The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, L.
Right arrow Articles by Roos, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, L.
Right arrow Articles by Roos, D.
The Journal of Immunology, 1998, 161: 3064-3070.
Copyright © 1998 by The American Association of Immunologists

Triple Role of Platelet-Activating Factor in Eosinophil Migration Across Monolayers of Lung Epithelial Cells: Eosinophil Chemoattractant and Priming Agent and Epithelial Cell Activator1

Lixin Liu*, Astrid E. M. Zuurbier*, Frederik P. J. Mul*, Arthur J. Verhoeven*, René Lutter{dagger}, Edward F. Knol* and Dirk Roos2,*

* Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology, and {dagger} Department of Pulmonology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Infiltration of eosinophils into the lung lumen is a hallmark of allergic asthmatic inflammation. To reach the lung lumen, eosinophils must migrate across the vascular endothelium, through the interstitial matrix, and across the lung epithelium. The regulation of this process is obscure. In this study, we investigated the migration of human eosinophils across confluent monolayers of either human lung H292 epithelial cells or primary human bronchial epithelial cells. Established eosinophil chemoattractants (IL-8, RANTES, platelet-activating factor (PAF), leukotriene B4, and complement fragment 5a (C5a)) or activation of the epithelial cells with IL-1ß induced little eosinophil transmigration (<7% in 2 h). In contrast, addition of PAF in combination with C5a induced extensive (>20%) transepithelial migration of unprimed and IL-5-primed eosinophils. Eosinophil migration assessed in a Boyden chamber assay, i.e., without an epithelial monolayer, was only slightly increased upon addition of PAF and C5a. Preincubation of eosinophils with the PAF receptor antagonist WEB 2086 only inhibited migration of unprimed eosinophils toward PAF and C5a, whereas preincubation of epithelial cells with WEB 2086 abolished migration of both IL-5-primed and unprimed eosinophils. This latter result indicated the presence of PAF receptors on epithelial cells. Indeed, addition of PAF to epithelial cells induced an increase in cytosolic free Ca2+, which was blocked by the PAF receptor antagonists WEB 2086 and TCV-309. Our results show that PAF induces permissive changes in epithelial cells, and that PAF acts as a chemoattractant and priming agent for the eosinophils.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Allergic asthma is characterized by massive infiltration of eosinophils into the lung. Eosinophils are considered to play an important role in the pathogenesis of allergic asthma (1, 2). After infiltration of the allergic reaction sites, eosinophils release toxic granule proteins, causing tissue damage, and generate lipid mediators that can influence the behavior of surrounding vasculature and airway smooth muscle cells. To reach the airway lumen, circulating eosinophils must first extravasate; i.e., the cells initially roll on the endothelium, followed by firm adhesion to and migration across endothelial cells (1, 3). Thereafter, the eosinophils migrate through the interstitial matrix and across the epithelium into the lung lumen (1, 4, 5). The migration to the sites of inflammation is presumed to be regulated at the levels of 1) adhesion receptors that mediate transient or firm adhesion to inflamed vascular endothelium and to extracellular matrix molecules, 2) activating factors (cytokines and chemokines) that induce migration and expression of adhesion molecules and their ligands, and 3) cells that are present at the inflammatory site and regulate the release of these activating factors.

The first step in the migration process is the interaction of circulating eosinophils with the vascular endothelium at the allergic reaction site. Endothelial cells actively regulate leukocyte infiltration in inflammatory tissues through different mechanisms, such as vasodilatation, expression of adhesion molecules, opening of intercellular junctions, and secretion of chemotactic factors (6). One can imagine that selective up-regulation and/or activation of adhesion molecules on eosinophils and endothelial cells by cytokines and the production of chemokines and/or chemoattractants may promote specific eosinophil migration and their subsequent accumulation in tissues. However, migration studies have shown that the so-called allergy-related cytokines or chemokines are not selective in attracting eosinophils (3, 7, 8, 9). These findings imply that the selective activation and attraction of eosinophils toward sites of allergic inflammation are the results of a complex interplay among different cell types, mediators, and adhesion molecules.

Recently, attention has focused on the role of the epithelium in leukocyte infiltration into inflammatory sites (10, 11, 12, 13). Besides the barrier function for protection against pathogen invasion, epithelial cells play an active role in the induction of inflammation through the expression of cellular adhesion molecules, such as ICAM-1 (14, 15, 16) and integrins (17). Another role of epithelial cells in lung inflammation involves the synthesis of a variety of proinflammatory mediators, including RANTES, PAF,3 and IL-8 after IL-1ß or TNF-{alpha} stimulation (18), and granulocyte-macrophage CSF after stimulation with eosinophil peroxidase (19).

Whereas eosinophil migration across endothelial monolayers has been described extensively (1, 3, 6, 8, 9), little is known about the mechanisms regulating eosinophil migration across epithelial monolayers. In the present study, we investigated human eosinophil migration across confluent monolayers of lung epithelial cell line cells (H292) and primary human bronchial epithelial cells (HBEC) in vitro. Substantial eosinophil transmigration was observed with combinations of PAF and C5a, or PAF and LTB4, but not with these chemoattractants alone. Besides the conventional potent chemotactic role of PAF and the priming effect of PAF on eosinophils, we also found that a permissive change in the epithelial monolayers induced by PAF is pivotal for efficient eosinophil transepithelial migration.


