mice (69), further supporting a key role for {alpha}5{beta}1 integrin in eosinophil function in allergic inflammation. To date, pharmacological intervention to prevent eosinophil adhesion in the airways has been directed at {alpha}4{beta}1 integrin. On the basis of our results, we speculate that {alpha}5{beta}1 integrin may provide a new target that could be modulated therapeutically to prevent eosinophil accumulation or activation in allergic inflammation.


   Acknowledgments
 
We thank Dr. Martin Hemler (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA) and Dr. Alison Humbles (Ina Sue Perlmutter Laboratory, Children’s Hospital, Harvard Medical School) for helpful discussion of this manuscript.


   Footnotes
 
1 A.B.-G. was supported by the National Asthma Campaign, K.B. was supported by the British Heart Foundation, and A.H. was supported by the Wellcome Trust. Back

2 Address correspondence and reprint requests to Dr. Anne Burke-Gaffney, Unit of Critical Care, National Heart and Lung Institute Division, Imperial College of Science, Technology and Medicine, Dovehouse Street, London, SW3 6LY, U.K. E-mail address: a.burke-gaffney{at}ic.ac.uk Back

3 Current address: Selective Genetics, San Diego, CA 92121. Back

4 Current address: Leukocyte Biology, Division of Biomedical Sciences, Imperial College of Science, Technology and Medicine, London, U.K. Back

5 Abbreviations used in this paper: HBEC, human bronchial epithelial cell; ACLB, albumin-coated latex bead; t, time; MFI, mean fluorescence intensity; PI, phosphatidylinositol. Back

Received for publication March 14, 2001. Accepted for publication November 20, 2001.


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 Results
 Discussion
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