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Published ahead of print on July 3, 2003, doi:10.1165/rcmb.2003-0053OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 261-270, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0053OC

Activation of Extracellular Regulated Kinases Is Required for the Increase in Airway Epithelial Permeability during Leukocyte Transmigration

Vladimir B. Serikov, Hyon Choi, Ken J. Chmiel, Reen Wu and Jonathan H. Widdicombe

Children's Hospital Oakland Research Institute, Oakland; and Department of Human Physiology and Department of Medicine, University of California Davis, Davis, California

Address correspondence to: Dr. J. H. Widdicombe, Department of Human Physiology, Tupper Hall, 4136, School of Medicine, University of California Davis, Davis, CA 95616. E-mail: jhwiddicombe{at}ucdavis.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The goal of this study was to determine whether the extracellular regulated kinases (ERK1/2) are involved in leukocyte transmigration across airway epithelium and the associated changes in epithelial permeability. In vitro, we used formyl-methionyl-leucyl-phenylalanine (fMLP) to induce migration of HL-60 cells (a human leukocyte cell line) across sheets of polarized Calu-3 airway epithelial cells and also to induce migration of human neutrophils across primary cultures of cow tracheal epithelial cells. In both systems, leukocyte migration decreased transepithelial electrical resistance (Rte), increased epithelial permeability to albumin (Palb), and increased ERK1/2 phosphorylation in epithelial cells. Leukocyte migration and the associated changes in Rte, Palb, and ERK1/2 phosphorylation were inhibited by calphostin C, a blocker of protein kinase C (PKC), and by PD98059 (a blocker of ERK1/2). Leukocyte transmigration in rat tracheas in vivo was induced with fMLP, and was associated with increased Palb and phosphorylation of epithelial ERK1/2. Again, migration and the associated changes were inhibited by luminal PD98059 or calphostin C though neither agent affected rat leukocyte migration in Boyden chambers in vitro. We conclude that PKC and ERK1/2 pathways are activated in airway epithelial cells during migration of leukocytes and are important regulators of airway epithelial permeability.

Abbreviations: bovine serum albumin, BSA • extracellular regulated kinases, ERK1/2 • fetal calf serum, FCS • formyl-methionyl-leucyl-phenylalanine, fMLP • mitogen-activated protein kinase, MAPK • permeability to albumin, Palb • phosphate-buffered saline, PBS • protein kinase C, PKC • transepithelial electrical resistance, Rte • tight junction, TJ


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inflammatory diseases of the upper respiratory tract are characterized by leukocyte migration and extravasation of plasma constituents into the airway lumen (1–3). Massive leukocyte migration leads to breakdown of epithelial barrier function and leakage of interstitial fluid into the airway lumen. The resulting changes in the volume and composition of the airway surface liquid presumably impair mucociliary transport. In addition, plasma factors, such as soluble CD14, when present in the airway lumen, may enhance responsiveness of epithelial cells to lipopolysaccharides or may directly damage the epithelium.

Following adherence to the epithelial basolateral membrane (4), leukocytes migrate across epithelia, producing focal disruptions in tight junctions (TJ) (5). It is currently unclear what initiates these disruptions (5–9). One possibility is mechanical forces, caused by extravasation and the resulting increase in subepithelial hydrostatic pressure (6, 9). Alternatively, activation of signaling pathways in the epithelium may be induced by interaction of migrating leukocytes with the epithelial cells and cross-linking of intercellular adhesion molecules (5, 8). The permeability of TJ is also modulated by cytokines and proteases (8, 10). However, the specific transduction pathways, by which the paracellular permeability of the airway epithelium is regulated during leukocyte migration, are poorly characterized.

The functional status of TJ is a major determinant of epithelial permeability, and it is regulated by multiple transduction pathways (11) involving a complex network of interactions between proteins and small molecules (12–14). In general, epithelial leakiness is mostly related to activation of protein kinase C (PKC) and its downstream effector pathways (15), though other pathways may also be involved (16–19). PKC-dependent signaling pathways include those leading to phosphorylation of mitogen-activated protein kinases (MAPK) (20). MAPK are a family of serine/threonine kinases, three of which are relatively well studied: extracellular signal–regulated kinases (ERKs), Jun amino-terminal kinases (JNK), and p38 MAPK (21). ERKs are activated predominantly in response to growth factors, but they also can be activated by environmental stress (21). The pathway involved in ERK stimulation requires activation of Ras, Raf, and MAPK. MAPK can phosphorylate and activate myosin-light chain kinase (22), thus mediating myosin-ATPase-dependent contraction of the perijunctional actomyosin belt.

