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The Journal of Immunology, 2006, 177: 6379-6387.
Copyright © 2006 by The American Association of Immunologists, Inc.

VCAM-1 Signals Activate Endothelial Cell Protein Kinase C{alpha} via Oxidation1

Hiam Abdala-Valencia and Joan M. Cook-Mills2

Allergy-Immunology Division, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lymphocyte binding to VCAM-1 activates endothelial cell NADPH oxidase, resulting in the generation of 1 µM H2O2. This is required for VCAM-1-dependent lymphocyte migration. In this study, we identified a role for protein kinase C{alpha} (PKC{alpha}) in VCAM-1 signal transduction in human and mouse endothelial cells. VCAM-1-dependent spleen cell migration under 2 dynes/cm2 laminar flow was blocked by pretreatment of endothelial cells with dominant-negative PKC{alpha} or the PKC{alpha} inhibitors, Rö-32-0432 or Gö-6976. Phosphorylation of PKC{alpha}Thr638, an autophosphorylation site indicating enzyme activity, was increased by Ab cross-linking of VCAM-1 on endothelial cells or by the exogenous addition of 1 µM H2O2. The anti-VCAM-1-stimulated phosphorylation of PKC{alpha}Thr638 was blocked by scavenging of H2O2 and by inhibition of NADPH oxidase. Furthermore, anti-VCAM-1 signaling induced the oxidation of endothelial cell PKC{alpha}. Oxidized PKC{alpha} is a transiently active form of PKC{alpha} that is diacylglycerol independent. This oxidation was blocked by inhibition of NADPH oxidase. In summary, VCAM-1 activation of endothelial cell NADPH oxidase induces transient PKC{alpha} activation that is necessary for VCAM-1-dependent transendothelial cell migration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Vascular cell adhesion molecule-1 expression is induced on endothelial cells in inflammatory tissues during atherosclerosis, immune challenge, and transplantation (1, 2, 3). VCAM-1 is required for eosinophil infiltration into the lung in experimental OVA-induced asthma (3) and T cell infiltration across the blood-brain barrier in experimental allergic encephalomyelitis (4). VCAM-1 also functions in combination with other adhesion molecules during chronic inflammation and tumor metastasis. Moreover, the VCAM-1 knockout is a mouse embryonic lethal (5). Therefore, understanding VCAM-1 signaling has important implications for disease intervention.

Leukocyte binding to VCAM-1 on endothelial cells activates endothelial cell signaling required for lymphocyte migration (6, 7, 8). We have reported that binding to VCAM-1 activates endothelial cell NADPH oxidase (6, 8, 9). NADPH oxidase generates superoxide that dismutates to hydrogen peroxide, yielding 1 µM H2O2 (9, 10). Extracellular H2O2 is generated by cell membrane NADPH oxidase as VCAM-1-mediated signals are blocked by exogenous addition of catalase (6, 11). H2O2 can then diffuse through membranes at 100 µm/s (12). The VCAM-1-induced generation of H2O2 activates localized endothelial cell actin structural changes and is required for VCAM-1-dependent lymphocyte migration (6). Mechanisms for reactive oxygen species (ROS)3-induced intracellular endothelial cell signals during VCAM-1-dependent lymphocyte migration have not been defined.

ROS can stimulate activation of protein kinase C (PKC). PKC{alpha} activity is most often described as requiring the cofactors Ca2+ and phosphatidylserine or diacylglycerol (DAG). PKC{alpha} can also become activated by H2O2 oxidation of its regulatory domain (13). Moreover, PKC{alpha} prepared from 5 mM H2O2-treated COS-7 cells did not require its cofactors Ca2+, phosphatidylserine, or DAG (14). However, this 5 mM H2O2 is much higher than the 1 µM H2O2 generated by VCAM-1 signaling (9, 10). It has not been reported whether PKC{alpha} is activated by VCAM-1-stimulated ROS production.

PKC activation by phorbol esters (PMA) or poly-L-arginine has also been shown to regulate cell shape and permeability in monolayers of endothelial or epithelial cells, respectively (15, 16, 17). Endothelial cell monolayer permeability is increased by PMA stimulation of PKC{alpha} in HUVECs (15). PMA stimulation induces contraction of bovine pulmonary artery endothelial cells and increases permeability to albumin (18, 19). Increases in vascular permeability and increases in leukocyte transendothelial migration occur in inflammatory sites. Whether VCAM-1 "outside-in" signals modulate PKC activity has not been reported.

In this study, we demonstrate that VCAM-1-stimulated endothelial cell NADPH oxidase activity results in transient activation of PKC{alpha} in endothelial cell lines and in cultures of human lung microvascular endothelial cells. In addition, we demonstrate that PKC{alpha} activity is required for VCAM-1-dependent transendothelial spleen cell migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells

The endothelial cell line mHEVa cells was previously derived from BALB/c mouse axillary lymph nodes and cultured as described (6, 9, 11, 20, 21, 22). The mHEVa cells have been spontaneously immortalized but are not transformed (20). Human microvascular endothelial cells from the lung (HMEC-Ls) (Clonetics) were grown in endothelial growth medium (Clonetics) plus 5% FCS and were used at passage 1–4. For spleen cells, single-cell suspensions were obtained from spleens of male 6- to 8-wk-old BALB/c mice (Harlan Industries) as previously described (6) and the RBC were lysed by hypotonic shock (20). The animal procedures were reviewed and approved by the Animal Care and Use Committee at Northwestern University (Chicago, IL).

