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The Journal of Immunology, 2003, 171: 6097-6104.
Copyright © 2003 by The American Association of Immunologists

Activation of Endothelial Extracellular Signal-Regulated Kinase Is Essential for Neutrophil Transmigration: Potential Involvement of a Soluble Neutrophil Factor in Endothelial Activation 1

Brian N. Stein*,{dagger}, Jennifer R. Gamble*,{dagger}, Stuart M. Pitson*, Mathew A. Vadas*,{dagger} and Yeesim Khew-Goodall2,*

* Hanson Institute, Institute of Medical and Veterinary Science, and {dagger} Department of Medicine, University of Adelaide, Adelaide, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During an inflammatory response induced by infection or injury, leukocytes traverse the endothelial barrier into the tissue space. Extravasation of leukocytes is a multistep process involving rolling, tethering, firm adhesion to the endothelium, and finally, transendothelial migration, the least characterized step in the process. The resting endothelium is normally impermeable to leukocytes; thus, during inflammation, intracellular signals that modulate endothelial permeability are activated to facilitate the paracellular passage of leukocytes. Using a static in vitro assay of neutrophil transmigration across human umbilical vein endothelium, a panel of inhibitors of intracellular signaling was screened for their ability to inhibit transmigration. PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK) 1/2 activation, inhibited both transmigration across TNF-{alpha}-activated endothelium and transmigration induced by the chemoattractant fMLP in a dose-dependent manner. PD98059 did not inhibit neutrophil chemotaxis in the absence of an endothelial barrier nor neutrophil adhesion to the endothelium, suggesting that its effect was on the endothelium, and furthermore, that endothelial ERK activation may be important for transmigration. We demonstrate in this study that endothelial ERK is indeed activated during neutrophil transmigration and that its activation is dependent on the addition of neutrophils to the endothelium. Further characterization showed that the trigger for endothelial ERK activation is a soluble protein of molecular mass ~30 kDa released from neutrophils after activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leukocyte extravasation is a multistep process involving tethering, rolling, firm adhesion, and finally, transendothelial migration into the subendothelial space (1, 2). The mechanisms by which tethering, rolling, and firm adhesion occur are relatively well characterized, particularly in comparison with our current understanding of the mechanisms by which leukocytes traverse the endothelium. Under noninflammatory conditions, the endothelium has low permeability to leukocytes, but when an inflammatory response is initiated, the paracellular permeability of the endothelium is increased to enable leukocytes to pass in between endothelial cells.

It is now widely accepted that most leukocyte extravasation occurs at interendothelial junctions and that cell-cell adhesion receptors not only maintain the architecture of the endothelium, but also play a role in regulating vascular permeability (3, 4). Of particular relevance to regulating leukocyte extravasation are the homophilic adhesion receptors, vascular endothelial (VE)3-cadherin (5, 6, 7), an adherence junction protein, and platelet-endothelial cell adhesion molecule-1 (PECAM-1) (7, 8, 9, 10, 11).

In order for leukocytes to transmigrate across a fully sealed endothelium, adhesion between endothelial cells has to be transiently released to create a gap for the leukocytes to pass through. In addition, it is also possible that some form of transient adhesion between the endothelial cells and leukocytes is established as the leukocyte migrates through. One would therefore predict that mechanisms that disrupt interendothelial adhesion are set into action either when endothelial cells become activated by inflammatory cytokines or when activated leukocytes marginate and interact with the endothelium. Some of the mechanisms elucidated to date include the cleavage of adhesion receptors by elastase bound to the surface of leukocytes (12, 13) and activation of endothelial intracellular signaling pathways by adherent leukocytes (14). However, surface-bound elastase is unlikely to be a universal or major mechanism because monocytes and some monocytic cell lines that do not have surface-bound elastase can aptly transmigrate (5). Furthermore, there is now evidence that adhesion molecules such as VE-cadherin moved away from the site of leukocyte passage rather than being disrupted, whereas endothelial-endothelial PECAM-1 adhesion was released to enable the leukocyte to pass through (6, 7). Both these adhesion molecules were found to be displaced very transiently and returned to their earlier positions within a short time after the passage of the leukocyte; the period was too short for de novo synthesis of intact receptors to replace the cleaved ones (7).

Activation of endothelial intracellular signaling pathways therefore is likely to be essential for releasing PECAM-PECAM interaction or moving VE-cadherin away to enable the paracellular passage of leukocytes. It has been reported that leukocyte adherence leads to increases of endothelial intracellular Ca2+ that is essential for leukocyte transmigration to proceed (15, 16). Activation of myosin L chain kinase (MLCK) has also been observed to be essential for leukocyte transmigration (17, 18). Ca2+-dependent phosphorylation of MLCK appears to be essential for the cytoskeletal remodeling events that correlate with increased endothelial cell permeability. Unifying all of these is that many of the interendothelial adhesion receptors are linked to the actin cytoskeleton either directly or through their interactions with a number of intermediary cytosolic proteins (19). It is therefore conceivable that leukocyte adhesion-dependent intracellular Ca2+ fluxes activate MLCK to reorganize the cytoskeleton, leading to alterations in interendothelial adhesion receptor function that facilitate leukocyte transmigration.