Here, we investigated the role of ERK1/2 signaling in leukocyte transmigration across airway epithelium and the associated increase in epithelial permeability. Migration of leukocytes across polarized sheets of human airway epithelium and primary cultures of cow tracheal epithelium in vitro and into the lumen of rat tracheas in vivo was induced with the common bacterial chemotactic agent formyl-methionyl-leucyl-phenylalanine (fMLP). Both in vivo and in vitro, migration of neutrophils or neutrophil-like HL-60 cells induced ERK1/2 phosphorylation and an increase in epithelial permeability via PKC-related pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
fMLP was purchased from Sigma (St. Louis, MO), and calphostin C and PD98059 from Calbiochem (San Diego, CA). All were dissolved in dimethyl sulfoxide at 1,000 times the final concentration, and stored at -20°C. Calceine-AM and Texas-Red albumin were from Molecular Probes (Eugene, OR), and the PKH26 labeling kit was from Sigma. Mouse monoclonal anti–phospho-p44/42 (ERK1/2) and anti-total p44/42 antibodies were from Cell Signaling Technology (Beverly, MA).

Cell Culture
The human lung adenocarcinoma-derived cell line Calu-3 (American Type Culture Collection, Rockville, MD) was grown in a 1:1 mix of Dulbecco's Modified Eagle Medium (DMEM) H-21 and Ham's F-12 medium (DME/F-12) supplemented with 15% (vol/vol) heat-inactivated fetal calf serum (FCS; Invitrogen, Carlsbad, CA), 100 U/ml penicillin (Invitrogen), and 100 µg/ml streptomycin (Invitrogen). Phenol red was omitted from the medium, as it interfered with fluorescence measurements. Cells (passages 10–30) were plated on the external surface of polycarbonate membranes (3.0-µm pore size, 6.5 mm diameter) at the base of inverted transwells (Costar, Cambridge, MA). The day after plating, the inserts were returned to their normal orientation and the cells were grown with air–liquid interface, i.e., with medium added only to the basolateral side (inside of the insert). By Day 18, cell sheets revealed transepithelial resistance (Rte) higher than 250 {Omega} x cm2, as measured with a "chopstick" voltmeter (Evommeter, model EVOMX-G; World Precision Instruments, Sarasota, FL). Confluence of cell sheets was also confirmed by Diff-Quik staining and by light and scanning electron microscopy. Permeability to albumin was in the range of 10–15 x 10-6 cm/s. Cells were used between 18 and 21 d after plating.

Isolated bovine tracheal epithelial cells were obtained by protease digestion as previously described (23). They were plated at 106 cells/cm2 on 3-µm pore polycarbonate membranes and grown in DME/F-12 medium with growth factors (23).

The HL-60 human leukocyte cell line (ATCC) was grown in suspension in RPMI-1640 medium with 10% FCS and was differentiated into neutrophil-like phenotype by treatment with retinoic acid (10 nM) for 4 d before use, as described (24).

Migration Experiments
To assess migration, the HL-60s or human neutrophils (107/ml) were labeled with 5 µg/ml calcein-AM (Molecular Probes, Eugene, OR) for 30 min at 37°C, according to the manufacturer's protocol. After labeling, the cells were washed three times with RPMI-1640 medium. Levels of calcein-AM were measured with a spectrofluorometer (model CytoFluor 2350; Millipore).

The outer (mucosal) compartment of epithelial cell cultures was filled with prewarmed medium with 10% FCS with or without 0.1 µM fMLP. The HL-60s or human neutrophils (5 x 105 cells in 0.2 ml of prewarmed incubation medium) were placed in the serosal compartment, and the transwells were incubated for 4–24 h at 37°C with 5% CO2 and at maximal humidity. The number of migrating cells was determined by flow cytometry (Flow-Calibur; Beckton-Dickinson, Oxnard, CA) and by total fluorescence of cell suspension. To determine migration of labeled cells we used an established technique (25). The percentage of transmigrated HL-60 was calculated from the amount of fluorescence detected in the fluid from the lower compartment in relation to the fluorescence of the total added calcein-AM–labeled HL-60. A calibration curve (fluorescence intensity versus number of labeled cells) was performed for each experiment and confirmed by flow cytometry or by cell counts with a hemocytometer. To correct for a possible presence of non–cell-associated fluorescence, medium with transmigrated cells was centrifuged at 1,000 rpm for 15 min, supernatant discarded, and the 50-µl pellet reconstituted to 0.5 ml. Random migration (no chemoattractant present) was subtracted from total migration. Results are expressed as the percent of total number of migrated cells in the presence of pharmacologic agent to control (no pharmacologic agent).

For pharmacologic studies, epithelial cell sheets were incubated with drugs of interest (calphostin C, 10-8 M, PD98059, 5 x 10-5 M) for 1 h before addition of leukocytes or fMLP. Drugs were added to both apical and basolateral sides in medium. Immediately before use, the medium containing drugs was removed, and the cell sheets were washed twice with a large volume RPMI-1640 medium.