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

Recombinant human (rh) TNF-{alpha} was a gift from Dr. A. Creasy (Cetus, Oakland, CA). PAF, C5a, FMLP, and LTB4 were purchased from Sigma (St. Louis, MO). rhRANTES was purchased from Life Technologies (Gaithersburg, MD). The PAF receptor antagonist WEB 2086 was a gift from Dr. H. Heuer (Boehringer Ingelheim, Ingelheim, Germany), and the PAF receptor antagonist TCV-309 was purchased from Takeda (Osaka, Japan) (20). RhIL-1ß and rhIL-5 were purchased from Pepro Tech (Rocky Hill, NJ), and rhIL-8 and rhIFN-{gamma} were obtained from Boehringer Mannheim (Mannheim, Germany). WEB 2086 was dissolved in DMSO at 1000 times the final concentration and was stored at -20°C. C5a, PAF, FMLP, RANTES, LTB4, IL-1ß, IL-5, IL-8, and IFN-{gamma} were dissolved in PBS supplemented with 0.5% (w/v) human serum albumin (HSA; Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands) and were stored at -20°C. HEPES medium contained 132 mM NaCl, 6.0 mM KCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 1.2 mM KH2PO4, 20 mM HEPES, 5.5 mM glucose, and 0.5% (w/v) HSA. Incubation medium used in the transmigration assay consisted of a 1/1 mixture of RPMI 1640 and medium 199 (Life Technologies, Paisley, U.K.) supplemented with 0.5% (w/v) HSA. ELISA sample buffer consisted of PBS supplemented with 0.1% (v/v) Tween-20 (Merck, Schuchardt, Germany), 0.2% (w/v) hexadecyl-trimethyl-ammonium bromide (Sigma), 0.2% (w/v) BSA (Sigma), and 20 mM EDTA.

Cell culture

The human lung adenocarcinoma-derived cell line H292 (American Type Culture Collection CRL 1848, Rockville, MD) (21) was grown in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS (Life Technologies), 100 U/ml penicillin (Life Technologies), 100 µg/ml streptomycin (Life Technologies), and 2 mM glutamine (Merck). The doubling time of the H292 cells was approximately 24 h. Primary HBEC were obtained from bronchial tissues with a microscopically normal appearance from patients undergoing thoracotomy. The cells were cultured in a keratinocyte medium (Keratinocyte-SKM, Life Technologies) (14). After confluence was reached, cell suspensions were obtained by proteolysis with trypsin/EDTA (Life Technologies). The activity of trypsin was inhibited immediately after detachment of the cells by means of soybean trypsin inhibitor type II (Sigma), and the cells were subsequently transferred for propagation. The 4th to 30th passages of H292 cells and the 3rd to 4th passages of HBEC were used for subculture on polycarbonate membranes (3.0-µm pore size, 12 mm diameter) of inverted transwells (Costar, Cambridge, MA), according to the method of Parkos et al. (22) with minor modifications (23). In brief, sterile polyoxymethylene polyacetal collars with an inner diameter equal to the outer diameter of the transwells and with a height of 13 mm were tightly fixed to the bottoms of the transwells. Epithelial cell suspensions were added to the inverted transwells and were allowed to attach for 18 h. Thereafter, the collars were removed, and the transwells were placed upright in culture dishes and incubated for 5 days. In this way, monolayers of epithelial cells hanging underneath the membranes were obtained. Complete confluence of the inverted epithelial cell monolayers was reached as determined by May-Grunwald/Giemsa staining and light microscopy, and the confluence of the monolayers was confirmed by [3H]inulin (Amersham, Aylesbury, U.K.) leakage experiments (24).

Granulocyte isolation

Blood was obtained from healthy volunteers. Granulocytes were purified from a buffy coat of 500 ml of blood by density gradient centrifugation over isotonic Percoll (Pharmacia, Uppsala, Sweden) (25). After lysis of the erythrocytes in the pellet fraction with a cold lysis buffer containing 155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA (pH 7.4), the granulocytes were washed twice in PBS and resuspended in HEPES medium without CaCl2.

Eosinophil purification

Human eosinophils were purified by means of the FMLP method (26). In brief, granulocytes in HEPES medium without CaCl2 were incubated for 30 min at 37°C to restore the initial density of the cells. The cells were washed, resuspended in PBS supplemented with 0.5% (w/v) HSA and 13 mM trisodium citrate, and incubated for 5 min in a shaking water bath at 37°C. The incubation was continued for 10 min after the addition of 10 nM FMLP to the cell suspension. Thereafter, the eosinophils were purified by centrifugation (15 min, 1000 x g) over isotonic Percoll (1.082 g/ml, pH 7.4), washed, and resuspended in HEPES medium. The purity and viability of the eosinophils were >95%. Contaminating cells were mostly neutrophils. In some experiments, eosinophils were isolated by means of immunomagnetic removal of CD16-expressing cells (27).

Eosinophil transmigration

Fresh medium was added to the transwells 4 h before the start of the transmigration assay, and the transwells were washed twice just before starting the experiment. The lower compartment was filled with prewarmed incubation medium with or without chemoattractants. Eosinophils (5 x 105 cells in 0.5 ml of prewarmed incubation medium) were placed in the upper compartment, and the transwells were incubated for 2 h at 37°C with 5% CO2 and maximal humidity. Whenever indicated, eosinophils and/or epithelial cells were preincubated with 10 µM WEB 2086 for 5 min before the start of the transmigration assay. When epithelial cells were treated with WEB 2086, WEB 2086 was added to the lower compartment. WEB 2086 remained present throughout the experiment. After the 2-h incubation, the upper and lower compartments were washed separately with incubation medium and ELISA sample buffer, respectively, and the fluids were collected. The cells in the collected fluids and in the excised membranes were lysed in ELISA sample buffer. The percentage of eosinophils that had transmigrated was determined by means of an ELISA for eosinophil cationic protein (ECP).

ECP quantification

The amount of ECP as a measure for the number of eosinophils in different cell preparations was determined by means of a slightly modified, previously described ELISA (28). In brief, specific polyclonal rabbit antiserum (poAb) against human ECP was obtained by immunization of a rabbit with highly purified human ECP (29). Human ECP was a gift from Prof. I. Olsson (Lund, Sweden). The Ig fraction of the rabbit serum was isolated by ammonium sulfate precipitation and was biotinylated (30).