We therefore set out to investigate whether other endothelial intracellular signaling pathways are essential for leukocyte transmigration. We report in this work that activation of the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) 1/2, in the endothelium facilitates neutrophil transmigration. In addition, we observed that a soluble factor derived from fMLP- or IL-8-stimulated neutrophils can activate endothelial ERK, and suggest that this neutrophil factor is the trigger for ERK activation in the endothelium during leukocyte transmigration.


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

Chemically synthesized IL-8 was a gift of I. Clark-Lewis (Centre for Biomedical Research, Vancouver, Canada). It was produced as a 72-aa form using automated solid-phase methods, as described elsewhere. fMLP was from Sigma-Aldrich (St. Louis, WA); human rTNF-{alpha} was from Genentech (South San Francisco, CA; batch 3056-55) or purchased from R&D Systems (Minneapolis, MN). Purified human fibronectin (Boehringer Mannheim, Indianapolis, IN) diluted in PBS, pH 7.3, to 50 µg/ml was used for coating surfaces, unless otherwise stated. Anhydrous cell culture grade DMSO (Sigma-Aldrich) was used as solvent for PD98059. The inhibitors that act on mitogen-activated protein/ERK kinase (MEK) PD98059 were from Calbiochem (San Diego, CA), and U0126 was from Promega (Madison, WI). Phospho-ERK Ab was obtained from Promega; anti-ERK Ab was a kind gift of A. Murray, Flinders University of South Australia, Australia; and the {beta}2 integrin functional blocking Ab, TS1/18, was a kind gift of T. Springer, Harvard Medical School (Boston, MA).

Culture of HUVEC

HUVEC were extracted by collagenase treatment according to a modified version of Wall et al. (20). Cells were grown in 25-cm2 gelatin-coated tissue culture flasks (Costar, Cambridge, MA) in endotoxin-free M199 medium (Cytosystems, Sydney, Australia) supplemented with 20% FCS (PA Biological, Sydney, Australia), 20 mm HEPES, sodium pyruvate, and nonessential amino acids at 37°C in a 5% CO2 atmosphere. Cells were replated 2–5 days after establishment of culture by harvesting with 0.05% trypsin-0.02% EDTA. Endothelial cell growth supplement (Multicel, Trace Biosystems, Castle Hill, NSW, Australia) at 25 mg/ml and heparin were added to cells that were passaged twice or more. In general, cells between passages 2 and 5 were used. All reagents used in the growth and passaging of HUVEC were made up under endotoxin-free conditions and contained <10 pg/ml endotoxin determined by the Limulus amebocyte assay.

Purification of human neutrophils

Neutrophils were purified from normal donors, as previously described (21), by dextran sedimentation, followed by density-gradient centrifugation with Lymphoprep (Nycomed, Oslo, Norway) and hypotonic lysis of erythrocytes. They were resuspended in assay medium (RPMI 1640 with 10 mM HEPES and 2.5% FCS) before use. Cytological examination of stained cytocentrifuged preparations showed >95% of the cells were neutrophils. Trypan blue staining confirmed over 98% of these cells were viable.

Transmigration assay

This was performed as previously described using Transwells (6.5 mm diameter, 3 µm pore size; Costar) on 24-well culture trays (21). Briefly, 5 x 104 HUVEC (between passages 2 and 5) were seeded in the upper chamber of each Transwell precoated with fibronectin (50 µg/ml for 30 min) and the cells were grown at 37°C in 5% CO2-supplemented air to form a confluent monolayer. Neutrophils were added at 5 x 105 cells/well to the top chamber and, where indicated, chemoattractant was added to the lower chamber. The neutrophils were incubated at 37°C for 1 h, after which the number that had transmigrated into the lower chamber was collected and counted. Transmigration is expressed as a percentage of neutrophils added.

Neutrophils were counted using one of two methods. Neutrophils retrieved from the lower compartment were counted directly using either a Coulter counter (Model ZF; Coulter, Herts, U.K.) or an indirect colorimetric assay based on the conversion of a tetrazolium salt (MTT) to a formazan. Briefly, MTT (0.2 mg/ml; Sigma-Aldrich) was added to the lower chamber and incubated for 4 h at 37°C. The neutrophils were pelleted by centrifugation, the pellets were resuspended in 200 µl acid isopropanol for 1 h, and the absorbance at 550 nm was determined. A standard curve was constructed by serial dilution of the neutrophil preparation, and the percentage of neutrophils transmigrating was calculated from this. The two methods used produced results of good fit (least squares fit regression analysis, >95% confidence) (data not shown).