Immunofluorescence
The human neutrophils used in these studies were stained with PKH26 (Sigma) according to the manufacturer's protocol. Following 12 h of neutrophil transmigration, primary tracheal epithelial cell cultures were washed with phosphate-buffered saline (PBS) and fixed for 10 min in 4% paraformaldehyde. Fixed cells were then washed with PBS, exposed to cold methanol (-20°C) for 5 min, washed twice, exposed to 0.2% Triton X-100 in PBS for 5 min, and then again washed twice with PBS. Cells were further blocked with 3% bovine serum albumin (BSA)/0.1% Tween 20 in 4x SSC for 20 min. Mouse anti-human anti–phospho-ERK1/2 antibody (Cell Signaling) (1:500 in 1% BSA/2x SSC/0.1% Tween 20) was added for 1 h at room temperature, and cells were washed three times with PBS and blocked with 3% BSA/0.1% Tween 20 in 4x SSC for 20 min. FITC-conjugated secondary antibody (goat anti-mouse, 1:100 in 1% BSA/2x SSC/0.1% Tween 20; Sigma) was added for 1 h. Control filters were stained with isotype antibody. Cell sheets were washed three times with PBS, co-stained with propidium iodide (0.5 µg/ml) for 5 min, and washed three times with PBS. Filters were mounted on glass slides in Fluoro-Guard antifade reagent (BioRad, Hercules, CA) and coverslips were applied. At least three different filters were analyzed for each experimental condition. Laser confocal fluorescence microscopy (Axiovert 100M; Zeiss, Germany) was performed at UC Davis School of Medicine Confocal Imaging Core Facility.

Permeability Measurements
The permeability of Calu-3 monolayers and primary cultures of bovine epithelium to Texas-red–labeled bovine serum albumin was estimated according to standard methods (26). Assuming that the tracer flux is due to diffusional transport, then the relationship between the concentration of tracer in the bottom chamber [Cb(t)] versus time and permeability (P) of the barrier is given by:

where Vb and Vt represent the volume in the top and bottom chambers (0.2 and 0.5 ml, respectively), T(t) is the total amount of tracer added to the system at time t, and A is the area of the monolayer (0.33 cm2). The permeability (P) of the membrane alone was measured so that the contribution of the cell monolayer (Pc) was separated from the contribution due to the membrane (Pm), according to the relationship 1/Ptotal = 1/Pc + 1/Pm, where Ptotal is the total permeability for the system.

Animal Experiments
In total, 40 male Sprague-Dawley rats weighing 350–400 g were used. Following anesthetization with Nembutal (35 mg/kg intraperitoneally), a PE50 catheter was placed into the carotid artery and a tracheotomy with PE200 tubing was performed three tracheal rings above the carina. A perfused tracheal segment with intact systemic blood supply was formed as described previously (27). In brief, a transverse incision was made through the ventral surface of the trachea just below the larynx and a second incision was made 14–16 mm caudal to the first. L-shaped catheters (1.5 mm OD) were tightly secured at both ends of the segment so formed and filled with saline at 38°C. One catheter was connected to a syringe. The other remained open, with its long limb pointing vertically. Saline was delivered to the lumen through the syringe, and intraluminal pressure (0 cm H2O unless otherwise stated) was set from the level in the vertical limb of the open cannula. The tracheal segment was covered with plastic lining to prevent drying and heated with a lamp to 38–39°C. To measure transepithelial albumin flux, Evans Blue dye, which binds to plasma albumin, was injected intravenously (25 mg/kg). The solution in the tracheal lumen was completely replaced at 1-h intervals, and its Evans Blue content was determined from the absorbance at 615 nm (Turner spectrophotometer SP-380; Barnstead, Dubuque, IA).

The leukocyte content of tracheal fluid or blood was measured with a hemocytometer. Differential cell counts were performed with Diff-Quik staining (American Scientific Products, McGraw Park, IL), following the formation of cell smears with a cytocentrifuge (Shandon Scientific, London, UK).

To induce leukocyte transmigration, 0.25 µM fMLP was added to the luminal perfusate of the tracheal segment. One hour after recovery from surgery, Evans Blue dye (25 mg/kg) was infused into the bloodstream and samples of plasma and tracheal fluid (sample volume 1.5 ml) were taken hourly for 4 h. Five rats received fMLP and five received vehicle alone.

In tests of pharmacologic blockers of transduction pathways, each animal was used as its own control. Thus, leukocyte migration was induced as described above and the period of induced leukocyte migration from 2–3 h was used as a baseline for each animal. After 3 h of tracheal perfusion with fMLP alone, the tracheal segment was perfused for another hour with fMLP plus pharmacologic agent (experimental period). The levels of leukocyte migration and albumin flux rate during the experimental period were compared with the same variables during the baseline period by paired t test. The following pharmacologic agents were added to the tracheal perfusate: calphostin C, a blocker of PKC (50 nM, n = 6), and PD98059, ERK1/2 blocker (50 µM, n = 6).