Culture plates with 96 wells (Maxisorb, Nunc, Roskilde, Denmark) were coated overnight at 4°C with 100 µl of 3 µg/ml rabbit anti-ECP poAb diluted in 0.1 mM NaHCO3. After each incubation step, the plates were washed three times with PBS containing 0.1% (v/v) BSA and Tween-20. The wells were blocked with blocking buffer, consisting of PBS supplemented with 0.2% (w/v) BSA and 0.1% (v/v) Tween-20, for 1 h at 37°C. Highly purified ECP and samples were diluted in ELISA sample buffer, added at a volume of 100 µl/well, and incubated for 2 h at 37°C. The wells were subsequently incubated with 100 µl of 1 µg/ml biotin-conjugated anti-ECP poAb diluted in block buffer for 90 min at 37°C. The biotin-labeled Abs were allowed to bind avidin-biotinylated alkaline phosphatase complex (Dako, Glostrup, Denmark) diluted in Tris-buffered saline supplemented with 0.1% (v/v) Tween-20 and 0.2% (w/v) BSA for 30 min at room temperature according to the manufacturer’s description. Enzymatic activity was detected with 1 mg/ml phosphatase substrate (Sigma 104) dissolved in 1 M diethanolamine, 0.5 mM MgCl2, and 0.02% NaN3 (pH 9.8). The absorbance was measured after 60 min in a Multiscan Multisoft microplate reader (Labsystems Oy, Helsinki, Finland) at 405 nm. The sensitivity of this assay ranged from 0.1 to 10 ng/ml of ECP. We confirmed that this ECP ELISA assay was highly specific for eosinophils, i.e., no reaction was measured in lysates of human monocytes, lymphocytes, or neutrophils. In addition, eosinophil migration quantified by means of the ECP ELISA and that determined by cell counting in the lower compartment were comparable. The total amount of ECP added to the transwell system as well as that in each compartment separately (upper compartment, lower compartment, and membrane) were determined. The percentage of recovery was always >85%. The percentage of eosinophils that had transmigrated was calculated from the amount of ECP detected in the lower compartment in relation to the total amount of added ECP. Alternatively, transepithelial migration of calcein-AM (Molecular Probes, Eugene, OR)-labeled eosinophils was measured by both ECP detection and calcein quantification.

Calcein labeling

Eosinophils were labeled with calcein-AM before the onset of the transmigration assay. The cells (10 x 106/ml) were labeled with 4 µg/ml calcein-AM diluted in HEPES medium for 45 min at 37°C. After labeling, the cells were washed three times with HEPES medium. The transmigration assay with calcein-AM-labeled eosinophils was performed as described above, except that HEPES medium was used instead of incubation medium. The concentrations of calcein-AM in the upper compartment, lower compartment, and membrane were measured with a spectrofluorometer (model RF-540, Shimadzu, Kyoto, Japan). The percentage of eosinophils that had transmigrated was calculated from the amount of fluorescence detected in the lower compartment in relation to the fluorescence of the total added calcein-AM-labeled eosinophils.

Chemotaxis assay

Chemotaxis in Boyden chambers was measured with a computerized image analyzer (Quantimet 600, Leica, Cambridge, U.K.) to quantify eosinophil migration into filters after an incubation period of 1 h (31).

Intracellular free Ca2+ concentration ([Ca2+]i) measurement

For the [Ca2+]i measurement, cells (6–10 x 106/ml in HEPES medium) in suspension were loaded with 1 µM indo-1/AM (Molecular Probes) for 40 min at 37°C (32). The cells were washed, resuspended in HEPES medium to the previous concentration, and kept in the dark at room temperature. Before being transferred to a cuvette, the indo-1/AM-loaded cells were diluted 10 times in HEPES medium and were prewarmed for 5 min at 37°C. Fluorescence changes in the magnetically stirred cells were monitored with a spectrofluorometer (model RF-540, Shimadzu), with 340 and 390 nm as excitation and emission wavelengths, respectively. To calibrate indo-1 fluorescence as a function of [Ca2+]i, all trapped indo-1 was saturated with Ca2+ by addition of 10 µM digitonin, after which indo-1 fluorescence was quenched with 0.5 mM MnCl2. A dissociation constant of 250 nM for the indo-1-Ca2+ complex was used to calculate [Ca2+]i (33).

Statistical analysis

Results were expressed as the mean ± SEM of the number of different experiments mentioned in the figure and table legends. Results were analyzed using Student’s t test. One-sided p values were calculated, and p values exceeding 0.05 were considered not significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Low eosinophil transepithelial migration induced by individual chemoattractants

Human eosinophil migration across confluent monolayers of lung epithelial H292 cell line cells was analyzed in the physiologic basolateral to apical direction (with the epithelial cells growing underneath a filter membrane). Eosinophil transepithelial migration reached its plateau after 2 h of incubation (our unpublished observations). The monolayers remained intact during the transmigration assay, as checked by examination of the filter by light microscopy and by determination of [3H]inulin diffusion through the monolayer before and after the transmigration assay.

Individual chemoattractants (C5a, PAF, IL-8, RANTES, or LTB4) induced little transmigration of IL-5-primed eosinophils, i.e., maximally 7% (Table IGo). Pretreatment of the epithelial monolayers with 5 ng/ml IL-1ß for 4 h (Table IGo) or with TNF-{alpha} and/or IFN-{gamma} for 4 or 24 h (not shown) did not induce this migration either. In these studies eosinophils that were isolated by means of removal of CD16-expressing cells showed a similar response. Moreover, quantification of eosinophil migration by means of ECP measurement yielded results similar to those obtained by calcein fluorescence measurement.


View this table:
[in this window]
[in a new window]
  		Table I. Eosinophil transepithelial migration induced by individual chemoattractants1

 
Enhancement of eosinophil transmigration by PAF

In general, we observed that neither individual chemoattractants nor most combinations of chemoattractants induced >10% of eosinophil transepithelial migration (Tables I and II). Only when PAF was combined with C5a, LTB4, or RANTES did higher percentages of eosinophils migrate across the monolayers (Table IIGo). The enhanced migration toward PAF/C5a (up to 25%) was not only observed with IL-5-primed eosinophils but also with unprimed cells (Fig. 1Go). In addition, PAF/C5a enhanced the migration of IL-5-primed eosinophils across primary HBEC (C5a, 6.5%; PAF, 9.5%; C5a/PAF, 34.6%). This synergistic response was not observed when C5a was combined with LTB4 (Table IIGo); thus, the enhancement must be due to the effect of PAF. Both PAF and C5a play a chemotactic role in these transmigration assays, because the addition of either of these agents to the upper compartment just before starting the migration assay resulted in a dose-dependent inhibition of eosinophil transmigration toward PAF/C5a in the lower compartment (not shown). PAF did not induce damage to the epithelial cell layers, as measured by light microscopy and determination of [3H]inulin diffusion through the monolayer.