Chemotaxis assay

Chemotaxis assays were performed using Transwells essentially as in the transmigration assay, except that the HUVEC monolayer was omitted. In addition, instead of precoating the upper chamber with fibronectin, the lower chamber was precoated with gelatin to prevent adhesion of neutrophils. Assay medium with or without added chemoattractants was added to the lower chamber, and 5 x 105 neutrophils/well were placed into the upper chamber of the Transwells. Neutrophils that had migrated through the filters after 1-h incubation at 37°C were counted. Counts are expressed as a percentage of the total number of cells added.

Adhesion assays

Adhesion assays were performed, as previously described (22), with the exception that neutrophils were used. Briefly, HUVEC were seeded on fibronectin (50 µg/ml)-coated 96-well flat-bottom plates at 5 x 104 cells/well and cultured for 2 days, as described above. After washing, neutrophils (5 x 105/well) were added to the confluent HUVEC monolayer and incubated for 30 min at 37°C in 5% CO2-supplemented air, after which nonadherent neutrophils were gently washed off. After washing, the cells were stained with Rose Bengal, and total numbers of adherent neutrophils were determined by densitometry. The number of adherent neutrophils was computed from a standard curve and expressed as a percentage of the neutrophils added.

In some cases in which the endothelial monolayer was omitted, the neutrophils were plated directly onto 96-well tissue culture dishes.

ERK activation assay

HUVEC (106 cells/well) were seeded in six-well tissue culture dishes. The confluent monolayers, either untreated or pretreated as indicated, were washed in PBS and lysed in 20 mM Tris-Cl, pH 8.0, containing 150 mM NaCl, 1 mM CaCl, 1% Triton X-100, 5 mM leupeptin, 10 mM PMSF, 25 mM benzamidine, 50 mM Na fluoride, 1 mM Na vanadate, and 50 mM {beta}-glycerophosphate (all from Sigma-Aldrich) for Western blotting analysis. Protein concentration was determined using the Bradford reagent (Bio-Rad, Hercules, CA), and equal amounts of protein were loaded onto a 7.5% SDS polyacrylamide gel. Western blots were conducted using an Ab specific to phosphorylated ERK, i.e., the activated form of ERK, and developed by ECL (Amersham, Arlington Heights, IL). Total ERK present was determined by stripping the filter and reblotting with an Ab against ERK 1/2.

Fractionation of neutrophil-conditioned medium

Neutrophils were prepared as described above. A total of 10 ml of conditioned medium was prepared by incubating neutrophils at 5 x 106/ml with 100 nM fMLP for 15 min at 37°C. The neutrophils were then spun down for 5 min at 1300 x g, and the supernatant was taken off, concentrated to 1 ml, and applied to a Superdex 75 HR 10/30 column (Pharmacia Biotech, Uppsala, Sweden) pre-equilibrated with 10 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 4.5 mM KCl at a flow rate of 0.5 ml/min. Proteins were eluted under the same conditions, and 1.0-ml fractions were collected. FCS (2.5%), glucose (5 mM), MgCl2 (1 mM), and CaCl2 (2 mM) were added to the fractions before assaying for their ability to activate ERK. The molecular mass of the eluted protein was estimated from this column by comparison with the elution volumes of RNase A, chymotrypsinogen A, OVA, and BSA. The V0 of the column was determined by the elution volume of blue dextran (molecular mass 2000 kDa).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neutrophil transmigration across endothelium is inhibited by inhibitors that act on MEK

To identify signaling pathways in the endothelium that are essential for neutrophil transmigration, a screen for their effects on transmigration was conducted using pharmacological inhibitors of various signaling pathways (data not shown). The studies were performed using an in vitro model of neutrophil transmigration across a confluent monolayer of cultured HUVEC. Transmigration induced by an exogenously added chemoattractant gradient and across TNF-{alpha}-activated endothelium were both examined. PD98059, an inhibitor that acts on MEK, the upstream activator of the extracellular regulated kinases (ERK) 1/2, was found to inhibit in a dose-dependent manner neutrophil transmigration induced by a chemoattractant (fMLP) gradient as well as transmigration across TNF-{alpha}-activated endothelium (Fig. 1, A and B). In general, at high doses of PD98059 (40 µM), transmigration across TNF-{alpha}-activated endothelium was inhibited almost completely, and transmigration across a gradient of fMLP was inhibited by 70–80% (Fig. 1B). The inhibitory effects of PD98059 were also confirmed using a second MEK inhibitor, U0126 (Fig. 1C). The difference in rates of basal transmigration between A and C in Fig. 1 is not statistically significant (p = 0.12). Differences between experiments in the rates of transmigration following stimulation most likely reflect differences in the properties of endothelial cells and neutrophils obtained from different donors.