Reagents

Apocynin was from Acros Organics. Diphenyleneiodonium chloride (DPI), Gö-6976, Rö-32-0432, and rabbit anti-PKC{alpha} (catalog no. SA-144) were obtained from Biomol. The [5, 6, 8, 9, 11, 12, 14, 15-[3H] (N)]-arachidonic acid (60–100 Ci/mM) was obtained from PerkinElmer. Catalase was from ICN Biomedicals. Rat anti-mouse VCAM-1 (clone MVCAM.A), mouse anti-human VCAM-1 (clone 51-10C9), and rat IgG (isotype Ab, clone R35-95) were obtained from BD Pharmingen. Goat anti-mouse IgG1 (catalog no. 1070-01) and goat anti-rat IgG (catalog no. 3050-01) were from Southern Biotechnology Associates. Rabbit anti-phospho-PKC{alpha}beta Thr638 (catalog no. 9375), and mouse anti-phosphotyrosine (catalog no. 9411) were from Cell Signaling Technology. Rabbit anti-phosphoserine (catalog no. 61-8100) were from Zymed Laboratories. Mouse anti-beta-actin (catalog no. ab6276) was obtained from Abcam. HRP-conjugated donkey anti-rabbit Ab was obtained from Amersham Biosciences. HRP-conjugated goat anti-mouse IgG was obtained from Bio-Rad. Dominant-negative (DN) PKC{alpha} in the plasmid pCMV (vector) was a gift from A. Descoteaux (University of Québec, Québec, Canada). This inactive transdominant mutant PKC{alpha} has the lysine in the ATP-binding domain replaced (23). Iodoacetamidofluorescein (IAF) (catalog no. I9271), anti-FITC (catalog no. F5636), DTT (catalog no. D-9779), DMSO (catalog no. 154938), and H2O2 (catalog no. H-1009) were obtained from Sigma-Aldrich.

Cell association and migration with laminar flow

The parallel plate flow chamber was used to examine migration under conditions of laminar flow. Spleen cells were used as a source of cells contiguous with the blood stream that could then migrate across endothelial cells. Spleen cell migration across the mHEV cell lines is stimulated by mHEV cell constitutive production of the chemokine MCP-1 (22) and is dependent on adhesion to VCAM-1 (6). We have previously reported that, after migration across the mHEV cells, the spleen cells are 65–70% B cells, 12–15% CD4+ cells, and 5–8% CD8+ cells (10). For this migration assay, endothelial cells were grown to confluence on slides and then the slide was placed in a parallel plate flow chamber (24). In vivo, in the absence of inflammation, the rapid fluid dynamics of the blood result in blood cells located midstream of the vascular flow (25). However, during inflammation, there is a change of fluid dynamics (25, 26, 27). With inflammation, vascular permeability increases yielding fluid flow from the blood into the tissues which likely contributes to contact of blood cells with the endothelium ("margination") (25, 27). There is also cell contact as the blood cells leave the capillaries and enter the postcapillary venules (26). Therefore, spleen cells (3 x 106) were added to the flow chamber (3.5 cm2) at 2 dynes/cm2. Next, to initiate spleen cell contact with the endothelial cells in vitro, the spleen cells were allowed to settle in the chamber as monitored by microscopy and then 2 dynes/cm2 was applied for the 15 min laminar flow assay. We have observed by microscopy that during the assay under laminar flow, the spleen cells in contact with the endothelial cells either roll, roll and detach, or roll, firmly attach, and migrate. After cell contact, the focus of the studies is on mechanisms of transendothelial migration under conditions of laminar flow. For this assay, the coculture was exposed to laminar flow at 2 dynes/cm2 at 37°C for 3 min to examine cell association or for 15 min to examine cell migration. After the 3 or 15 min at 2 dynes/cm2, the cells are washed at 2 dynes/cm2 with PBS supplemented with 0.2 mM CaCl2 and 0.1 mM MgCl2 because cations are required for cell adhesion. These cells were fixed with 3% paraformaldehyde for 1 h. To quantify migrated spleen cells at 15 min, phase contrast microscopy was used to count migrated cells that are phase dark (28). It has been reported that the transendothelial migration of an individual leukocyte, after it has rolled to a site of migration, occurs in 2 min (20). However, transendothelial migration of leukocytes is asynchronous. In the laminar flow assay, spleen cell migration is detected by 15 min. The number of spleen cells that were associated but not migrated (phase light cells) at 15 min is low because in 15 min, the majority of nonmigrating cells roll off the monolayer of endothelial cells as determined by microscopy (data not shown). Therefore, the number of spleen cells associated with the endothelial cells at 3 min of laminar flow are those cells that mediated cell-cell contact.

Transfection

mHEVa were grown to 90% confluence in 12-well plates or on slides. The cells were transfected with 1 µg of DN PKC{alpha} or the vector pCMV per well. Each of these transfections were performed in the presence of 2 µl/well LipofectAMINE 2000 Reagent (Invitrogen Life Technologies) according to instructions in culture medium without gentamicin, penicillin, or streptomycin. After 3.5 h, the medium was removed and replaced with medium containing antibiotics for 24 h.

Immunoprecipitation (29)

Endothelial cells in 12-well plates were washed with PBS and lysed in 100 µl/well radioimmunoprecipitation buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.2% SDS, 150 mM NaCl, 10 mM, HEPES (pH 7.3), 2 mM EDTA, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 100 mg/ml iodoacetamide, and 1 mM PMSF). Triplicate samples were combined, and lysates were incubated on ice for 20 min. Lysates were sonicated and then centrifuged for 10 min at 15,000 x g at 4°C. Equal volumes of this lysate were incubated with 50 µl of protein G beads and 5 µg/ml of indicated Ab overnight at 4°C with gentle rocking. These beads were washed three times with radioimmunoprecipitation buffer and one time each with 10 mM NaCl followed by 10 mM Tris (pH 7.4). Beads were heated for 5 min at 100°C in SDS sample buffer.