View this table:
[in this window]
[in a new window]
  		Table II. Eosinophil transepithelial migration induced by combinations of chemoattractants1

 


View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 1. Human eosinophil migration across monolayers of lung H292 epithelial cells induced by PAF and/or C5a. Unprimed eosinophils (hatched bars) or IL-5-primed eosinophils (solid bars; 106 cells/ml) were added to the upper compartment of the transwell system, and chemotactic solutions of PAF (10-6 M), C5a (10-8 M), or PAF plus C5a were added to the lower compartment. Eosinophil transmigration was measured after a 2-h incubation at 37°C. Asterisks indicate that eosinophil transmigration toward the combination of PAF and C5a was significantly greater than the sum of the results with PAF and C5a alone (p < 0.05). Data are the mean ± SEM of four experiments.

 
Eosinophil chemotaxis in a modified Boyden chamber assay

To investigate the effect of PAF on eosinophil migration toward C5a in the absence of epithelial cells, chemotaxis of eosinophils toward PAF, C5a, or combinations was assessed. The total distance migrated by eosinophils in filters was determined in a Boyden chamber assay. Addition of PAF or C5a to the lower compartment as well as IL-5 priming of eosinophils were found to enhance migration, but these three factors together appeared to render the strongest migration stimulus (Fig. 2Go).



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 2. Migration of eosinophils in the Boyden chamber chemotaxis assay. Unprimed (hatched bars) or IL-5-primed (solid bars) eosinophils (2 x 106/ml) were added to the upper chambers, and chemotactic solutions of C5a (10-8 M), PAF (10-6 M) or PAF plus C5a were placed in the lower chambers. A 150-µm thick filter (8-µm pore size) and a "stop" filter (0.45-µm pore size) were placed between the two chambers. After 1 h at 37°C, the 8-µm filter was removed, fixed in butanol/ethanol (20/80%, v/v), and stained with Weigert solution. Eosinophil migration was analyzed with a computerized Image Analyzer (Quantimet 600) by measuring the total distance of all eosinophils that had migrated >10 µm into the 150-µm filter. Results are mean ± SEM of three experiments.

 
Effect of WEB 2086 on eosinophil transepithelial migration toward PAF and C5a

The role of PAF in inducing eosinophil migration across epithelial monolayers toward C5a was further analyzed with the PAF receptor antagonist WEB 2086. When eosinophils were treated with WEB 2086, transmigration of unprimed eosinophils was inhibited, whereas transmigration of IL-5-primed cells remained unaffected, even when higher concentrations (up to 25 µM) of WEB 2086 were used (Fig. 3GoA). In contrast, the transmigration of both IL-5-primed and unprimed eosinophils was impaired when the epithelial monolayers were treated with WEB 2086 (Fig. 3GoA). WEB 2086 treatment of both eosinophils and epithelial cells did not yield more inhibition than WEB 2086 treatment of the epithelial cells alone. Eosinophil migration across monolayers of primary HBEC was comparably affected by WEB 2086 (Fig. 3GoB).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 3. The role of PAF in eosinophil migration across lung epithelial monolayers toward mixed chemotactic solutions of PAF (10-6 M) and C5a (10-8 M). Unprimed (hatched bars) and IL-5-primed (solid bars) eosinophils were tested for their response against this mixture (indicated by P/C). The PAF receptor antagonist WEB 2086 (10 µM) was incubated with eosinophils, epithelial cells, or both and remained present during the 2-h transmigration assay. Lung H292 epithelial cells (A; mean ± SEM; n = 4) and primary HBEC (B; mean; n = 2) were analyzed in the same experimental set-up. In A, asterisks mark the significant difference as compared with the control groups (C5a/PAF): * Indicates p < 0.05, and ** indicates p < 0.01. In a control experiment, 0.8% DMSO had no inhibitory effect.

 
Increase in cytosolic free Ca2+ concentration in epithelial cells induced by PAF

The inhibitory effect of WEB 2086 treatment of epithelial cells on transmigration of eosinophils indicated the existence of PAF receptors on lung epithelial cells. To investigate the response of epithelial cells to PAF binding, we measured the change in the intracellular free Ca2+ concentration in epithelial cells. Both H292 epithelial cells and primary HBEC showed a rapid [Ca2+]i increase upon addition of 10-6 M PAF (Fig. 4Go), and this response was completely abolished by pretreatment of the cells with two distinct PAF receptor antagonists, i.e., 10 µM WEB 2086 (Fig. 4Go) and 0.1 µM TCV-309 (not shown). Addition of C5a (10-8 M), IL-8 (10-8 M), FMLP (10-8M), WEB 2086 (10 or 25 µM), or TCV-309 (0.1 µM) to epithelial cells did not cause a Ca2+ response (not shown). Moreover, HUVEC did not show a Ca2+ response upon addition of PAF, revealing a difference in this respect between epithelial and endothelial cells.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 4. Changes in the cytosolic free Ca2+ concentration of epithelial cells in suspension in response to PAF. H292 epithelial cells (A) and primary HBEC (B) were suspended in HEPES medium, and [Ca2+]i was measured as described in Materials and Methods. Epithelial cells in suspension showed a rapid increase in [Ca2+]i after PAF (10-6M) addition (arrow). In the lower graphs, 10 µM WEB 2086 had been incubated with the epithelial cells 1–4 min (37°C) before PAF addition.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Experimental set-up

In this study, we investigated the migration of human eosinophils across monolayers of human lung epithelial cell line H292 cells and primary HBEC. The H292 epithelial cell line was chosen because these cells form polarized confluent monolayers and functional tight junctions in a reproducible manner (24). Moreover, H292 cells resemble differentiated primary bronchial epithelial cells, for instance in the expression of mucins and cytokeratins (our unpublished observations). In a previous study, we demonstrated that human neutrophil migration across lung epithelial cells is strongly dependent on the polarity of the epithelial cells, i.e., much more pronounced in the physiologic basolateral to apical direction than in the opposite direction (23). Eosinophils exhibit the same preferential transepithelial migration, but to a lesser extent (our unpublished observations). Therefore, the epithelial cells were routinely cultured in the inverted position, with the apical side toward the lower compartment of the transwell system. The integrity of the epithelial monolayers was checked after each eosinophil migration assay, for eosinophils may release toxic components upon activation (7, 34, 35, 36, 37) and thus may damage the epithelial monolayers. However, we did not detect a decreased integrity of the epithelial cell monolayer after eosinophil passage or after incubation with PAF.