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  		FIGURE 1. Inhibitors that act on MEK inhibit neutrophil transmigration across endothelium. A, Confluent HUVEC monolayers plated (5 x 104 cells/well) on Transwells were either unstimulated or activated with TNF-{alpha} (4 ng/ml) for 4 h. Thirty minutes before the assay, some monolayers were treated with PD98059 (25 µM) or DMSO control. Neutrophils (5 x 105 cells/well) were added with either PD98059 or DMSO. Transmigration was assayed across TNF-{alpha}-activated endothelium (TNF) and unstimulated endothelium with (fMLP) or without (Nil) a gradient of 10 nM fMLP. The mean and SEM from three separate experiments, each conducted in triplicate, are shown. An ANOVA was performed looking for an effect of PD98059 compared with DMSO on transmigration: in this experiment, p < 0.0001, and in the overall series, p < 0.0001. B, Endothelial monolayers were treated as in A, but the concentration of PD98059 was varied, as indicated. The data are presented as percentage of transmigration relative to that across unstimulated endothelium. One representative experiment of seven, each performed in triplicate, is shown. ANOVA performed looking for an effect of PD98059 compared with DMSO on transmigration showed p < 0.001 for TNF-{alpha}-activated endothelium and p < 0.001 for an fMLP gradient. C, Endothelial cells on Transwells were left unstimulated or activated with TNF-{alpha}, as in A. Using the standard protocol, UO126 or its solvent, DMSO, was added to the monolayers 30 min before the experiment, and then added with the neutrophils. The mean and SEM from three separate experiments, each performed in triplicate, are shown. ANOVA performed showed p < 0.001 when looking for an effect of U0126 compared with DMSO on transmigration.

 
PD 98059 does not inhibit transmigration by inhibiting neutrophil chemotaxis or adhesion

Two important properties of leukocytes governing their ability to transmigrate across an endothelial barrier are their ability to migrate and to adhere to the endothelium. Because the neutrophils were exposed to the MEK inhibitor throughout the duration of the transmigration assay, the effect of PD98059 on both neutrophil migration and adhesion was examined. To investigate the effect of PD98059 on neutrophil migration, a chemotaxis assay (in the absence of an endothelial monolayer) across a chemoattractant gradient was used. In addition to fMLP, IL-8 was also used as a chemoattractant to mimic the resultant IL-8 chemoattractant gradient generated when TNF-{alpha}-activated endothelium is used in the transmigration assay (23). Both fMLP- and IL-8-stimulated neutrophil chemotaxis were not significantly affected by PD 98059 when it was included with the assay (Fig. 2A). This suggests that the inhibitor had no effect on the ability of neutrophils to sense a chemotactic gradient or their ability to migrate toward it.



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  		FIGURE 2. PD98059 did not inhibit neutrophil chemotaxis or adhesion to endothelium. A, Chemotaxis assay using neutrophils pretreated for 20 min at room temperature with 30 µM PD98059 or DMSO. Chemotaxis was stimulated by the presence of 10 nM fMLP, or 1 nM IL-8 in the lower chamber; medium alone served as a control (Nil). The data represent one of three experiments, each performed in triplicate. ANOVA looking for an effect of PD98059 compared with DMSO on chemotaxis gave p = 0.359. B, Confluent HUVEC monolayers were stimulated with TNF-{alpha} for 4 h, with PD98059 or DMSO added in the final 30 min. The extent of neutrophil adhesion to the endothelium was determined using a standard adhesion assay. The data are presented as the percentage of neutrophil remaining adherent relative to that added (% adhesion). The results represent one of three experiments, each performed in triplicate (p = 0.238, t test comparing DMSO + TNF with PD + TNF).

 
To determine whether exposure of neutrophils to PD98059 affected neutrophil adhesion to TNF-{alpha}-activated endothelium, an adhesion assay was conducted on TNF-{alpha}-activated endothelium in the presence of the inhibitor or its vehicle. TNF-{alpha} treatment of endothelium stimulated neutrophil adhesion, but adhesion was not significantly affected by the inclusion of PD98059 (Fig. 2B), suggesting that the inhibitor did not affect the ability of neutrophils to adhere to the endothelium.