In tests for ERK1/2 phosphorylation in rats, the perfused tracheal segment was divided into two subsegments. In one group of animals, one subsegment was filled with saline and the other with fMLP, and in the other group one subsegment was filled with fMLP and the other one with fMLP and PD98059 or fMLP and calphostin C (n = 4 in each group).

As a positive control for blockade of transudation, in six experiments a pressure head of 10 cm H2O was applied during the experimental period; we have previously shown that this blocks transudation (27).

Scanning Electron Microscopy
At the end of the experiment, pieces of trachea were removed and fixed in 2.3% glutaraldehyde/0.05 M Na cacodylate, dehydrated in ethanols, critical point dried (Polaron, UK), mounted on stubs, sputter-coated with platinum (Polaron), and viewed in a conventional scanning electron microscope (Phillips).

Cytologic Staining of Isolated Rat Tracheal Epithelial Cells
In each of the six rats, we perfused the lumens of the two separate tracheal segments with PBS alone or PBS with 0.25 µM fMLP. In three experiments, fMLP was added to the distal, and in three to the proximal segment. After 4 h, each section of the rat trachea was placed in a separate microtube with 300 µl of PBS containing 40 mg/ml protease XIV (Sigma). The microtubes were then placed in an incubator at 37°C for 2 h, and then shaken briefly with a vortex to dislodge cells from the surface of the tracheas. The digested pieces of trachea were discarded and the media were spun down at 1,000 rpm for 10 min. The protease solution was removed from each tube and each pellet was resuspended in 100 µl of DME/F-12 with 5% FCS. Cells were deposited on glass microslides with a cytocentrifuge for 5 min at 1,000 rpm. Following air-drying, cells were stained in Diff-Quik (Dade Behring, Switzerland), according to the manufacturer's instructions. Stained slides were again air-dried and viewed with an Olympus light microscope; cells were differentially counted at x40.

Western Blot Analysis
Western blots were performed for phosphorylated ERK1/2 and total ERK1/2. After 1 h, the perfused segments of rat tracheas were separately removed, and the epithelial cells were scraped off into lysis buffer (4°C) composed of 1% Triton X-100, 2 mM EDTA, 2 mM dithiotheritol, 0.25 mg/ml leupeptin, 0.25 mg/ml pepstatin A, 0.4 mg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride. In cell culture experiments, six cell sheets were combined in 100 µl of ice-cold buffer. Protein content of the lysate was measured by the BCA method (Pierce, Rockford, IL). Samples (20 µg total protein) were run on 10% SDS-polyacrylamide gels, and the separated proteins transferred to Nylon membranes, where they were blocked overnight at 4°C with 5% nonfat dry milk in TBST (10 mM TrisCl, pH 8.0; 150 mM NaCl; 0.1% Tween 20). The blot was rinsed twice with TBST and incubated for 2 h at room temperature with mouse monoclonal anti–phospho-p44/42 (ERK1/2) antibody in TBST containing 5% BSA. The membrane was washed for 15 min with TBST and incubated with goat antimouse IgG conjugated with horseradish peroxidase in TBST plus 5% milk for 1 h, and then washed three times with TBST and developed with enhanced chemiluminescence reagent (Amersham, Arlington, IL). For total ERK, membranes were washed and re-probed with mouse monoclonal anti-p44/p42 in the same way. Staining was quantified using NIH Image v.1.6 software.

Leukocyte Isolation and Boyden Chamber Studies
Leukocytes were obtained from citrate-buffered human blood. Blood was diluted 1:2 with Ca2+/Mg2+–free Hanks balanced salt solution (HBSS) and cells were pelleted at 2,000 rpm for 20 min. The pellet was suspended in HBSS and layered on a discontinuous gradient of 1.119 and 1.077 g/ml Histopaque (Sigma). After centrifugation at 700 g for 30 min, the granulocyte band at the interface of the two densities was harvested and cells washed twice. Contaminating erythrocytes were removed by a standard hypotonic lysis step, and the granulocytes were resuspended in RPMI-1640 medium with 10% FCS to a final concentration of 5 x 10 6 cells/ml. The final suspension was more than 95% neutrophils. Leukocyte migration was studied in a standard 48-well chemotaxis chamber (Neuroprobe, Inc., Gaithersburg, MD) with 3 µm polycarbonate filter according to the manufacturer's protocol. Chemoattractant (0.25 µM fMLP) was placed in the lower wells of the chamber, and neutrophil suspension (40 µl), mixed with the drug of interest, was placed in the top wells and allowed to migrate through the filter for 1 h in a humid atmosphere of 5% CO2 at 38°C. Studies were performed in triplicate with three suspensions of neutrophils from different subjects. Filters were fixed and stained with Diff-Quik. Migration was assessed from the total number of neutrophils inside the filter. Random migration (no chemoattractant present) was subtracted from total migration. Results are expressed as the percent of cells migrated in the presence of pharmacologic agent as compared to control (no pharmacologic agent).