Eosinophil migration was detected by means of an ECP ELISA (28). Quantification of this specific eosinophil marker excludes neutrophil interference in the determination of eosinophil migration. This is necessary because the eosinophil suspensions are generally contaminated with 0 to 4% neutrophils. Detection of eosinophil migration by cell counting or by calcein fluorescence measurement (38) yielded similar results as ECP measurement. Thus, we found no indication for ECP release during eosinophil migration or selective migration of ECP-rich or ECP-poor eosinophils.

Chemotactic and priming role of PAF

Transepithelial migration of primed eosinophils was low. Even PAF, the most potent chemoattractant, attracted <7% of the eosinophils. Resnick et al. (39) also observed low eosinophil migration across human intestinal epithelium toward individual chemoattractants, but, in contrast, relatively high migration toward PAF. Apparently, PAF induces more eosinophil migration across intestinal epithelium than across bronchial epithelium.

We show that eosinophil migration across bronchial epithelium toward C5a, LTB4, or RANTES is synergistically enhanced by PAF. This stimulatory effect was specific for PAF, but did not occur when PAF was combined with other chemoattractants. On the basis of these results, we predict that in human eosinophils, PAF activates a signaling pathway that differs from those activated by C5a, LTB4, and RANTES.

Eosinophils need to be primed (e.g., by IL-5) for induction of migration across epithelial cells; i.e., unprimed eosinophils hardly migrated across epithelial monolayers. However, massive migration of unprimed eosinophils across epithelial monolayers was induced by PAF/C5a, and this migration could be inhibited by treatment of the eosinophils with the PAF receptor antagonist WEB 2086. In contrast, treatment of IL-5-primed eosinophils with WEB 2086 did not inhibit transepithelial migration toward PAF/C5a. This reveals the priming role of PAF that had diffused from the lower compartment to unprimed eosinophils in the upper compartment. In addition, PAF and C5a both induce chemotaxis of eosinophils in a Boyden chamber assay. Thus, C5a is a chemoattractant for eosinophils, and PAF is not only a chemoattractant but also a primer for eosinophils.

Epithelium-activating role of PAF

Our results indicate that the synergistic transepithelial migration of eosinophils toward C5a/PAF is partly due to the direct action of PAF on the epithelial cells. We found that the synergistic enhancement by PAF was not observed when the eosinophils migrated in the absence of epithelial cells. Moreover, treatment of the epithelial monolayer with the PAF receptor antagonist WEB 2086 strongly decreased the transepithelial migration toward C5a/PAF. Expression of PAF receptors in bronchial epithelial cells was confirmed by the finding that PAF induces a rapid increase in [Ca2+]i in epithelial cells, a response that was prevented by pretreatment of epithelial cells with the PAF receptor antagonists WEB 2086 and TCV-309. PAF receptors have previously been identified in epithelial cells derived from the chinchilla middle ear (40); feline, canine, guinea pig, and cow trachea (41, 42, 43, 44); and rabbit cornea (45). PAF induces an increase in [Ca2+]i, acts as a mucous secretagogue, and decreases the ciliary beat frequency of the tracheal epithelial cells (41, 42, 43, 46). Moreover, PAF receptors have been identified on human primary bronchial epithelial cells (47), and it has been shown that PAF induces up-regulation of the nuclear transcription factor activator protein-1 in these cells (47). Binding of PAF to the receptors in bronchial epithelial cells may cause transduction of signals leading to functional changes, such as the ability to permit eosinophil transmigration.

Together, these data strongly suggest that PAF induces permissive changes in epithelial cells that favor eosinophil transmigration. However, the exact nature of the epithelial changes that allow eosinophil transmigration remains to be elucidated. The role of PAF is currently under investigation. Preliminary results suggest that the morphology of bronchial epithelial cells is unaffected by PAF, and that the monolayers do not become more "leaky" when PAF is present. Moreover, we found that ICAM-1 expression is not up-regulated by PAF (4-h incubation with PAF; unpublished observation). Thus, PAF-treated bronchial epithelial cells do not appear to be activated or morphologically changed. PAF possibly induces more subtle changes in the epithelial cells. One possible explanation might be that tight junction resistance is decreased as a result of the PAF-induced [Ca2+]i increase. Regulation of tight junction resistance by [Ca2+]i elevation has been shown in human intestinal epithelial T84 cell line cells (48). Tight junction resistance in T84 cells is unaffected by PAF (39), because these epithelial cells do not express PAF receptors (49). However, inulin leakage experiments did not show increased permeability of the monolayers. Another possibility is that PAF induces changes in cortical actin, which affects cell-cell adhesion of epithelial cells and may result in augmented transmigration (50).

Differences between endothelial and epithelial cells

PAF did not induce an increase in [Ca2+]i in endothelial cells from umbilical veins (HUVEC), and PAF did not enhance eosinophil migration across HUVEC toward C5a (6, 51) (not shown). Thus, HUVEC probably lack a functional PAF receptor. However, endothelial cells are capable of PAF production after incubation with IL-1ß or TNF-{alpha} (52, 53), in contrast to epithelial cells (23). These results confirm the importance of the stimulatory effects of PAF on eosinophil transepithelial migration. Moreover, this demonstrates that different mechanisms control eosinophil migration across the endothelium and epithelium.

PAF did not enhance eosinophil transepithelial migration toward IL-8. It is known that IL-8 is a very poor chemoattractant for eosinophils (9). We have previously shown that lung epithelial cells generate IL-8 after activation with IL-1ß (23, 54). In accordance, eosinophil migration across IL-1ß-stimulated epithelial monolayers was not enhanced by PAF. These results suggest that IL-8 is too weak to induce eosinophil migration even in the case of primed eosinophils and PAF-activated epithelium.