Endothelial ERK is activated by neutrophils

Data presented in Fig. 2 suggested that the decreased transmigration caused by inhibitors of ERK activation was not due to their effect on neutrophil function per se. This, in turn, suggested that endothelial ERK activation may be essential for transmigration to occur. We therefore investigated whether ERK activation in the endothelium may be occurring under the conditions of the transmigration assay and which parameter(s) present in the assay system was responsible for its activation. Initially, the role of the inducers, TNF-{alpha} and fMLP, used in the transmigration assay was assessed. Endothelial monolayers were treated with TNF-{alpha} or fMLP, as well as a number of cytokine and noncytokine activators of the endothelium, and ERK activation was determined by Western blotting with an Ab specific for the MEK-phosphorylated form of ERK, i.e., activated ERK. fMLP did not activate endothelial ERK (Fig. 3A, right panel). TNF-{alpha} (Fig. 3, A, left panel (0.4 ng/ml for 15 min), and B, lanes 1 and 3 (4 ng/ml for 4 h)) and IL-4 marginally activated ERK (Fig. 3A, left panel). This is consistent with our earlier observations showing a 1.5- to 2.0-fold activation of ERK (24). However, the degree of ERK activation was small in comparison with other activators such as oncostatin-M and PMA (Fig. 3A). Furthermore, because both fMLP (which did not activate ERK)- and TNF-{alpha}-induced transmigration were inhibited by PD98059, we explored the possibility that the endothelial ERK activation occurring during transmigration might be triggered by other factors.



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  		FIGURE 3. The presence of neutrophils is essential for activation of endothelial ERK. A, Endothelial cells treated with 0.4 ng/ml TNF-{alpha} (TNF), 30 ng/ml IL-4 (IL-4), 20 ng/ml oncostatin-M (OsM), 100 ng/ml PMA, or medium alone (Nil) for 15 min were assayed for ERK activation. Western blots shown were conducted using an anti-phospho-ERK Ab to detect activated ERK. Equal amounts of protein were loaded onto each lane. B, HUVEC were activated with 4 ng/ml TNF-{alpha} for 4 h or left unstimulated. After washing, either medium containing fMLP (-) or neutrophils pretreated with 10 nM fMLP (+) were added at a 10:1 ratio of neutrophils to HUVEC, and ERK activation was assayed after 15 min at 37°C. Western blots shown were conducted using Abs to phospho-ERK (upper panel) or reprobed with an anti-ERK polyclonal Ab after membranes were stripped (lower panel).

 
The role of the neutrophils in activating endothelial ERK was therefore investigated. An endothelial monolayer was set up as in the transmigration assay. The endothelial monolayer was either prestimulated with TNF-{alpha} or left unstimulated with fMLP included at the time of neutrophil addition. Following incubation with the endothelial monolayer, neutrophils were removed and the endothelial monolayer washed extensively to ensure complete removal of neutrophils before lysis and Western blotting with the phospho-ERK Ab to detect activated ERK. Addition of neutrophils to both resting and TNF-{alpha}-activated endothelium resulted in a dramatic increase in ERK activation (Fig. 3B), much greater (at least 10-fold) than that observed when TNF-{alpha} was added alone. In addition, there was no significant difference in the degree of ERK activation between unstimulated and TNF-{alpha}-stimulated endothelium, indicating that preactivation of the endothelium by TNF-{alpha} was not necessary for the neutrophils to trigger endothelial ERK activation. Although nonadherent neutrophils were removed from the endothelial monolayer before assaying for endothelial ERK activation and although ERK was not activated in the neutrophils that were removed (data not shown), we cannot completely exclude the possibility that the ERK activation observed is from a small proportion of neutrophils that remained adherent to the endothelial monolayer after washing. This will be addressed by data presented in Fig. 5.



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  		FIGURE 5. Conditioned medium from chemoattractant-stimulated neutrophils activates ERK in endothelial cells. Resting HUVEC monolayers incubated with medium (Nil), neutrophils (N), or neutrophil-conditioned medium (CM) were analyzed for ERK activation by Western blotting with an anti-phospho-ERK Ab. A, Neutrophils were stimulated for 15 min with 1 nM fMLP and then divided into two aliquots. One aliquot was centrifuged, and the top two-thirds were taken, carefully avoiding the cellular pellet, and added as conditioned medium (CM). The other aliquot was resuspended, and two-thirds were taken and added as the equivalent amount of neutrophil preparation (N). B, Conditioned medium from neutrophils treated with 1 or 100 nM fMLP (prepared as in A) was added to HUVEC either undiluted (neat) or after diluting 1 in 3 (1/3) in medium. C, Conditioned medium from unstimulated neutrophils or neutrophils stimulated with 10 nM IL-8 for either 15 or 45 min were added to HUVEC monolayers. Lower panel, Shows membrane reblotted with an anti-ERK Ab after stripping. D, Confluent HUVEC monolayers (5 x 104 cells/well) were pretreated with varying concentrations of PD98059 for 30 min before incubation with fMLP (10 nM)-stimulated neutrophil-conditioned medium. After 15-min incubation with neutrophil-conditioned medium, the cells were lysed and Western blotted with an anti-phospho-ERK Ab to detect ERK activation. The blots were then stripped and reblotted with anti-ERK Ab to detect total ERK present in each lane. The ratio of phospho-ERK to total ERK was determined by quantitating the Western blots using a Molecular Dynamics (Sunnyvale, CA) Typhoon Variable Mode Imager. The data are plotted relative to the amount of ERK activation in the absence of PD98059 (100%). E, Conditioned medium was made from neutrophils (106/ml) that were either preincubated for 30 min with 30 µM PD98059 or not, before stimulation by 10 nM fMLP. Conditioned medium from neutrophils that were unstimulated was used as a control. All conditioned medium was desalted by passage through PD10 columns (Amersham Biosciences) to remove PD98059 from the samples, where it was added. HUVEC monolayers were incubated with the desalted conditioned medium, and endothelial Erk activation was assayed after 15 min. The figure shows a representative Western blot for phospho-ERK and for total ERK from the same blot after stripping and reblotting from one of two experiments, and also a plot of the ratio of phospho-ERK to total ERK quantitated as described in D.