Western blotting

Cell lysates or immunoprecipitates in SDS sample buffer were analyzed by 7.5 or 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes according to manufacturer’s instructions (Bio-Rad) (300 mA for 3 h). The membranes were blocked in 5% nonfat dried milk or in 5% BSA for phosphoserine Abs in TBS plus 0.1% Tween 20 (TBST) for 1 h at room temperature and washed three times for 5 min in TBST. Membranes were incubated with primary Abs in TBST plus 5% BSA overnight, washed three times for 5 min in TBST, incubated with secondary Abs in TBST plus 5% BSA for 1 h, washed three times for 10 min in TBST, and examined for detection with ECL (Amersham Biosciences) and autoradiography. Equal protein loading was verified by stripping the membrane with Restore Western Blot Stripping Buffer (Pierce) for 15 min and then labeling with rabbit anti-PKC{alpha} or mouse anti-beta-actin. Densitometry was performed using Image J software from the National Institutes of Health. The data are presented as the fold increase in the ratio of relative intensity of the band/the relative intensity of band for the loading control (total PKC{alpha} or beta-actin).

IAF reactivity (30)

Endothelial cells were stimulated with anti-VCAM-1, isotype control Ab, or 1 µM H2O2. These cells were lysed in the presence of 10 µM IAF (pH 5.5–6) and PKC{alpha} was immunoprecipitated. Fluorescein or total PKC{alpha} was detected by Western blot. IAF reacts with nonoxidized cysteines (30). Thus, loss of IAF reactivity indicates oxidation. Controls include 10 mM DTT-reduced lysates for 10 min or lysates oxidized with 200 µM H2O2 for 20 min before addition of IAF.

Free DAG

The mHEV cells were prelabeled with 1.5 µCi/well [3H]arachidonic acid for 48 h. Where indicated, the cells were pretreated for 20 min with inhibitors. Cells were stimulated with H2O2 or by cross-linking VCAM-1 with 27 µg/ml rat anti-mouse VCAM-1 plus 15 µg/ml goat anti-rat IgG Ab. The stimulants and inhibitors were added to medium from the endothelial cell monolayers so that there was no addition of fresh serum-containing medium which stimulates phospholipase C activity. The H2O2 or VCAM-1 stimulation of endothelial cells was not done in serum-free conditions because serum is required for the mHEVa cell viability and the endothelial cell promotion of spleen cell migration (data not shown). The H2O2 or VCAM-1 stimulation was terminated with ice-cold methanol/concentrated HCl (100:1, v/v) and lipids were extracted by the method of Bligh and Dyer (31). After centrifugation at 2000 x g for 20 min, the lower chloroform phase was collected and evaporated under nitrogen. Samples as well as standards for DAG, phosphatidic acid, and arachidonic acid were resuspended in 15 µl of chloroform and spotted onto Silica Gel 60 TLC plates (200 µm; catalog no. 10028; Selecto Scientific). Plates were developed in a solvent system of hexane:ether:acetic acid (70:30:1) for the separation of DAG. The lipids were visualized by iodine vapor and then individual spots that comigrated with lipid standards were scraped and quantified by liquid scintillation spectrometry.

Statistics

Data were analyzed by a one-way ANOVA followed by Tukey’s multiple comparisons test (SigmaStat; Jandel Scientific).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Endothelial cell PKC{alpha} is required for VCAM-1-dependent spleen cell migration across endothelial cells

VCAM-1 is a member of the Ig superfamily. Another member of the Ig superfamily, ICAM-1, signals through PKC{alpha} in endothelial cells (32, 33). It is not known whether PKC{alpha} is involved in VCAM-1 signaling for VCAM-1-mediated lymphocyte migration. Therefore, we have used cultures of endothelial cells to determine whether endothelial PKC{alpha} is required for VCAM-1-dependent spleen cell migration under laminar flow. It has been previously reported that VCAM-1 can support leukocyte rolling, firm adhesion, and migration (6, 21, 34). Spleen cells were chosen as a source of cells contiguous with the blood. We chose an endothelial cell model that provides a method to examine VCAM-1-mediated lymphocyte migration, while avoiding endothelial cell outside-in signals from lymphocyte binding to other adhesion molecules on endothelial cells (6, 9, 10, 20). In this model, the cell line mHEVa constitutively expresses VCAM-1 and does not express other ligands for leukocyte migration such as ICAM-1, PECAM-1, and E-selectin as determined by immunofluorescence labeling (21), cDNA microarray analysis or leukocyte adhesion assays with blocking Abs (data not shown) (21). Spleen cell migration across these endothelial cells requires adhesion to VCAM-1 (6, 21). The endothelial cell line also constitutively produces MCP-1 for the stimulation of lymphocyte chemotaxis (22). To examine PKC{alpha} function, confluent monolayers of mHEVa cells were untreated or pretreated for 20 min with two inhibitors of PKC{alpha}, Rö-32-0432 (100 nM), or Gö-6976 (2.3 nM). The IC50 for Rö-32-0432 is 18–72 nM for purified rat brain PKC{alpha}, beta, {gamma} (35). Gö-6976 inhibits PKC{alpha} and PKCbetaI with IC50 values in the nanomolar range, whereas up to 3 µM Gö-6976 has no effect on the activity of PKC {delta}, {epsilon}, {zeta} (36, 37). After treatment with the PKC{alpha} inhibitors, the cells were washed five times. The last wash was added to a set of untreated cells to determine whether effective concentrations of free inhibitor were removed. Spleen cells were added at 2 dynes/cm2 laminar flow. After 15 min, the monolayers were washed and fixed. Spleen cell migration was quantified by phase contrast microscopy (28). Rö-32-0432 and Gö-6976 blocked spleen cell migration under laminar flow (Fig. 1A).


Figure 1
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  		FIGURE 1. Inhibition of endothelial cell PKC{alpha} blocks VCAM-1-dependent spleen cell migration. A, Confluent monolayers of mHEVa cells were nontreated (NT) or treated for 20 min with the PKC{alpha}-selective inhibitors 2.3 nM Gö-6976 and 100 nM Rö-32-0432 and then washed five times. In addition, medium from the last washes was added to nontreated spleen cells to ensure that the inhibitor was sufficiently removed. Spleen cells were added to the endothelial monolayer in the presence or absence of anti-VCAM-1 (14 µg/cm2 endothelial cell monolayer) at 2 dynes/cm2, allowed to briefly settle to mediate cell contact and then exposed to 2 dynes/cm2 laminar flow for 15 min. Cells were washed and fixed in 3% paraformaldehyde for 1 h. Spleen cell migration was examined by phase contrast microscopy (28 ). Nonmigrated spleen cells are phase-light and migrated spleen cells appear as phase dark (28 ). The cells that migrated are >88% lymphocytes (10 ). Gö-6976 and Rö-32-0432 had no affect on cell viability, as determined by trypan blue exclusion (data not shown). Data are from three to five experiments. *, p < 0.05 compared with nontreated, DMSO treated, and last washes. B, Confluent monolayers of mHEVa cells were treated as in A with the inhibitors indicated, 2 dynes/cm2 laminar flow was applied for 3 min and then the cells were washed and fixed. The total number of spleen cells associated with the endothelial monolayer was determined. Data are from three experiments. *, p < 0.05 compared with cells without anti-VCAM-1 Abs.

 
The last washes, that were added to nontreated cells, had no effect on spleen cell migration (Fig. 1A). The inhibitors did not affect cell viability as determined by trypan blue exclusion (data not shown). Gö-6976 did not affect the total number of spleen cells that were associated with the endothelial cells under laminar flow (Fig. 1B). Furthermore, Gö-6976 did not affect VCAM-1-mediated interaction of spleen cells with the endothelial cells (Fig. 1B). The order of magnitude of cells migrated/cm2 in 15 min is similar to previous studies by us and others (6, 38, 39, 40, 41) when the data are adjusted for the experimental parameters such as well area and time course (data not shown). In summary, although there was no effect of inhibition of PKC{alpha} on association of the spleen cells with the endothelial cells, the spleen cells were unable to complete migration across the endothelial cells.

It was then determined whether transient transfection of endothelial cells with a DN PKC{alpha} blocks VCAM-1-dependent spleen cell migration. mHEVa cells on slides were Lipofectamine-transfected with 1 µg of DN PKC{alpha} plasmid or vector pCMV for 3.5 h, washed, and cultured for 24 h. These cells were examined for increased PKC{alpha} expression by Western blot as well as examined for their ability to promote spleen cell migration. Migration was examined in the parallel plate flow chamber assay at 2 dynes/cm2 laminar flow for 15 min (28). The transfection did not affect mHEVa cell expression of VCAM-1 as determined by immunolabeling and flow cytometry (data not shown). As expected, transfection with the DN PKC{alpha} increased total PKC{alpha} expression (Fig. 2A). Importantly, transfection with the DN PKC{alpha} inhibited spleen cell migration as compared with the vector control (Fig. 2B). The DN PKC{alpha} did not alter the number of spleen cells associated with the endothelial cells (Fig. 2C). Therefore, taking together the data, VCAM-1-dependent spleen cell migration across monolayers of the mHEVa cell lines used endothelial cell PKC{alpha} activity.


Figure 2
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  		FIGURE 2. DN PKC{alpha} blocks VCAM-1-dependent spleen cell migration. mHEVa were grown to 90% confluence and transfected with 1 µg of vector with DN PKC{alpha} or vector pCMV for 3.5 h, washed, and cultured for 24 h. A, The cells were examined for total PKC{alpha} expression by Western blot. B, Spleen cells were added to the endothelial cell monolayer and the coculture was exposed to 2 dynes/cm2 laminar flow for 15 min as in Fig. 1. Cells were washed and fixed in 3% paraformaldehyde for 1 h. Spleen cell migration was examined by phase contrast microscopy. C, Spleen cells were added to the endothelial cell monolayer and the coculture was exposed to 2 dynes/cm2 laminar flow for 3 min as in Fig. 1. Cells were washed and fixed in 3% paraformaldehyde for 1 h. Total number of spleen cells associated with the endothelial cells was examined by microscopy. The DN PKC{alpha} had no affect on cell viability, as determined by trypan blue exclusion (data not shown). Data are from three experiments. *, p < 0.05 compared with nontreated.

 
VCAM-1 outside-in signals activate endothelial cell PKC{alpha}

We previously reported that anti-VCAM-1 cross-linking of VCAM-1 mimics spleen cell activation of endothelial cell NADPH oxidase (6, 9, 11). Anti-VCAM-1 Abs are also used to demonstrate that binding to VCAM-1 can stimulate the signal rather than signals generated by a subsequent cell-cell interaction. Therefore, we determined whether anti-VCAM-1 stimulates endothelial cell PKC{alpha} activity. This was examined in endothelial cell lines (mHEVa cells) and cultures of nonimmortalized endothelial cells (HMEC-L). The endothelial cell lines constitutively express VCAM-1 whereas the HMEC-L cells require cytokine stimulation for expression of adhesion molecules. We stimulated adhesion molecule expression on HMEC-L cells with TNF-{alpha} overnight. After TNF-{alpha} stimulation, adhesion molecules including VCAM-1 were expressed by HMEC-L cells as determined by immunolabeling and fluorescence microscopy (data not shown). VCAM-1 on monolayers of mHEVa cells or TNF-{alpha}-treated HMEC-Ls was cross-linked with anti-VCAM-1 plus a secondary Ab for 10 min. Anti-CD98 was used as a control primary Ab because it binds to the mHEVa cells (data not shown) but CD98 does not signal through PKC{alpha} (42, 43, 44). After stimulation, the cells were washed, lysed, and examined by Western blot for the phosphorylation of PKC{alpha} Thr638. This Thr638 is an autophosphorylation site indicative of activation of PKC{alpha} (45). At the optimal time of 10 min (Fig. 3A), Ab cross-linking of VCAM-1 significantly increased the phosphorylation of PKC{alpha} Thr638 as compared with nontreated cells, isotype Ab-treated cells, or anti-CD98-treated cells in mHEVa cells (Fig. 3, B and C). Anti-VCAM-1 also stimulated phosphorylation of PKC{alpha} Thr638 in cytokine-treated cultures of HMEC-L cells (Fig. 3D). Furthermore, VCAM-1 stimulation did not increase total PKC{alpha} (Fig. 3 and see Fig. 6). In summary, stimulation of VCAM-1 increased PKC{alpha} activity in an endothelial cell line and in cultures of HMEC-L cells.


Figure 3
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  		FIGURE 3. Anti-VCAM-1 stimulation induces an increase in phosphorylation of PKC{alpha} Thr638 that is dependent on ROS generation. Confluent monolayers of A–C mHEVa cells and D TNF-{alpha} (1 ng/ml)-treated HMEC-L cells in 12-well plates were nontreated (NT) or incubated for 30 min with apocynin (4 mM), with catalase (5000 U/ml), or with DPI (5 µM). These endothelial cells were stimulated with 27 µg/ml anti-VCAM-1 Ab or control Ab plus 15 µg/ml of a secondary Ab for (A) 5–60 min or (B–D) 10 min. In D, the HMEC-L cells were also stimulated with 1 µM H2O2 for 10 min. Upper micrographs in each panel are representative Western blots using rabbit anti-phospho PKC{alpha} Thr638 (1/1000), rabbit anti-PKC{alpha} (1/100), or anti-beta-actin (1/5000) followed by HRP-conjugated donkey anti-rabbit secondary Ab (1/4000) and ECL detection. The phosphorylation status of PKC{alpha} Thr638 is presented as the fold increase in the ratio of the relative intensity of phospho-PKC{alpha} Thr638/the relative intensity of the loading control (total PKC{alpha} or beta-actin). The apocynin, catalase, DPI, and H2O2 had no effect on endothelial cell viability as determined by trypan blue exclusion (data not shown). Data presented are the mean ± SE from three experiments. *, p < 0.05 compared with NT, isotype Ab, or inhibitor-treated cells.

 

Figure 6
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  		FIGURE 6. Anti-VCAM-1 and H2O2 induces oxidation of PKC{alpha}. mHEVa cells were treated with apocynin and then treated with anti-VCAM-1 or control Ab for 10 min as described in Fig. 3. A, The lysates were nontreated or reduced with 10 mM DTT for 10 min. As a positive control for oxidation, a lysate from NT cells was oxidized with 200 µM H2O2 for 20 min. The lysates were then examined for cysteine oxidation status by determining reactivity with 10 µM IAF (20 min), which reacts with nonoxidized cysteines. PKC{alpha} was immunoprecipitated and examined by Western blot using anti-fluorescein or anti-total PKC{alpha} Abs (1/1000). There was no change in total PKC{alpha}. B, mHEVa cells were stimulated for the time indicated with anti-VCAM-1 and examined for IAF reactivity as described in A. Data presented are the mean ± SD from three experiments. *, p < 0.05 as compared with NT. **, p < 0.05 for the comparison indicated.

 
VCAM-1 activation of endothelial cell PKC{alpha} is mediated by endothelial cell NADPH oxidase-generated ROS

Adhesion to VCAM-1 stimulates endothelial cell H2O2 generation that is dependent on endothelial cell NADPH oxidase (9, 11) but not NO synthase, xanthine oxidase, or cytochrome P450 (6). NADPH oxidase generates superoxide that dismutates to H2O2. The VCAM-1-stimulated H2O2 generation is dependent on endothelial cell NADPH oxidase as it is blocked by apocynin (an NADPH oxidase inhibitor), by DPI (an irreversible inhibitor of flavoproteins such as NADPH oxidase) or by antisense to the catalytic subunit of NADPH oxidase (6, 9, 11). Therefore, we determined whether the anti-VCAM-1-stimulated generation of ROS activates PKC{alpha}. mHEVa cells were pretreated for 30 min with the H2O2 scavenger catalase (5000 U/ml), with apocynin (4 mM), or with DPI (5 µM). In addition, TNF-{alpha}-activated HMEC-Ls were pretreated for 30 min with 4 mM apocynin. We have previously reported that these concentrations of inhibitors block VCAM-1-stimulated generation of ROS and block VCAM-1-dependent lymphocyte migration without affecting lymphocyte adhesion to endothelial cells or endothelial cell viability (6, 10). After pretreatment, the cells were stimulated with anti-VCAM-1 or a control Ab and a secondary Ab. Anti-CD98 or isotype control Abs did not stimulate PKC{alpha} (Fig. 3). DPI, apocynin, and catalase inhibited anti-VCAM-1-stimulated phosphorylation of PKC{alpha} Thr638 in the mHEVa cells (Fig. 3, B and C). Apocynin also blocked VCAM-1-stimulated PKC{alpha} phosphorylation in cultures of HMEC-L cells (Fig. 3D). In summary, PKC{alpha} has a function downstream of NADPH oxidase that is required for endothelial cell promotion of VCAM-1-dependent spleen cell migration.

Exogenous H2O2 activates endothelial cell-associated PKC{alpha}

Binding to VCAM-1 stimulates endothelial cell NADPH oxidase, resulting in the generation of 1 µM H2O2 (10). H2O2 diffuses through cell membranes at 100 µm/s (12), suggesting that the H2O2 produced at the endothelial cell membrane upon VCAM-1 stimulation may modulate intracellular signaling pathways. Therefore, it was determined whether exogenous H2O2 (0.1–5 µM) activated autophosphorylation of endothelial cell PKC{alpha} Thr638. At 10 min, 1 µM H2O2 significantly increased autophosphorylation of PKC{alpha} Thr638 in mHEVa cells (Fig. 4) and cultures of cytokine-treated HMEC-L cells (Fig. 3D). A 5-fold increase in H2O2 (5 µM) had less activation of PKC{alpha} (Fig. 4). In summary, VCAM-1 activates endothelial cell NADPH oxidase resulting in the generation of low concentrations of H2O2 that then can activate endothelial cell PKC{alpha}. This H2O2 activation of PKC{alpha} was transient (Fig. 4B). Therefore, H2O2, at concentrations that are generated by VCAM-1-outside-in signals (10), stimulated a significant increase in PKC{alpha} Thr638 phosphorylation.


Figure 4
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  		FIGURE 4. Exogenous 1 µM H2O2 activates endothelial cell-associated PKC{alpha}. A, mHEVa cells were incubated with low concentrations of exogenous H2O2 (0.1, 1, and 5 µM) for 10 min. Phospho-PKC{alpha} Thr638 was examined by Western blot and analyzed as described in Fig. 3. *, p < 0.05 as compared with 0, 0.1 and 5 µM H2O2. **, p < 0.05 as compared with 0 and 0.1 µM H2O2. B, mHEVa cells were incubated with 1 µM H2O2 for 1, 5, 10, or 15 min. *, p < 0.05 as compared with 1, 5, and 15 min. **, p < 0.05. A significant decrease compared with 10 min. Data presented are the mean ± SE from three experiments.

 
VCAM-1-mediated activation of PKC{alpha} does not increase levels of free DAG

PKC{alpha} is a classical PKC that uses calcium and DAG as cofactors for activity. During cell stimulation of DAG-dependent PKC{alpha}, phospholipase generation of DAG is increased (46, 47). PKC{alpha} can also be activated by oxidation with 4 mM H2O2 and this activity is DAG independent (13, 14). Therefore, we determined whether binding to VCAM-1-stimulated generation of DAG. mHEV cells were prelabeled with 1.5 µCi/well [3H]arachidonic acid for 48 h during which the endothelial cells grew to confluence. Fresh culture medium was not added during anti-VCAM-1 or 1 µM H2O2 stimulation, as we observed that fresh serum in culture medium stimulates phospholipase activity in the endothelial cells (data not shown). An increase in free [3H]DAG was not detected after stimulation of endothelial cells by Ab cross-linking of VCAM-1 or treatment with 1 µM H2O2 (Fig. 5). Ab cross-linking of VCAM-1 or 1 µM H2O2 induced a slight decrease in DAG (Fig. 5). In summary, binding to VCAM-1 or 1 µM H2O2 did not stimulate an increase in DAG.


Figure 5
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  		FIGURE 5. Anti-VCAM-1 and H2O2 stimulation does not increase endothelial cell DAG. Phospholipase C activity was assessed by measuring the production of [3H]DAG by mHEV cells prelabeled with (A) 1.5 µCi/well or (B) 0.5 µCi/well [3H]arachidonic acid for 48 h. A, Endothelial cells were stimulated with 27 µg/ml rat anti-mouse VCAM-1 Ab plus 15 µg/ml goat anti-rat IgG Ab for 10 min. B, Endothelial cells were stimulated with 1 µM H2O2 for 30–600 s. *, p < 0.05. Data presented are the mean ± SE from three experiments.

 
VCAM-1 induces oxidation of PKC{alpha}

Oxidation of PKC{alpha} has been reported to induce a transient activation of PKC{alpha} that is independent of DAG (13, 14). Because binding to VCAM-1 activated a transient increase in phosphorylation of PKC{alpha} Thr638 (Fig. 3A) and no increase in free DAG in the cells (Fig. 5), it was determined whether binding to VCAM-1 induced oxidation of PKC{alpha}. Confluent monolayers of mHEVa cells were nontreated, treated with apocynin or treated with the 0.1% DMSO solvent control. The cells were nonstimulated, stimulated with anti-VCAM-1 or an isotype Ab plus a secondary Ab, or stimulated with 1 µM H2O2 for 10 min. The lysates from these cells were nontreated or as a control, they were reduced with 10 mM DTT for 10 min. A positive control for oxidation was a NT cell lysate incubated with 200 µM H2O2 for 20 min. To examine oxidation of cysteines, the lysates were incubated for 20 min with 10 µM IAF which reacts with nonoxidized cysteines (30). PKC{alpha} was immunoprecipitated and examined by Western blot for fluorescein or total PKC{alpha}. Anti-VCAM-1 induced a decrease in IAF reactivity (Fig. 6), indicating an oxidation of PKC{alpha} (30). PKC{alpha} from 1 µM H2O2-treated cells also had reduced reactivity with IAF (Fig. 6). These decreases in IAF reactivity were blocked by apocynin and were reversed by reduction of lysates with DTT (Fig. 6). As expected, the positive control lysates that were exposed to high levels of H2O2 exhibited reduced IAF reactivity (Fig. 6). The oxidation of PKC{alpha} in anti-VCAM-1-stimulated cells was transient (Fig. 6B), which is consistent with the transient nature of PKC{alpha} activity after oxidation and the transient autophosphorylation at PKC{alpha} Thr638 (Fig. 3). In summary, anti-VCAM-1 and the VCAM-1-signaling intermediate, 1 µM H2O2, induced oxidation of PKC{alpha} and transient activation of PKC{alpha}.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrate that endothelial cell PKC{alpha} activity is an intracellular signal during VCAM-1-dependent cell migration. We previously reported that lymphocyte migration across mHEVa cells is blocked by Ab inhibition of VCAM-1 binding (6, 10) or by inhibition of endothelial cell NADPH oxidase activity (6, 9). We have shown here that this VCAM-1-dependent migration is blocked by inhibition of PKC{alpha}. The activation of PKC{alpha} is downstream of VCAM-1-stimulated NADPH oxidase in endothelial cell lines and cultures of HMEC-L cells. The endothelial cell NADPH oxidase-generated ROS oxidize and activate endothelial cell PKC{alpha}. Moreover, the exogenous addition of 1 µM H2O2, which corresponds to the levels of H2O2 produced after VCAM-1 stimulation (6, 10, 20), mimicked VCAM-1-induced phosphorylation of PKC{alpha} Thr638 and the oxidation of PKC{alpha}. The phosphorylation of PKC{alpha} is required for enzyme activity and Thr638 is an autophosphorylation site (45, 48). Thus, we demonstrate that VCAM-1 stimulates ROS-mediated activation of PKC{alpha} that is required for VCAM-1-dependent spleen cell migration. This is the first report on VCAM-1 signaling through PKC{alpha} and we identified an oxidation mechanism for this activation.

PKC{alpha} is a classical PKC that uses the cofactors calcium and DAG. We have previously reported that VCAM-1 stimulates release of intracellular calcium and calcium channels (9). However, in addition to DAG activation of PKC{alpha}, PKC{alpha} can also be activated independent of DAG when PKC{alpha} is oxidized by H2O2. The majority of studies on ROS modulation of PKC activity or endothelial cell function focus on high levels of ROS for damage to endothelial cells (49). It has been reported that 5–10 mM H2O2 induces translocation of PKC{alpha} to the membrane of human saphenous vein endothelial cells (50) or induces an increase in activity of an oxidatively modified PKC{alpha} that is independent of Ca2+ and phospholipids in transfected COS-7 cells, NIH3T3 cells, C6 glioma, or B16 melanoma cells (13, 14). H2O2 (5 mM) activates PKC{alpha} by oxidation of the PKC regulatory domain in 3–10 min (13). However, beyond 10 min, these high concentrations of H2O2 induce inactivation of the PKC{alpha}, presumably through additional oxidation (13). In addition, it has been reported that, when PKC{alpha} is oxidized by high concentrations of H2O2, it then induces activation of PLD for generation of phosphatidic acid in leukemia cells (51). They also reported that <200 µM H2O2 does not induce PKC{alpha}-mediated activation of PLD in the leukemia cells (51). Consistent with this, in our studies, PLD activity, as measured by free phosphatidic acid generation, was not increased in 1 µM H2O2-treated endothelial cells or in anti-VCAM-1-stimulated endothelial cells that generate 1 µM H2O2 (data not shown). We did demonstrate that low concentrations of H2O2 (1–5 µM) activate and oxidize PKC{alpha} in 10 min. However, in contrast to the later oxidative inactivation of PKC{alpha} in studies with high concentrations of H2O2, we found that, after 10 min, PKC{alpha} oxidation was reduced to baseline and its enzymatic activation was reversed. Therefore, with low concentrations of H2O2, there is reversible oxidation/activation of PKC{alpha}. We also demonstrated that there was not an increase in generation of free DAG under conditions that avoided addition of fresh serum-containing medium because fresh serum induced an increase in DAG (data not shown). Therefore, these data are consistent with transient DAG-independent PKC{alpha} activity downstream of VCAM-1 activation of NADPH oxidase. This transient activation of PKC{alpha} is in concordance with the rapid transendothelial migration of leukocytes as after migration, endothelial cell junctions are rapidly reformed. Furthermore, it is likely that the VCAM-1-stimulated production of a maximum of 1–2 µM H2O2 is tightly controlled so that leukocyte migration is obtained without vascular damage.

Localization of signals during leukocyte transendothelial migration is important as endothelial cells retract only at the site of contact with a leukocyte. Therefore, it is interesting to discuss how extracellular production of H2O2 could activate localized signals for endothelial cells. Cell membrane NADPH oxidase generates extracellular superoxide which rapidly dismutates to H2O2. Therefore, how would extracellular H2O2, which rapidly diffuses as well as passes through cell membranes at 100 µm/s (12), result in stimulation of localized endothelial function to allow endothelial cell shape changes at the site of leukocyte contact and VCAM-1-dependent leukocyte passage? Also, is not extracellular ROS just washed away by blood flow? Our current and previous studies suggest that, because exogenous addition of catalase scavenges the H2O2 and blocks VCAM-1-dependent responses (6, 11), VCAM-1 engagement results in extracellular H2O2. Specifically, we have reported that catalase scavenging of extracellular H2O2 blocks VCAM-1-dependent lymphocyte migration (6) and VCAM-1/ROS-dependent activation of endothelial cell-associated matrix metalloproteinases (MMPs) (11). Furthermore, this VCAM-1-dependent endothelial cell ROS oxidation and activation of endothelial cell-associated MMPs was not altered under static conditions verus conditions of laminar flow (11). Thus, a working model is that ROS produced by localized endothelial cell membrane NADPH oxidase: 1) oxidize localized proteins such as membrane-associated MMPs or 2) diffuse into the cell at 100 µm/s and act on localized intracellular PKC{alpha}, thereby affecting localized endothelial cell functions. In the cell, as the concentration of H2O2 is only 1 µM (9, 10), H2O2 likely oxidizes localized targets but, as the H2O2 diffuses, it would be too dilute to have a functional effect. In addition, extracellular ROS that is washed away by the blood flow would also be diluted and therefore have little functional effect at distant sites. The detection of localized VCAM-1-dependent changes in endothelial cell junctions is an exciting target recently under investigation in our laboratory and beyond the scope of this manuscript.

When addressing oxidative activation of PKC, it is important to include that PKC{alpha} activity is also regulated by phosphatases. Inhibition of Ser/Thr phosphatases (PPs) increases PKC{alpha} phosphorylation in bovine pulmonary microvascular endothelial cells (52). An inhibition of Ser/Thr PPs can occur by oxidation of the phosphatase active site (53, 54, 55, 56). Furthermore, the sensitivity of PKC{alpha} to Ser/Thr PPs is reversible (57), providing an additional potential mechanism for transient PKC activity during endothelial cell retraction. Alternatively, active phosphatases can dephosphorylate PKC{alpha} and thus may participate in down-regulation of PKC{alpha} activity, thereby contributing to the transient nature of the PKC{alpha} activity during VCAM-1 signaling. Whether VCAM-1-stimulated PKC{alpha} is also modulated by Ser/Thr PPs is an interesting area for future research. Nevertheless, we identified that ROS generated by anti-VCAM-1-stimulation induced oxidation of PKC{alpha} and activation of PKC{alpha} autophosphorylation. Both the oxidation and phosphorylation of PKC{alpha} was transient. Furthermore, the oxidative activation of PKC{alpha} was required for VCAM-1-dependent spleen cell migration.

There are many stimulants that increase vascular permeability through the regulation of PKC{alpha} and phosphatases. During inflammation, there is an increase in leukocyte migration across endothelium and an increase in vascular permeability. DN PKC{alpha} blocks histamine-induced increases in endothelial permeability (58). PKC{alpha}-dependent hyperpermeability of endothelium is also induced by the chemokine CCL2 (59) or the cytokine TNF-{alpha} (16). In addition, the inhibition of phosphatases induces cell rounding, cytoskeletal disorganization, and cellular detachment in smooth muscle cells, HUVECs, and HMEC-L cells (60, 61). Thus, during inflammatory processes, PKC{alpha} regulates increases in endothelial cell permeability and VCAM-1 signaling in endothelial cells.

During leukocyte passage into inflamed tissue, the migration of leukocytes across the endothelium can be inhibited by Abs against a limited number of endothelial cell adhesion molecules or by a combination of Abs against multiple endothelial cell adhesion molecules. With regards to VCAM-1-dependent inflammation in vivo, anti-VCAM-1 Abs block infiltration of eosinophils into the lung in experimental asthma and block T cell infiltration into the brain in experimental allergic encephalomyelitis (3, 4). Furthermore, Abs against the ligand for VCAM-1, {alpha}4 integrin, have been used in multiple sclerosis and inflammatory bowel disease patients (62, 63, 64, 65). Therefore, in our studies on identification of VCAM-1-dependent signals, we focused on VCAM-1-mediated models. The mHEVa cells are a model for lymphocyte triggering of VCAM-1 whereas examination of lymphocyte triggering of a complex mix of adhesion molecules on TNF-{alpha}-activated HMEC-L cells does not facilitate identification of signaling mechanisms for VCAM-1. Furthermore, we have reported that in vivo treatment with the ROS scavenger bilirubin blocks VCAM-1-dependent leukocyte migration but not the migration of leukocytes that use multiple other endothelial cell adhesion molecules (66). Therefore, in these studies, the VCAM-1 on HMEC-L cells expressing multiple adhesion molecules was specifically triggered by anti-VCAM-1 Abs rather than by lymphocyte binding as lymphocytes would trigger a complex set of intracellular signals through multiple adhesion molecules on the TNF-{alpha}-activated HMEC-L cells. For the same reasons, the migration of spleen cells across cytokine-activated HMEC-L cells was not examined to address VCAM-1-mediated signals as the HMEC-L cells express multiple receptors that support leukocyte migration and these receptors have different signaling pathways (8, 66). Nevertheless, our studies performed with HMEC-L cells demonstrate that VCAM-1 on microvascular endothelial cells activates PKC{alpha} through VCAM-1-triggered endothelial cell NADPH oxidase and ROS. The identification of mechanisms for VCAM-1 signaling is important for proposing intervention of VCAM-1-dependent processes in vivo such as VCAM-1-dependent lung eosinophilia, VCAM-1-dependent T cell migration into the brain in multiple sclerosis, or VCAM-1-dependent T cell migration into the bowel in inflammatory bowel disease (3, 4, 62, 63, 64, 65).

In summary, during VCAM-1 signaling, ROS oxidize, and transiently activate PKC{alpha}. This activation of PKC{alpha} is required for VCAM-1-dependent spleen cell migration. Thus, we have defined a novel mechanism for VCAM-1 signaling and identified a function for oxidized PKC{alpha} during VCAM-1-dependent spleen cell migration.


   Acknowledgments
 
This manuscript is dedicated in memory of Norman Cook who constructed the parallel plate flow chamber and was an excellent teacher, builder, pilot, friend, and father.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant RO1 HL68171 (to J.M.C.-M.). Back

2 Address correspondence and reprint requests to Dr. Joan M. Cook-Mills, Allergy-Immunology Division, Feinberg School of Medicine, Northwestern University, McGaw-304, 240 East Huron, Chicago, IL 60611. E-mail address: j-cook-mills{at}northwestern.edu Back

3 Abbreviations used in this paper: ROS, reactive oxygen species; PKC, protein kinase C; DAG, diacylglycerol; HMEC-L, human microvascular endothelial cells from the lung; DPI, diphenyleneiodonium chloride; IAF, iodoacetamidofluorescein; DN, dominant negative; MMP, matrix metalloproteinase; PP, phosphatase. Back

Received for publication March 28, 2006. Accepted for publication August 15, 2006.


   References
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 Abstract
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 Materials and Methods
 Results
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 Disclosures
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