Conclusion

Together, our results show that human eosinophils migrate massively across lung epithelial monolayers in response to a chemotactic gradient of PAF in combination with a potent chemoattractant (C5a or LTB4). In this process, PAF acts as a priming agent and as a chemoattractant for the eosinophils. In addition, PAF induces transmigration-permissive changes of the epithelial cells. Clarification of this epithelial activation may help in the development of drugs to prevent eosinophil migration into the lungs and concomitant damage to the epithelium.


   Acknowledgments
 
We thank Dr. P. S. Hiemstra and S. van Wetering for providing the primary human bronchial epithelial cells, and Drs. Arne Egesten and Anton T. J. Tool for helpful discussions and technical assistance. We also thank Prof. Inge Olsson (Lund University, Lund, Sweden) for providing purified ECP.


   Footnotes
 
1 This study was supported by The Netherlands Asthma Foundation (Grant 32.93.13) and Nederlandse Fondsenwervingsacties Volksgezondheid (Grant 93.044/VG). Back

2 Address correspondence and reprint requests to Dr. Dirk Roos, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail address: Back

3 Abbreviations used in this paper: PAF, platelet-activating factor; HBEC, human bronchial epithelial cells; C5a, complement fragment 5a; LTB4, leukotriene B4; rh, recombinant human; HSA, human serum albumin; ECP, eosinophil cationic protein; [Ca2+]i, intracellular free Ca2+ concentration. Back