 
Blocking {beta}2 integrin-mediated neutrophil adhesion does not reduce ERK activation

A crucial step in leukocyte extravasation is firm adhesion of the leukocytes to the endothelium mediated by the binding of the leukocyte {beta}2 (CD18) integrins to their receptors on the endothelium (reviewed in Refs. 1 and 2)). To investigate whether adhesion to the endothelium was essential for activation of endothelial ERK by neutrophils, the neutrophils were either preincubated with a functional blocking Ab to {beta}2 integrin (TS 1/18) or not before being added to the endothelial monolayer in the presence of fMLP. After 15 min, the neutrophils and medium were removed, and the endothelial monolayer was washed and lysed. Western blots to detect ERK activation showed that ERK activation occurred only in the presence of neutrophils as expected and was not reduced by pretreatment with the functional blocking anti-{beta}2 integrin Ab (Fig. 4A). Parallel adhesion assays conducted in the presence or absence of fMLP confirmed that pretreatment of neutrophils with the anti-{beta}2 integrin Ab did indeed block fMLP-stimulated neutrophil adhesion, but not to basal adhesion (Fig. 4B). These data indicate that neutrophil-stimulated ERK activation was not dependent on {beta}2 integrin-mediated adhesion.



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  		FIGURE 4. Neutrophil adhesion is not a requirement for endothelial ERK activation. A, Neutrophils, untreated or incubated with a {beta}2 integrin functional blocking Ab, TS1/18 (50 µg/ml), for 20 min at room temperature were added to HUVEC in the presence of 1 nM fMLP for 15 min. HUVEC monolayers were then analyzed for ERK activation by Western blotting with an anti-phospho-ERK Ab. B, Neutrophils, untreated or treated with the TS1/18 Ab, as in A, were added to 96-well tissue culture dishes in the presence or absence of 1 nM fMLP, and neutrophil adhesion was determined. The data are presented as the percentage of neutrophil remaining adherent relative to that added (% adhesion). By ANOVA comparing ± TS1/18, p = 0.0002, n = 3.

 
A soluble neutrophil factor activates endothelial ERK

Because neutrophil adhesion to the endothelium was not essential for endothelial ERK activation, the possibility that the inducer may be a soluble factor produced by the neutrophils was examined. Conditioned medium from neutrophils stimulated with fMLP was harvested and used to stimulate endothelial monolayers. Care was taken to ensure that no neutrophils were carried over into the supernatants. As controls, an equivalent proportion of the neutrophils used to generate the conditioned medium as well as medium with only fMLP added were used to stimulate endothelial monolayers. fMLP alone had no effect on endothelial ERK activation, but both the neutrophils and conditioned medium activated ERK (Fig. 5A), suggesting that a soluble neutrophil factor is the active agent in activating endothelial ERK. This also confirms that it is activated endothelial and not neutrophil ERK that is detected by the assay. ERK activation induced by the conditioned medium was less than that induced when neutrophils were added, but was still significantly greater than the basal level of activated ERK present in resting endothelium or endothelium treated with fMLP alone. This may be attributed to the possibility that the neutrophils were continuing to produce the factor during the period of incubation with HUVEC, resulting in a higher local concentration of factor than when only the conditioned medium was added.

The dosage of fMLP required to induce production of the factor and the dosage of conditioned medium required to activate endothelial ERK were also investigated. Neutrophil-conditioned medium was prepared after incubation of neutrophils with 1 or 100 nM fMLP. At each concentration of conditioned medium (neat or one-third dilution) added to HUVEC, increasing the fMLP concentration used to stimulate neutrophils from 1 to 100 nM, led to an increase in the ERK-activating factor produced; the effect of fMLP dosage is more marked at the lower concentration of conditioned medium used (Fig. 5B). At each fMLP concentration used to induce the ERK-activating factor, increasing the amount of conditioned medium added to HUVEC resulted in an increase in endothelial ERK activation, with the dose dependency being more marked when the lower fMLP concentration was used to induce the ERK-activating factor (Fig. 5B).