Statistical Analyses
Data are presented as mean ± SEM. Comparison between means was performed by ANOVA followed by two-tailed Student's t test with Bonferroni correction and regression analyses where appropriate. P < 0.05 was regarded as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transepithelial Migration of HL-60 across Cultures of Calu-3
In a series of preliminary experiments, we determined that the optimal period of migration of neutrophil-like HL-60 across Calu-3 layers was 4 h, during which time ~ 15–25% of cells transmigrated in the serosal-to-mucosal direction across the epithelial layer. Both spontaneous and induced migrations were 3- to 4-fold less in the mucosal-to-serosal direction, as compared with the serosal-to-mucosal direction (data not shown), as described before (28). For each experimental treatment at least six different layers from two to three different cultures were used. The mean initial Rtes (200–400 {Omega} x cm2) were not different among different treatment groups.

fMLP by itself did not cause changes in Rte or albumin permeability of epithelial layers (Table 1). Over 4 h, migration in the absence of fMLP was 18.8 ± 0.9% and in the presence of fMLP was 39.9 ± 1.6%. Therefore, induced migration was 20.1 ± 1.8%. Rte did not change significantly when medium alone was added to the upper well (93 ± 7% of initial), or when the neutrophil-like HL-60 were added to the upper well without fMLP in the lower well (124 ± 21%). Rte significantly decreased to 43.6% of control when neutrophil-like HL-60s migrated toward fMLP. Changes in permeability to albumin showed the same pattern. In control cells, permeability was 15.8 ± 2.3 x 10-6 cm/s and it did not change significantly when cells or fMLP were added separately (Table 1). However, stimulation of neutrophil-like HL-60 migration by fMLP significantly increased permeability to albumin by 206%.


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  		TABLE 1 Spontaneous and initiated migration of neutrophil-like HL-60 across confluent layers of Calu-3 cells

 
ERK1/2 phosphorylation was minimal when medium alone or HL-60 were added to the upper well (Figure 1A). However, ERK1/2 phosphorylation increased in epithelial cells after 4 h of transmigration of neutrophil-like HL-60s toward fMLP. Transmigrated HL-60, collected from the bottom of the well, showed no detectable phospho-ERK1/2 (p-ERK) (data not shown). Further, when cell layers were stained with Diff-Quik after 4 h of HL-60 migration, the percentage of HL-60 was < 5% of the total cell number.



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  		Figure 1. Leukocyte migration is associated with ERK1/2 phosphorylation of Calu-3 cells. Epithelial cells were lysed and were subjected to SDS-PAGE and Western blot with anti-phosphotyrosine antibody to ERK1/2 (upper panel) and antibody to total ERK1/2 (lower panel). Molecular weight markers (not shown) indicated that both bands were of ~ 42 kDa. Results of two experiments. (A) ERK1/2 phosphorylation was minimal when fMLP or HL-60 alone were added. ERK phosphorylation increased in epithelial cells after 4 h of transmigration of neutrophil-like HL-60s toward fMLP. (B) ERK1/2 phosphorylation, increased by leukocyte migration toward fMLP was diminished by PD98059 and calphostin C. The term "vehicle" indicates that the epithelium was treated with DMSO, the vehicle for both calphostin C and PD98059.

 
We tested the effects of the specific PKC blocker, calphostin C, and specific ERK1/2 activation blocker, PD98059. Both agents were without effect on Rte and permeability to albumin of epithelial layers. When tested for the effect on migration of HL-60 across empty filters (without epithelium), PD98059 did not change the fMLP-induced migration of neutrophil-like HL-60s across the filters (28.8 ± 1.4%). Both PD98059 and calphostin C significantly reduced the epithelial ERK1/2 phosphorylation induced by transmigration of HL-60 (Figure 1B). The effects of pharmacologic agents on migration rate through the epithelial layers, Rte, permeability to albumin, and quantitative assessment of ERK1/2 phosphorylation are shown in Figure 2. Blockade of changes in Rte by PD98059 were not significantly different for concentrations of 5 x 10-5 M and 10-4 M (data not shown).



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  		Figure 2. PD98059 and calphostin C prevent changes in permeability due to leukocyte migration induced by fMLP. The rate of induced migration (A), Rte (B), permeability to albumin (C), and relative degree of ERK1/2 phosphorylation (D) in Calu-3 monolayers 4 h after neutrophil-like HL-60 migration induced by fMLP. Induced migration was calculated as total migration minus spontaneous migration.

 
Thus, in epithelial layers calphostin C significantly reduced neutrophil-like HL-60 migration compared to that seen with fMLP alone (P < 0.05) and prevented the migration-induced changes in albumin permeability and Rte. PD98059 slightly but significantly reduced the rate of migration and blocked the migration-related increases in Rte and albumin permeability.