Received for publication June 13, 1997. Accepted for publication April 30, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Knol, E. F., D. Roos. 1996. Mechanisms regulating eosinophil extravasation in asthma. Eur. Respir. J. 9:136s.
  2. Weller, P. F., K. Lim, H. C. Wan, A. M. Dvorak, D. T. W. Wong, W. W. Cruikshank, H. Kornfeld, D. M. Center. 1996. Role of eosinophil in allergic reactions. Eur. Respir. J. 9:109s.
  3. Moser, R., J. Fehr, T. Olgiati, P. L. B. Bruijnzeel. 1992. Migration of primed human eosinophils across cytokine-activated endothelial cell monolayers. Blood 79:2937.[Abstract/Free Full Text]
  4. Lukacs, N. W., R. M. Strieter, S. L. Kunkel. 1995. Leukocyte infiltration in allergic airway inflammation. Am. J. Respir. Cell Mol. Biol. 13:1.[Abstract]
  5. Roman, J.. 1996. Extracellular matrix and lung inflammation. Immunol. Res. 15:163.[Medline]
  6. Ebisawa, M., M. C. Liu, T. Yamada, M. Kato, L. M. Lichtenstein, B. S. Bochner, R. P. Schleimer. 1994. Eosinophil transendothelial migration induced by cytokines. II. Potentiation of eosinophil transendothelial migration by eosinophil-active cytokines. J. Immunol. 152:4590.[Abstract]
  7. Rot, A., M. Krieger, T. Brunner, S. C. Bischoff, T. J. Schall, C. A. Dahinden. 1992. RANTES and macrophage inflammatory protein 1{alpha} induce the migration and activation of normal human eosinophil granulocytes. J. Exp. Med. 176:1489.[Abstract/Free Full Text]
  8. Kitayama, J., M. W. Carr, S. J. Roth, J. Buccola, T. A. Springer. 1997. Contrasting responses to multiple chemotactic stimuli in transendothelial migration. J. Immunol. 158:2340.[Abstract]
  9. Ebisawa, M., T. Yamada, C. Bickel, D. Klunk, R. P. Schleimer. 1994. Eosinophil transendothelial migration induced by cytokines. III. Effect of the chemokine RANTES. J. Immunol. 153:2153.[Abstract]
  10. Bloemen, P. G. M., M. C. van den Tweel, P. A. J. Henricks, F. Engels, M. J. V. Van de Velde, F. J. Blomjous, F. P. Nijkamp. 1996. Stimulation of both human bronchial epithelium and neutrophils is needed for maximal interactive adhesion. Am. J. Physiol. 270:L80.[Abstract/Free Full Text]
  11. Godding, V., J. M. Stark, J. B. Sedgwick, W. W. Busse. 1995. Adhesion of activated eosinophils to respiratory epithelial cells is enhanced by tumor necrosis factor-{alpha} and interleukin-1ß. Am. J. Respir. Cell Mol. Biol. 13:555.[Abstract]
  12. Rennard, S. I., D. J. Romberger, J. H. Sisson, S. G. Von Essen, I. Rubinstein, R. A. Robbins, J. R. Spurzem. 1994. Airway epithelial cells: functional roles in airway disease. Am. J. Respir. Crit. Care Med. 150:S27.
  13. Simon, R. H., III. R. Paine. 1995. Participation of pulmonary alveolar epithelial cells in lung inflammation. J. Lab. Clin. Med. 126:108.[Medline]
  14. Bloemen, P. G. M., M. C. van den Tweel, P. A. J. Henricks, F. Engels, S. S. Wagenaar, A. A. Rutten, F. P. Nijkamp. 1993. Expression and modulation of adhesion molecules on human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 9:586.
  15. Cunningham, A. C., J. A. Kirby. 1995. Regulation and function of adhesion molecule expression by human alveolar epithelial cells. Immunology 86:279.[Medline]
  16. Li, X. C., A. M. Jevnikar, D. R. Grant. 1997. Expression of functional ICAM-1 and VCAM-1 adhesion molecules by an immortalized epithelial cell clone derived from the small intestine. Cell. Immunol. 175:58.[Medline]
  17. Sheppard, D.. 1996. Epithelial integrins. BioEssays 18:655.[Medline]
  18. Stellato, C., L. A. Beck, G. A. Gorgone, D. Proud, T. J. Schall, S. J. Ono, L. M. Lichtenstein, R. P. Schleimer. 1995. Expression of the chemokine RANTES by a human bronchial epithelial cell line: modulation by cytokines and glucocorticoids. J. Immunol. 155:410.[Abstract]
  19. Motojima, S., T. Adachi, K. I. Manaka, M. Arima, T. Fukuda, S. Makino. 1996. Eosinophil peroxidase stimulates the release of granulocyte-macrophage colony-stimulating factor from bronchial epithelial cells. J. Allergy Clin. Immunol. 98:(Suppl.):S216.[Medline]
  20. Terashita, Z., M. Kawamura, M. Takatani, S. Tsushima, Y. Imura, K. Nishikawa. 1992. Beneficial effects of TCV-309, a novel potent and selective platelet activating factor antagonist in endotoxin and anaphylactic shock in rodents. J. Pharmacol. Exp. Ther. 260:748.[Abstract/Free Full Text]
  21. Banks-Schlegel, S. P., A. F. Gazdar, C. C. Harris. 1985. Intermediate filament and cross-linked envelope expression in human lung tumor cell lines. Cancer Res. 45:1187.[Abstract/Free Full Text]
  22. Parkos, C. A., C. Delp, M. A. Arnaout, J. L. Madara. 1991. Neutrophil migration across a cultured intestinal epithelium: dependence on a CD11b/CD18-mediated event and enhanced efficiency in physiological direction. J. Clin. Invest. 88:1605.
  23. Liu, L., F. P. J. Mul, R. Lutter, D. Roos, E. F. Knol. 1996. Transmigration of human neutrophils across lung epithelial cell monolayers is preferentially in the physiological basolateral to apical direction. Am. J. Respir. Cell Mol. Biol. 15:771.[Abstract]
  24. van Schilfgaarde, M., L. van Alphen, P. Eijk, V. Everts, J. Dankert. 1995. Paracytosis of Haemophilus influenzae through cell layers of NCI-H292 lung epithelial cells. Infect. Immun. 63:4729.[Abstract]
  25. Roos, D., M. de Boer. 1986. Purification and cryopreservation of phagocytes from human blood. Methods Enzymol. 132:225.[Medline]
  26. Koenderman, L., M. L. Hamelink, P. T. M. Kok, A. J. Verhoeven, P. L. B. Bruijnzeel. 1988. An improved method for the isolation of eosinophilic granulocytes from peripheral blood of normal individuals. J. Leukocyte Biol. 44:79.[Abstract]
  27. Hansel, T. T., I. J. M. De Vries, T. Iff, S. Rihs, M. Wandzilak, S. Betz, K. Blaser, C. Walker. 1991. An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils. J. Immunol. Methods 145:105.[Medline]
  28. Reimert, C. M., P. Venge, A. Kharazmi, K. Bendtzen. 1991. Detection of eosinophil cationic protein (ECP) by an enzyme-linked immunosorbent assay. J. Immunol. Methods 138:285.[Medline]
  29. Olsson, I., P. Venge. 1974. Cationic proteins of human granulocytes. II. Separation of the cationic proteins of the granules of leukemic myeloid cells. Blood 44:235.[Abstract/Free Full Text]
  30. Gretch, D. R., M. Suter, M. F. Stinski. 1987. The use of biotinylated monoclonal antibodies and streptavidin affinity chromatography to isolate herpesvirus hydrophobic proteins or glycoproteins. Anal. Biochem. 163:270.[Medline]
  31. Schweizer, R. C., B. A. C. Van Kessel-Welmers, R. A. J. Warringa, T. Maikoe, J. A. M. Raaijmakers, J. W. J. Lammers, L. Koenderman. 1996. Mechanisms involved in eosinophil migration: platelet-activating factor-induced chemotaxis and interleukin-5-induced chemokinesis are mediated by different signals. J. Leukocyte Biol. 59:347.[Abstract]
  32. Vossebeld, P. J. M., J. Kessler, A. E. G. K. Von dem Borne, D. Roos, A. J. Verhoeven. 1995. Heterotypic Fc{gamma}R clusters evoke a synergistic Ca2+ response in human neutrophils. J. Biol. Chem. 270:10671.[Abstract/Free Full Text]
  33. Bijsterbosch, M. K., K. P. Rigley, G. G. Klaus. 1986. Cross-linking of surface immunoglobulin on B lymphocytes induces both intracellular Ca2+ release and Ca2+ influx: analysis with indo-1. Biochem. Biophys. Res. Commun. 137:500.[Medline]