TNF-{alpha} activation of endothelial cells results in the production of IL-8. We therefore investigated whether neutrophils exposed to IL-8 would also be induced to produce the ERK-activating factor. Incubation of neutrophils with IL-8, like fMLP, also induced the ERK-activating factor (Fig. 5C).

We had shown above that the inhibitor of ERK activation, PD98059, inhibited neutrophil transmigration. The endothelial ERK activation induced by conditioned medium derived from fMLP (10 nM)-activated neutrophils was also inhibited by PD98059 with a similar dose response (Fig. 5D) to that required for inhibition of transmigration (Fig. 1B). This further confirms that the soluble factor released by activated neutrophils is a likely candidate for activating endothelial ERK during neutrophil transmigration. Because the neutrophils were exposed to PD98059 in the transmigration assay, there is a possibility that the inhibition of transmigration by PD98059 was due to its effect on the release of the soluble ERK-activating factor by neutrophils. Neutrophil-conditioned medium obtained from neutrophils preincubated with PD98059 before stimulation with fMLP, followed by desalting to remove the PD98059, induced endothelial ERK activation to the same degree as that from neutrophils that had not been exposed to PD98059 (Fig. 5E). This confirms that the PD98059 did not inhibit transmigration by inhibiting the release of the soluble neutrophil factor.

Characterization of the ERK-activating factor produced by neutrophils

Initial characterization was aimed at determining whether the factor produced by neutrophils is a protein. Boiling the conditioned medium led to a complete loss of activity (Fig. 6A), suggesting that the factor is likely to be a protein. Centrifugation of the conditioned medium through a membrane with a pore limit of 10 kDa resulted in retention of most of the activity, indicating that it has a molecular mass greater than 10 kDa (data not shown).



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  		FIGURE 6. Characterization of the soluble ERK-activating neutrophil factor. A, Neutrophils stimulated with 100 nM fMLP (N), or conditioned medium prepared from fMLP-stimulated neutrophils (CM) that was either untreated or boiled for 30 min in a water bath were added to a resting confluent monolayer. ERK activation was detected by Western blotting with an anti-phospho-ERK Ab (top panel). The membrane was reblotted with an anti-ERK Ab after stripping (bottom panel). B, Fractionation of fMLP (100 nM)-stimulated neutrophil-conditioned medium by gel filtration using FPLC. The protein elution profile detected by absorbance at 280 nm is shown. Inset, Shows the column calibration curve obtained with known molecular mass standards. C, One-milliliter fractions of protein eluting from the gel filtration column shown in B were collected and added to HUVEC monolayers. After 15-min exposure to the eluates, the monolayers were lysed and the fractions with ERK-activating properties were ascertained by Western blotting the HUVEC lysates with an anti-phospho-ERK Ab.

 
Further characterization of the factor was conducted by fractionating neutrophil-conditioned medium on a gel filtration FPLC column. To determine the elution volume of the ERK-activating factor, fractions collected from the column were added to HUVEC monolayers and endothelial ERK activation was monitored. The endothelial ERK-activating activity eluted essentially as a single peak, mostly contained within two fractions (Fig. 6, B and C), and from the calibration curve generated (Fig. 6B, inset), the molecular mass of the ERK-activating factor was estimated to be ~30 kDa.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By using a range of pharmacological inhibitors of intracellular signaling pathways to study the late steps in transendothelial migration, we have found that activation of the mitogen-activated protein kinase, ERK 1/2, in the endothelium is essential for neutrophils to traverse the endothelial barrier. The inhibitor of MEK activation, PD98059, which consequently inhibits ERK activation, did not inhibit the ability of neutrophils to migrate toward a chemoattractant gradient in the absence of an endothelial monolayer nor their ability to adhere to activated endothelium. These data suggest that the adverse effect of PD98059 on neutrophil transmigration is most likely to be on signaling pathways within the endothelium rather than the neutrophil. In the multistep paradigm of transmigration (1, 2), leukocytes roll, tether, and then firmly adhere to the endothelium via the {beta}2 integrins before the final steps of traversing the endothelium. Thus, because PD98059 did not affect {beta}2 integrin-dependent adhesion of neutrophils to the endothelium, ERK activation is likely to be necessary for the steps postadhesion.

Intuitively, one would predict the activation of multiple signaling pathways within the endothelial cells in the vicinity of transmigrating leukocytes that would lead to alterations in cell-cell adhesion to allow leukocytes to pass between endothelial cells. There have not been, to our knowledge, previous reports of endothelial ERK activation associated with neutrophil transmigration, and because few signaling pathways that mediate the late steps in transmigration have been elucidated, our findings that endothelial ERK activation is essential for transmigration are a significant step forward.

Initial studies to identify the trigger for endothelial ERK activation during neutrophil transmigration suggest that it is not the fMLP or TNF-{alpha} added to induce transmigration, but rather the neutrophils themselves that activate endothelial ERK. Activation of endothelial ERK was not dependent upon adhesion of neutrophils to the endothelium. This is in contrast to previously identified endothelial intracellular signaling pathways involved in transmigration, which were up-regulated in a manner requiring neutrophil adhesion to the endothelium (15, 25). This finding is of particular interest because it supports the notion that multiple parallel signaling pathways are activated within the endothelium during transmigration. Further support for this comes from our preliminary studies indicating that ERK activation is not downstream of intracellular Ca2+-MLCK activation as ERK activation was still observed in the presence of the Ca2+ chelator, BAPTA-AM (B. Stein and Y. Khew-Goodall, unpublished observations).

Because PD98059 inhibited transmigration induced by an exogenously added chemoattractant as well as across TNF-{alpha}-activated endothelium, its effect is not likely to be on TNF-{alpha} signaling or on necessary downstream functions associated with endothelial activation such as IL-8 production. We have alluded, above, to alterations in cell-cell adhesion being important for transmigration to occur. Activation of MLCK, as a consequence of increasing intracellular Ca2+, upon firm adhesion of neutrophils to the endothelium, is one endothelial intracellular signaling pathway that has been reported to be activated during transmigration (15, 17, 18). It is thought that one downstream consequence of activation of this pathway is alterations in the cytoskeleton that serves to stabilize cell-cell interactions. This, in turn, may lead to transient increases in endothelial permeability caused by changes in the interactions between cell-cell adhesion complexes and the cytoskeleton. Although ERK activation is commonly associated with mitogenic signaling, it has a large array of substrates, including a number of microtubule-associated proteins (26) that when phosphorylated by ERK results in destabilization of the microtubules (27). The interaction between the microtubular and actin cytoskeletons suggests that ERK activation could also be involved in alterations of cell-cell adhesion during transmigration, although other mechanisms of action cannot be ruled out at this stage. Recent findings show that multiple cell adhesion receptors are modulated during transmigration, and moreover that different mechanisms are used to regulate different receptors to increase paracellular permeability (6, 7). It would therefore not be unexpected to find multiple signaling pathways activated to regulate different aspects of cytoskeletal function associated with cell-cell adhesion.

Further characterization of the trigger for activating endothelial ERK suggests that it is a soluble protein factor of molecular mass ~30 kDa that is produced by activated neutrophils. Production of the soluble factor, although independent of neutrophil-endothelium interactions, is dependent on chemoattractant activation of neutrophils. Although ERK activation induced by the soluble neutrophil factor appears to be adhesion independent in our static in vitro transmigration assay, in vivo in the presence of blood flow, neutrophil adhesion may be necessary to facilitate binding of locally released factor to the endothelium. Maximum levels of the factor are achieved within 10–15 min of fMLP or IL-8 stimulation. This suggests that de novo synthesis is not the mechanism of its up-regulation and that it is likely to be a product of neutrophil degranulation. Whether the soluble neutrophil factor is a novel molecule or a known molecule performing a novel role, identification of the factor and its mode of action remain the key to establishing its importance in transmigration. Further work is also underway to determine whether this is a universal mechanism used by all transmigrating leukocytes or whether it is confined to subsets of leukocytes.


   Acknowledgments
 
We thank Jenny Drew and Anna Sapa for the preparation and culture of HUVEC, and the staff of the Women’s and Children’s Hospital and Burnside Hospital (Adelaide, South Australia) for the collection of umbilical cords. We also thank Drs. A. Murray, Flinders University of South Australia, and T. Springer, Harvard Medical School, for their generous gifts of Abs, and Dr. I. Clark-Lewis for kindly providing us with IL-8.


   Footnotes
 
1 This work was supported by National Health and Medical Research Council, Australia Program Grant (to J.R.G., M.A.V., and Y.K.-G.), and a National Health and Medical Research Council Postgraduate Research Award to B.N.S. Back

2 Address correspondence and reprint requests to Dr. Yeesim Khew-Goodall, Hanson Institute, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia. E-mail address: yeesim.khew-goodall{at}imvs.sa.gov.au Back

3 Abbreviations used in this paper: VE, vascular endothelial; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein/ERK kinase; MLCK, myosin L chain kinase; PECAM, platelet-endothelial cell adhesion molecule. Back

Received for publication January 30, 2003. Accepted for publication September 23, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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