Migration of Human Neutrophils across Primary Cultures of Cow Tracheal Epithelium
Primary cultures of cow tracheal epithelial cells were characterized by higher initial Rte (800–1200 {Omega} x cm2) and slower rate of neutrophil transmigration than Calu-3 cells. Therefore, instead of 4 h of migration, Rte and albumin permeability were assessed 12 h after addition of leukocytes and fMLP. fMLP significantly increased transepithelial migration (2.5-fold), which resulted in significant decrease in Rte (38.3 ± 13%, P = 0.03) (Figure 3). As for Calu-3 cells, changes in Rte induced by migrating cells were blocked by both calphostin C and PD98059. Neither PD98059 nor calphostin C changed spontaneous migration of neutrophils across epithelial layers. They were also without significant effect on Rte and permeability to albumin of epithelial layers without neutrophils (data not shown).



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  		Figure 3. PD98059 and calphostin C prevent changes in Rte due to migration of human neutrophils across primary cultures of bovine tracheal epithelium. The migration rate (A) and Rte (B) 12 h after neutrophil migration induced by fMLP. *P < 0.05 compared with medium control; +P < 0.05 compared to fMLP alone.

 
Immunostaining for ERK1/2
Immunostaining of cow tracheal epithelial cells as well as Calu-3 cells demonstrated that during neutrophil migration induced by fMLP, epithelial cells became positively stained for p-ERK1/2 (Figure 4). To distinguish staining of epithelial cells from staining of transmigrating neutrophils, neutrophils were stained with PKH26 (red fluorescence). Figure 4 shows that early during migration, ERK1/2 fluorescence was focal and often closely associated with migrating leukocytes. Later, virtually all cells were positive for ERK1/2.



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  		Figure 4. Activation of epithelial ERK1/2 during neutrophil migration induced by fMLP. (A–D) Confocal microscopy images of primary cultures of bovine tracheal epithelium immunostained for phosphorylated ERK1/2 (green fluorescence). Neutrophils are labeled with PKH26 (red fluorescence). (A) fMLP only; (B) human neutrophils without fMLP; (C) 1 h after initiation of migration with fMLP; (D) 12 h after initiation of migration. Bars = 40 µm or 20 µm (C). (E–F) Cultures of Calu-3 cells, stained for pERK1/2 (green fluorescence) and propidium iodide (red fluorescence) for DNA. (E) Epithelial layer without HL-60 migration (control). (F) Layer after HL-60 migration. Bars = 25 µm.

 
Transepithelial Migration In Vivo Rat Tracheas
When perfused with saline alone, there was no increase in transudation or leukocyte transmigration over the experimental period of 4 h (Figure 5). However, addition of fMLP to the tracheal perfusate led to a large increase in both transudation and leukocyte migration, which reached maximum levels at 3 h. Differential cell counts showed that 83 ± 9% of the cells transmigrated after 4 h were PMNs, 5 ± 1% were monocytes, 11 ± 4% were lymphocytes, and 1% were eosinophils. Transudation increased in parallel with the appearance of leukocytes, though we observed a 50% rise in transudation by the end of the first hour of exposure to fMLP that was unaccompanied by any significant increase in leukocyte migration. The maximal rate of leukocyte migration in individual experiments correlated significantly with the corresponding rate of transudation (r = 0.9).



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  		Figure 5. Induction of leukocyte migration into rat trachael lumen by fMLP. (A) The time course of leukocyte migration into the tracheal lumen. (B) The time course of albumin appearance (tracheal fluid/plasma ratio) in the tracheal lumen. *P < 0.05 compared with control by nonpaired Student's t test.

 
SEM studies of control rats showed that the epithelial lining of the trachea remained intact over the duration of the experiment (Figure 6A). By contrast, on the surface of tracheas perfused for 4 h with fMLP, we observed leukocytes and erythrocytes with occasional areas of epithelial damage. Figure 6B illustrates the trachea, in which these changes were most marked. Cytologic staining of cells recovered from digested rat tracheal epithelium revealed that before migration 3 ± 1% of cells in epithelium were granulocytes, and 4 h later this number increased to 6 ± 2% (P = 0.03).



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  		Figure 6. Scanning electron microscopy view of the rat tracheal surface. (A) Control study. (B) Four hours after fMLP. Portions of damaged epithelium are visible with migrating leukocytes (arrows) and erythrocytes (stars).

 
We applied a 10-cm H2O luminal hydrostatic pressure as a positive control to block appearance of plasma markers in the tracheal lumen. Pressure reduced transudation essentially to zero, although leukocyte migration was not significantly changed (Figure 7). Calphostin C and PD98059 reduced both transudation and leukocyte migration in vivo. However, these agents are small enough to diffuse across tight junctions and affect the function of leukocytes beneath the epithelium. Thus, their action on transmigration could reflect their influence on either leukocytes or epithelium. To distinguish between these possibilities, we tested the effects of these drugs on leukocyte migration in vitro and found that neither PD98059 (110 ± 10% of control) nor Calphostin C (102 ± 15% of control) significantly altered migration in Boyden chambers.



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  		Figure 7. Blockers of PKC or ERK reduce leukocyte migration (A) and changes in epithelial permeability (B) in rat tracheal epithelium in vivo. Values are percent of baseline ± SEM. *P < 0.05 compared with baseline by paired Student's t test.

 
In nine additional experiments, tracheas were divided into two perfused segments. In three rats, one segment was filled with PBS and the other with PBS and fMLP (0.25 µM). In three rats, one segment was filled with PBS plus fMLP and the other with PBS plus fMLP plus PD98059 (50 µM). In three rats, one segment was filled with PBS plus fMLP and the other with PBS plus fMLP plus calphostin C (50 nM). Sixty minutes after addition of pharmacologic agents, the tracheal segments were excised and epithelial lysates prepared. Western blot analyses demonstrated that 60 min exposure to fMLP significantly increased the levels of p-ERK1/2, as illustrated in Figure 8. PD98059 and calphostin C inhibited the fMLP-induced changes in levels of p-ERK. Levels of total ERK1/2 (t-ERK) were not significantly different between the various experimental conditions. Immunoblot images were analyzed by densitometry for ERK1 and ERK2. Migration induced by fMLP increased the mean ratio of pERK/tERK for p42 from 0.9 to 2.5 (P = 0.03), and for p44 from 1.3 to 3.2 (P = 0.01). Both p42 and p44 phosphorylation were significantly reduced by PD98059 (2.8-fold, P = 0.02) and by calphostin C (6-fold, P = 0.01).



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  		Figure 8. ERK1/2 phosphorylation by Western blot of rat tracheal epithelial cell lysates. Three separate experiments. (A) fMLP-induced leukocyte migration results in ERK1/2 phosphorylation. (B) PD98059, the specific blocker of ERK1/2, reduced the level of ERK phosphorylation. (C) Calphostin C, blocker of PKC, blocked ERK1/2 phosphorylation. Bands were ~ 42 kDa.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In three separate systems, we have shown that induction of leukocyte migration across airway epithelium is associated with a breakdown in epithelial barrier function and with phosphorylation of ERK1/2 in epithelial cells. Further, blockage of PKC (with calphostin C) or ERK1/2 activation (with PD98059) reduced leukocyte migration and the associated increase in epithelial permeability and ERK1/2 phosphorylation. We conclude that transmigrating leukocytes activate the ERK1/2 signaling cascade via PKC in airway epithelial cells, resulting in breakdown of epithelial barrier function.

ERK activation is in general downstream of PKC, and our results are consistent with this idea. Thus, block of PKC with calphostin or of ERK1/2 with PD98059 both stabilized the epithelium, and inhibited leukocyte migration and the associated breakdown in epithelial barrier function. That inhibition of PKC was acting ultimately by inhibiting ERK1/2 was indicated by reduced levels of ERK1/2 phosphorylation in the presence of the PKC inhibitor, calphostin C. In Ras-transformed MDCK cells, PD98059 restored recruitment of occludin, claudin, and ZO-1 to the cell membrane and increased transepithelial electrical resistance. Thus, downregulation of the MAPK signaling pathway restored morphology and function of tight junctions (29). During chemotaxis and cell migration, interaction of a cell with adhesive proteins or growth factors initiates Ras/MAPK signaling. Inhibition of MAPK-ERK activity causes decreased MLCK function, MLC phosphorylation, and cell migration (22, 30). However, our results provide the first evidence for a major role of ERK in regulation of permeability of airway epithelium (both Rte and albumin permeability) during leukocyte migration.

In many of our studies, we measured migration of HL-60 cells across Calu-3 cells. HL-60 cells are a human promyelocytic cell line that can be induced to differentiate into neutrophils (and other types of leukocytes), and have been previously used for transepithelial migration studies (31). Calu-3 cells are a spontaneously transformed human airway epithelial cell line that has shown remarkably constant electrical properties over many passages and between many different laboratories (32–34). Calu-3 cells differ from primary cultures of human tracheal epithelium in their high levels of CFTR, lack of amiloride-sensitive Na absorption, and absence of pseudostratified ciliated morphology (33). Primary tracheal cultures and Calu-3 cells both resemble native epithelium in the presence of mucous cells, and in a transepithelial electrical resistance of several hundred {Omega} · cm2. Therefore, though not ideal, Calu-3 cells represented the best available immortal cell line for the study of neutrophil migration across human airway epithelium.

Recognizing the potential limitations of Calu-3 cells, we also repeated key experiments using freshly isolated human neutrophils migrating across primary cultures of bovine tracheal epithelium. Results with Calu-3 cells and primary bovine cultures were essentially identical, strongly indicating that the same results would have been obtained on native human tracheal epithelium.

In both systems, leukocyte migration decreased Rte, increased permeability to albumin, and increased epithelial ERK1/2 phosphorylation. Furthermore, transepithelial leukocyte migration, the decrease in Rte, the increase in permeability to albumin, and the phosphorylation of ERK1/2 were all inhibited by calphostin C and PD98059. To rule out actions of calphostin C and PD98059 on leukocytes, the epithelial cell sheets were pretreated with these agents and thoroughly washed before adding leukocytes.

We performed several controls to determine whether the HL-60 or neutrophils in epithelial layers were the possible source of elevated p-ERK1/2. First, even during maximal levels of transmigration, we observed that leukocytes accounted for less than 5% of the total cells within the epithelium. Second, HL-60s collected after migration did not show appreciable phosphorylation of ERK1/2. Finally, our data on immunostaining of epithelial layers clearly demonstrate phosphorylation of ERK1/2 in epithelial cells, but not leukocytes.

Within each epithelial cell type (Calu-3 or bovine trachea) we observed a strong dependence of migration on Rte, as others have before (35). The same was true between cell types. Thus, the primary cultures of bovine tracheal epithelium had a much higher average Rte than Calu-3 cells and showed a lower rate of leukocyte migration. Again consistent with previous observations (28), we observed preferential migration in serosal-to-mucosal direction, and we investigated migration in this physiologically relevant direction. The rates of migration that we observed across Calu-3 cells—75,000–125,000 cells per cm2 in 4 h—is close to that described by others for migration of leukocytes across the lung epithelium (19, 28, 35).

Similar results were obtained in vivo. Thus, perfusion of the rat tracheal lumen with fMLP stimulated both leukocyte migration and the appearance of plasma albumin in the lumen. In addition, ERK phosphorylation in the epithelium was increased. The maximal number of migrating leukocytes (~ 105 cells/h/cm2) was similar to that previously reported for dog trachea perfused in vivo with medium conditioned by P. aeruginosa (36). Once transudation and transmigration were maximal, we tested the effects of applying luminal hydrostatic pressure and of pharmacologic modification of intracellular signaling pathways. Positive pressure blocked transudation, but had no effect on leukocyte transmigration. By contrast to hydrostatic pressure, calphostin C and PD98029 decreased both transudation and migration. Both agents also reduced the increases in ERK1/2 phosphorylation associated with leukocyte migration. It seemed possible that these agents were diffusing across the epithelium and directly inhibiting the leukocyte migration. Our Boyden chamber studies, however, argued against this possibility. We conclude, therefore, that the in vivo effects of calphostin C and PD98059 are primarily targeted at the epithelium, and blockade of PKC and ERK signaling pathways prevented the leukocyte-induced increase in paracellular permeability.

There was a lack of correlation between the effects of ERK inhibitors on leukocyte migration, which was small (Figure 2A), as compared with their normalization of barrier function, which was essentially complete (Figures 2B and 2C). However, the rate of leukocyte migration and changes in permeability are not necessarily linearly correlated, and a critical level of leukocyte migration may be needed before epithelial barrier function is reduced. It also is possible that the route taken by migrating leukocytes may be altered by inhibition of ERK1/2.

The effects of inhibiting ERK1/2 or PKC were not identical (Figure 2). Calphostin C had greater effects on leukocyte migration, and smaller effects on barrier function, than PD98059. This suggests that there are pathways in addition to ERK1/2 that are activated by PKC to influence migration and barrier function.

During leukocyte transmigration, epithelial ERK1/2 may be activated by contact with leukocytes or by release of mediators from leukocytes or epithelial cells. To establish definitively a role for contact activation would require the use of neutralizing antibodies to adhesion molecules such as CD11, CD18, and ICAM-1 (35). However, soon after inducing migration with fMLP, the activation of ERK1/2 in the epithelia was patchy and often colocalized with migrating leukocytes (Figure 4C), suggesting that the initial activation of epithelial ERK1/2 is by contact with leukocytes. Later, epithelial ERK activation became global (Figure 4D), indicative of the involvement of cytokines. Supernatant from leukocyte suspensions treated with fMLP failed to alter epithelial ERK1/2 activation (data not shown), suggesting that any cytokines involved in epithelial ERK1/2 activation are derived from the epithelium itself.

In conclusion, we have identified ERK1/2 activity as an important proximal signaling event involved in changes of bronchial epithelial permeability during leukocyte migration. Specific targeting of epithelial ERK pathways may lead to therapies aimed at restoring bronchial epithelial barrier function, or preventing excess leukocyte trafficking to the airway lumen.


   Acknowledgments
 
This study was supported by HL60288 and AI50496. The authors are thankful to Beate Illek for help with in vitro migration studies.

Received in original form February 14, 2003

Received in final form June 11, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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