  34. Kay, A. B.. 1991. Asthma and inflammation. J. Allergy Clin. Immunol. 87:893.[Medline]
  35. Eda, R., H. Sugiyama, R. J. Hopp, C. Okada, A. K. Bewtra, R. G. Townley. 1993. Inhibitory effects of formoterol on platelet-activating factor induced eosinophil chemotaxis and degranulation. Int. Arch. Allergy Immunol. 102:391.[Medline]
  36. Eda, R., R. G. Townley, R. J. Hopp. 1994. Effect of terfenadine on human eosinophil and neutrophil chemotactic response and generation of superoxide. Ann. Allergy 73:154.[Medline]
  37. Takafuji, S., T. Ohtoshi, H. Takizawa, K. Tadokoro, K. Ito. 1996. Eosinophil degranulation in the presence of bronchial epithelial cells: effect of cytokines and role of adhesion. J. Immunol. 156:3980.[Abstract]
  38. Sunder-Plassmann, G., R. Hofbauer, G. Sengoelge, W. I. I. Horl. 1996. Quantification of leukocyte migration: improvement of a method. Immunol. Invest. 1-2:49.
  39. Resnick, M. B., S. P. Colgan, C. A. Parkos, C. Delp-Archer, D. McGuirk, P. F. Weller, J. L. Madara. 1995. Human eosinophils migrate across an intestinal epithelium in response to platelet-activating factor. Gastroenterology 108:409.[Medline]
  40. Wu, T., R. D. Rieves, C. Logun, J. H. Shelhamer. 1995. Platelet-activating factor stimulates eicosanoid production in cultured feline tracheal epithelial cells. Lung 173:89.[Medline]
  41. Kahhak, L., A. Roche, C. Dubray, C. Arnoux, J. Benveniste. 1996. Decrease of ciliary beat frequency by platelet activating factor: protective effect of ketotifen. Inflamm. Res. 45:234.[Medline]
  42. Kondo, M., J. Tamaoki, K. Isono, S. Takeuchi, Y. Ozawa, A. Chiyotani, K. Konno. 1994. Effect of platelet-activating factor on intracellular free calcium in cow tracheal epithelium. Am. J. Respir. Cell Mol. Biol. 3:278.
  43. Rieves, R. D., J. Goff, T. Wu, P. Larivee, C. Logun, J. H. Shelhamer. 1992. Airway epithelial cell mucin release: immunologic quantitation and response to platelet-activating factor. Am. J. Respir. Cell Mol. Biol. 2:158.
  44. Tamaoki, J., N. Sakai, K. Isono, T. Kanemura, I. Yamawaki, T. Takizawa. 1991. Effects of platelet-activating factor on bioelectric properties of cultured tracheal and bronchial epithelia. J. Allergy Clin. Immunol. 6:1042.
  45. Tao, Y., H. E. Bazan, N. G. Bazan. 1996. Platelet-activating factor enhances urokinase-type plasminogen activator gene expression in corneal epithelium. Invest. Ophthalmol. Vis. Sci. 37:2037.[Abstract/Free Full Text]
  46. Lin, J., S. K. Juhn, G. L. Adams, G. S. Giebink, Y. Kim. 1997. Dexamethasone inhibits mucous glycoprotein secretion via a phospholipase A2-dependent mechanism in cultured chinchilla middle ear epithelial cells. Acta Otolaryngol. 117:406.[Medline]
  47. LeVan, T. D., J. W. Bloom, D. G. Adams, J. L. Hensel, M. Halonen. 1998. Platelet-activating factor induction of activator protein-1 signaling in bronchial epithelial cells. Mol. Pharmacol. 1:135.
  48. Tai, Y. H., J. Flick, S. A. Levine, J. L. Madara, G. W. G. Sharp, M. Donowitz. 1996. Regulation of tight junction resistance in T-84 monolayers by elevation in intracellular Ca2+: a protein kinase C effect. J. Membr. Biol. 149:71.[Medline]
  49. Travis, S. P. L., B. Crotty, D. P. Jewell. 1995. Site of action of platelet-activating factor within the mucosa of rabbit distal colon. Clin. Sci. 88:51.[Medline]
  50. Hordijk, P. L., J. P. Ten Klooster, R. A. van der Kammen, F. Michiels, L. C. Oomen, J. G. Collard. 1997. Inhibition of invasion of epithelial cells by Tiam1-Rac signaling. Science 5342:1464.
  51. Warringa, R. A. J., H. J. J. Mengelers, J. A. M. Raaijmakers, P. L. B. Bruijnzeel, L. Koenderman. 1993. Upregulation of formyl-peptide and interleukin-8-induced eosinophil chemotaxis in patients with allergic asthma. J. Allergy Clin. Immunol. 91:1198.[Medline]
  52. Kuijpers, T. W., B. C. Hakkert, M. H. L. Hart, D. Roos. 1992. Neutrophil migration across monolayers of cytokine-prestimulated endothelial cells: a role for platelet-activating factor and IL-8. J. Cell Biol. 117:565.[Abstract/Free Full Text]
  53. Smart, S. J., T. B. Casale. 1994. TNF-{alpha}-induced transendothelial neutrophil migration is IL-8 dependent. Am. J. Physiol. 266:L238.[Abstract/Free Full Text]
  54. Liu, L., F. P. J. Mul, T. W. Kuijpers, R. Lutter, D. Roos, E. F. Knol. 1996. Neutrophil transmigration across monolayers of endothelial cells and airway epithelial cells is regulated by different mechanisms. Ann. NY Acad. Sci. 796:21.[Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
L. Liu, L. Hakansson, P. Ridefelt, R. C. Garcia, and P. Venge
Priming of Eosinophil Migration Across Lung Epithelial Cell Monolayers and Upregulation of CD11b/CD18 Are Elicited by Extracellular Ca2+
Am. J. Respir. Cell Mol. Biol., June 1, 2003; 28(6): 713 - 721.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. H. Shen, S. I. Ochkur, M. P. McGarry, J. R. Crosby, E. M. Hines, M. T. Borchers, H. Wang, T. L. Biechelle, K. R. O'Neill, T. L. Ansay, et al.
A Causative Relationship Exists Between Eosinophils and the Development of Allergic Pulmonary Pathologies in the Mouse
J. Immunol., March 15, 2003; 170(6): 3296 - 3305.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
V. E. L. Stubbs, P. Schratl, A. Hartnell, T. J. Williams, B. A. Peskar, A. Heinemann, and I. Sabroe
Indomethacin Causes Prostaglandin D2-like and Eotaxin-like Selective Responses in Eosinophils and Basophils
J. Biol. Chem., July 12, 2002; 277(29): 26012 - 26020.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
W. J. Howat, J. A. Holmes, S. T. Holgate, and P. M. Lackie
Basement Membrane Pores in Human Bronchial Epithelium : A Conduit for Infiltrating Cells?
Am. J. Pathol., February 1, 2001; 158(2): 673 - 680.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
J. S. ERJEFÄLT and C. G. A. PERSSON
New Aspects of Degranulation and Fates of Airway Mucosal Eosinophils
Am. J. Respir. Crit. Care Med., June 1, 2000; 161(6): 2074 - 2085.
[Full Text]

Home page
 J. Immunol.Home page
L. Liu, P. Ridefelt, L. Hakansson, and P. Venge
Regulation of Human Eosinophil Migration Across Lung Epithelial Monolayers by Distinct Calcium Signaling Mechanisms in the Two Cell Types
J. Immunol., November 15, 1999; 163(10): 5649 - 5655.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
N. Merendino, M. B. Dwinell, N. Varki, L. Eckmann, and M. F. Kagnoff
Human intestinal epithelial cells express receptors for platelet-activating factor
Am J Physiol Gastrointest Liver Physiol, October 1, 1999; 277(4): G810 - G818.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. K. Marathe, S. S. Davies, K. A. Harrison, A. R. Silva, R. C. Murphy, H. Castro-Faria-Neto, S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre
Inflammatory Platelet-activating Factor-like Phospholipids in Oxidized Low Density Lipoproteins Are Fragmented Alkyl Phosphatidylcholines
J. Biol. Chem., October 1, 1999; 274(40): 28395 - 28404.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, L.
Right arrow Articles by Roos, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, L.
Right arrow Articles by Roos, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS