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Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45267
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4 integrin,
suggesting that VCAM-1 adhesion was required for migration. To
determine whether signals within the endothelial cells were required
for migration, irreversible inhibitors of signal transduction molecules
were used to pretreat the endothelial cell lines. Inhibitors of NADPH
oxidase activity (diphenyleneiodonium and apocynin) blocked migration
>65% without affecting adhesion. Because NADPH oxidase catalyzes the
production of reactive oxygen species (ROS), we examined whether ROS
were required for migration. Scavengers of ROS inhibited migration
without affecting adhesion. Furthermore, VCAM-1 ligand binding
stimulated NADPH oxidase-dependent production of ROS by the endothelial
cells lines and primary endothelial cell cultures. Finally, VCAM-1
ligand binding induced an apocynin-inhibitable actin restructuring in
the endothelial cell lines at the location of the lymphocyte or
anti-VCAM-1-coated bead, suggesting that an NADPH oxidase-dependent
endothelial cell shape change was required for lymphocyte migration. In
summary, VCAM-1 signaled the activation of endothelial cell NADPH
oxidase, which was required for lymphocyte migration. This suggests
that endothelial cells are not only a scaffold for lymphocyte adhesion,
but play an active role in promoting lymphocyte
migration. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite significant advances in our understanding of adhesion events, much less is known about the mechanism(s) for migration, especially regarding the role of the endothelial cell in this process. A few studies suggest that intracellular signaling pathways in endothelial cells are required for endothelial cell promotion of lymphocyte migration. For example, neutrophil migration across HUVECs requires activation of endothelial cell calcium/calmodulin-dependent myosin light chain kinase (5), but the receptor(s) that mediates this signal is not known. It has also recently been shown that lymphocyte adhesion to ICAM-1 on brain endothelial cells activates the small GTP binding protein, Rho, and this Rho activity is required for lymphocyte migration across the endothelial cells (6). A signaling pathway has also been reported for VCAM-1. Ab cross-linking of VCAM-1 on the surface of HUVEC induces a calcium flux (7). However, it is not known whether this VCAM-1-mediated signaling is important for lymphocyte migration.
Signaling via adhesion molecules may be important for changes in cell shape during transendothelial migration. It has been reported that lymphocyte migration across rat HEV cell layers is an active process, requiring cytoskeletal structural changes, whereas the lymphocyte-HEV cell binding events are passive (8, 9). Furthermore, inhibition of actin filament polymerization by cytochalasin D and inhibition of tubulin elongation by colchicine block spleen lymphocyte migration across monolayers of a rat lymph node HEV cell line (10). It is well established that cytoskeletal proteins, such as actin, vinculin, and tubulin, regulate cell shape, suggesting that these proteins may regulate cell shape changes during lymphocyte migration across endothelial cells. It is not known whether changes in endothelial cell shape are activated by lymphocyte binding to endothelial cell adhesion molecules.
To examine whether VCAM-1 ligand binding activates changes in
endothelial cell shape and whether these signals are involved in the
regulation of lymphocyte migration, we used continuously cultured
endothelial cell lines that were previously developed in our laboratory
(11). These endothelial cell lines, mHEVa and mHEVc cells,
were derived from BALB/c mouse axillary and cervical lymph nodes,
respectively, by spontaneous immortilization. They exhibit contact
inhibition and do not form transformed foci. These endothelial cell
lines bind resting lymphocytes and then promote the migration of the
lymphocytes under the endothelial cell monolayer (11).
During lymphocyte migration across the mHEV cell lines, the endothelial
cells change shape by retracting their membrane at the site of the
lymphocyte, the lymphocyte migrates between adjacent endothelial cells
within a few minutes, and the endothelial cells reform their cell-cell
junction (11). The mechanism for lymphocyte adhesion to
the mHEV cell lines is by lymphocyte 4
integrin binding to VCAM-1 on the mHEV cells and not to any other known
adhesion molecule (12). The expression of VCAM-1 by the
mHEV cell lines is constitutive (11), in contrast to other
endothelial cells that require cytokine activation of VCAM-1
expression. Importantly, this constitutive VCAM-1 expression by the
mHEV cell lines enables the examination of intracellular signals
transduced by VCAM-1 without the complications of signals for
activation of VCAM-1 expression. Thus, these cell lines were used as a
simplified model to examine lymphocyte interactions with VCAM-1 on
endothelial cells. In this report we demonstrate that adhesion to
VCAM-1 on these endothelial cell lines induced endothelial cell NADPH
oxidase activity, which was required for lymphocyte migration and
endothelial cell actin-mediated cell shape changes. In addition, the
VCAM-1-mediated activation of NADPH oxidase was not required for the
initial lymphocyte adhesion to the endothelial cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male BALB/c mice, 4–6 wk old (Harlan Industries, Indianapolis, IN), were the source of resting splenic lymphocytes.
Reagents
The following inhibitors were obtained from Biomol (Plymouth
Meeting, PA): wortmannin, diphenyleneiodonium chloride (DPI),
herbimycin A, fluphenazine, phenoxybenzamine, methoxsalin, and
troleandomycin. Other inhibitors included apocynin and allopurinol
(Acros Organics, Pittsburgh, PA);
NG-methyl-L-arginine
(L-NMMA), and
N5-(1-iminoethyl)-L-ornithine
(Molecular Probes, Eugene, OR); and superoxide dismutase and catalase
(Sigma, St. Louis, MO.) Purified rat anti-mouse VCAM-1 (clone
MVCAM.A), rat IgG2a, rat anti-mouse 5
integrin (clone MFR5), FITC-conjugated rat anti-mouse CD45, and
mouse anti-human VCAM-1 (clone 51-10C9) were obtained from
PharMingen (San Diego, CA). Rat anti-mouse VLA-4 (clone PS-2) and
rat anti-mouse CD44 (clone KM201) were obtained from BioDesign
International (Kennebunk, ME). Mouse anti-human PECAM-1 (clone
JC/70A) was purchased from Dako (Carpinteria, CA). Biotin-conjugated
goat anti-rat Ig and biotin-conjugated goat anti-mouse IgG1
were obtained from Southern Biotechnology Associates (Birmingham, AL).
FITC- and tetramethylrhodamine isothiocyanate (TRITC)-phalloidin were
purchased from Sigma. Dihydrorhodamine 123 (DHR) was obtained from
Molecular Probes. Streptavidin-conjugated 10.4-µm beads were
purchased from Bangs Laboratories (Fishers, IN).
Cells and culture medium
Two endothelial cell lines, mHEVa and mHEVc, were previously derived from BALB/c mice axillary or cervical lymph nodes (11). Spleen cells were isolated as previously described (11). B78H1 cells (derived from B16 mouse melanoma) were a gift from Dr. Lloyd Graff (Department of Biochemistry, University of Illinois, Chicago, IL). The cells were incubated at 37°C and 6% CO2 in culture medium consisting of RPMI 1640 (Fisher Scientific, Cincinnati, OH) supplemented with 20% FCS, 2 mM glutamine (Sigma), 1 mM HEPES (Sigma), 10 mM sodium bicarbonate (Sigma), 100 U/ml penicillin (Fisher Scientific), 100 µg/ml streptomycin (Fisher), and 50 µg/ml gentamicin (Life Technologies, Grand Island, NY). For phenol red-free culture medium, phenol red-free RPMI 1640 (Life Technologies) was substituted for RPMI 1640 in the above culture medium. HUVECs were obtained from Clonetics (Walkersville, MD) and grown in endothelial growth medium (Clonetics) plus 5% FCS. The HUVECs were used at passage 4 or 5.
Lymphocyte migration assay
mHEVa cells or mHEVc cells were plated and grown to confluence in the upper chamber of Transwells with 12-µm pores (Costar Cambridge, MA). Inhibitors were added to both the upper and lower chambers of the Transwells as described in Results. After pretreatment with the irreversible inhibitors, the mHEV monolayers were washed by placing the Transwell in a new plate with 1.2 ml of fresh medium in the bottom chamber and 500 µl of fresh medium in the top chamber. After five washes, the last wash was added to a set of untreated wells to determine whether the washes were sufficient to remove effective concentrations of the inhibitor from the medium. This wash had no effect on migration (data not shown). Splenic lymphocytes (4 x 106 optimal dose) and 4–8 x 106 splenic RBC were added to the upper chamber on top of the mHEV monolayer. The cells were incubated at 37°C under static (nonflow) conditions. RBC served as a control for confluence of the monolayer. The RBC are smaller than the lymphocytes and do not migrate. Therefore, on the rare occasion that RBC were in the bottom chamber, the monolayer was not confluent, and the Transwell was discarded. Lymphocytes were collected from the bottom chamber and counted at the indicated times. Asynchronous lymphocyte migration occurs up to 48 h, whereas migration by a particular lymphocyte occurs in minutes (11). The effects of treatments on migration are only compared within an experiment, as the number of migrated lymphocytes varies among experiments but varies little among triplicate determinations within an experiment. Each experiment was performed at least twice. The inhibitors at the concentrations used had no affect on lymphocyte viability or mHEV cell viability, as determined by trypan blue exclusion (data not shown).
Lymphocyte adhesion assay
As previously described (11), the mHEV cell lines and the control cell line B78H1 were grown to confluence in 96-well, tissue culture-treated plates (Corning, Corning, NY). RBCs in spleen cell preparations were lysed by hypotonic shock with water and washed with culture medium. RBCs were not lysed by hypotonic shock with the ammonium chloride method (13), because it increased nonspecific binding in the adhesion assay. The splenic lymphocytes were then labeled with calcein acetoxymethyl ester (AM) (1 µM) for 15 min at 37°C and washed twice with PBS. Calcein-acetoxymethyl ester (AM) is a vital dye that is membrane permeable but becomes membrane impermeable and fluorescent when cleaved by intracellular esterases. Lymphocytes (1 x 106) were added to each well, then were then gently centrifuged onto the monolayers at 500 rpm without the brake. Centrifugation is not required for the adhesion, but it initiates simultaneous interactions between lymphocyte and cell monolayers. The cells were then incubated for 30 min at 37°C. To remove the nonadherent cells, the plates were gently vortexed twice for 2 s on a Genie Vortex I (Fisher) at setting 5. The medium containing the nonbound cells was then removed, and 100 µl of PBS supplemented with 0.2 mM CaCl2 and 0.1 mM MgCl2 was added. The plates were read on a microplate fluorometer (Cambridge Technology International, Watertown, MA).
Reactive oxygen species
mHEV cells or HUVECs were grown to confluence on 35-mm petri
dishes constructed with bottoms of 22-mm round glass coverslips. In
experiments with HUVECs, the endothelial cells were stimulated
overnight with 1 ng/ml TNF- to induce VCAM-1 expression. As
expected, the TNF--treated HUVECs expressed VCAM-1 as determined by
immunofluorescent labeling of HUVECs and fluorescent microscopy (data
not shown). The mHEV cells and HUVECs were preloaded with DHR (1 or 1.5
µM) in phenol red-free culture medium 15 min before stimulation of
the endothelial cells. DHR fluoresces when oxidized (14, 15). The mHEV cells were incubated with 5 x
106 lymphocytes, with anti-VCAM-1-coated
10.4-µm beads, or with anti-CD44-coated 10.4 µm beads as
controls. To Ab-coat beads, 40 µl of streptavidin-conjugated 10.4
µm beads (Bangs Laboratories) were labeled with 6 µg of
biotin-conjugated goat anti-rat Ig in 75 µl of PBS with
gentle rocking for 1 h at 4°C and then washed. These beads were
incubated with 8 µg of rat anti-mouse VCAM-1 or a rat control Ab
(anti-mouse CD44) in 40 µl of PBS for 30 min at 4°C with gentle
rocking and then washed. For experiments with the HUVECs, 40 µl of
streptavidin-conjugated 10.4-µm beads (Bangs Laboratories) were
incubated with 12 µg of biotin-conjugated goat
anti-mouse IgG1 in 75 µl of PBS with gentle rocking
for 1 h at 4o C. These beads were washed and
incubated with 8 µg of mouse anti-human VCAM-1 or a control Ab
(mouse anti-human PECAM-1) in 40 µl of PBS for 30 min at
4o C with gentle rocking and then washed. HUVECs,
preloaded with DHR, were incubated with the anti-human
VCAM-1-coated beads. Rhodamine 123 fluorescence was examined as
previously described (14) at 0–40 min using a
confocal microscope equipped with a x100 objective (Leica TCS 4D
microscope/SCANware system, Heidelberg, Germany) equipped with an
Omnichrome krypton-argon laser (Chino, CA). After each time point, the
field of cells was changed to avoid laser-induced rhodamine 123
fluorescence in the next image.
Actin localization
Mouse HEV cells were grown to confluence on Permanox eight-well chamber slides (Corning). Lymphocytes were added to the wells and incubated 0, 5, 15, or 30 min at 37°C, and the nonbound cells were removed. The bound cells were fixed with 3% paraformaldehyde/2% sucrose and then permeabilized for 5 min on ice with 0.1% Triton X-100. Lymphocyte and mHEV cell actin was incubated with 0.1 µg/ml TRITC-phalloidin for 40 min on ice and washed. Lymphocytes were labeled with 2.5 µg/ml FITC-conjugated rat anti-mouse CD45. Fluorescence was examined by confocal microscopy.
To examine anti-VCAM-1-coated bead induction of actin coalescence, the mHEV cells were incubated with 5 x 106 anti-VCAM-1-coated beads for 30 min at 37°C. The nonbound beads were removed, and the mHEV cells were fixed, permeabilized, and labeled with FITC-phalloidin as described above. Fluorescence was examined by confocal microscopy. The bead location was examined using reflected light and the confocal microscope.
Statistics
Data were analyzed by ANOVA followed by a multiple comparisons test (SigmaStat, Jandel Scientific, San Ramon, CA). The specific statistical tests are indicated in the figure legends.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lymphocytes bind to endothelial cells and then migrate between
adjacent endothelial cells. We have reported that lymphocytes bind and
migrate across monolayers of the endothelial cell lines, mHEVa and
mHEVc (11). We have also previously determined by Ab
inhibition studies that adhesion of resting lymphocytes to these
endothelial cell lines is dependent on lymphocyte
4 integrin binding to VCAM-1 on the mHEV cells
(12). Therefore, we determined whether adhesion via
4 integrin to VCAM-1 was required for
lymphocyte migration. Mouse HEV cells were grown to confluence in the
upper chamber of Transwells, and resting spleen cells were added. The
upper chamber was treated every 4 h with either 4 µg of
anti-4 integrin, 4 µg of
anti-VCAM-1, 4 µg of an isotype control Ab, or no Ab (Fig. 1
). In nontreated controls, 2–10% of
the lymphocytes migrated in 24 h, which is similar to other
studies with endothelial cell lines or cytokine-activated microvascular
endothelial cells (16, 17, 18, 19). Furthermore, because about
half of the lymphocytes bind to the mHEV cells (11), this
results in about 4–25% of the bound cells migrating.
Anti-4 integrin and anti-VCAM-1
significantly blocked lymphocyte migration across the mHEVa and mHEVc
cells, whereas the isotype control Ab had no effect on migration (Fig. 1). This indicates that adhesion was required for migration across
these cell lines.
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4
integrin or anti-VCAM-1 Abs to determine whether these molecules
are also involved in migration, because these Abs reversed lymphocyte
adhesion to the mHEV cell lines (data not shown). Furthermore, CD44,
which mediates migration of activated lymphocytes into inflammatory
sites as well as across cytokine-activated endothelial cells in vitro
(20, 21), did not inhibit resting lymphocyte migration
across the mHEV cell lines, as determined using an anti-CD44
blocking Ab (clone IM7; data not shown) (11). It is also
possible that lymphocytes migrate between the endothelial cells on
fibronectin. Inhibition of one fibronectin receptor VLA-5
(5ß1 integrin) with
blocking anti-5 integrin (clone MFR5) had
no effect on lymphocyte migration (data not shown). This could not be
tested further with CS-1 peptides that block integrin binding to
fibronectin, because these peptides also block binding to VCAM-1
(22). Furthermore, RGD-containing peptides, which block
binding to fibronectin, reversed endothelial cell attachment to the
solid support, causing the monolayer to dissociate from Transwells or
96-well plates (data not shown). In summary, resting lymphocyte
4 integrin binding to VCAM-1 on the mHEV cells
was required for lymphocyte migration. Lymphocyte migration across endothelial cell lines requires endothelial cell NADPH oxidase-catalyzed production of ROS
The role of VCAM-1 as a receptor for 4
integrin during lymphocyte adhesion has been clearly established
(1, 2). However, it is not known whether ligand binding to
VCAM-1 initiates intracellular signals in the endothelial cell that are
required for endothelial cell promotion of lymphocyte migration.
Therefore, our approach was to determine whether inhibitors of signal
transduction molecules blocked lymphocyte migration and then determine
whether VCAM-1 activated these signal transduction molecules in
endothelial cells. Because VCAM-1 is a member of the Ig gene
superfamily and other members of this superfamily, such as the Ig
receptor and the TCR, use tyrosine kinases to initiate signal
transduction, we first examined endothelial cell tyrosine kinase
involvement in lymphocyte migration. To determine whether tyrosine
kinase activity in the endothelial cells was required for lymphocyte
migration, an irreversible inhibitor of tyrosine kinase (herbimycin A)
was preincubated with the mHEV cells, the cells were washed, and then
lymphocyte migration across monolayers of the mHEV cells was examined.
This allowed the examination of the effect of the inhibitor on one cell
type during coculture of the lymphocytes with the mHEV cells.
Lymphocyte migration was not affected when mHEV cells were pretreated
with the irreversible tyrosine kinase inhibitor herbimycin A (1–5
µM; Fig. 2) (23, 24). We
then examined whether calmodulin was required for lymphocyte migration.
It has been reported that Ab cross-linking of VCAM-1 initiates an
endothelial cell calcium flux, but it is not known whether this calcium
flux is required for lymphocyte migration. Furthermore, HUVEC
calcium/calmodulin-dependent myosin light chain kinase is required for
neutrophil migration (5), but the receptor(s) that
mediates this signal is not known. Therefore, we examined whether
irreversible inhibitors of calmodulin blocked endothelial cell
promotion of lymphocyte migration. Pretreatment of the endothelial
cells with the irreversible calmodulin inhibitors phenoxybenzamine
(10–50 µM) (25) or fluphenazine (25–50 µM)
(26) did not inhibit lymphocyte migration (Fig. 2).
Pretreatment of the endothelial cells with the irreversible
phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin (0.5–1
µM) (27, 28) also did not block lymphocyte migration
(Fig. 2). In contrast, pretreatment of lymphocytes with these
inhibitors significantly decreased lymphocyte migration across the mHEV
cells (Fig. 2), but did not affect lymphocyte adhesion to the mHEV cell
lines (data not shown), suggesting that, subsequent to the initial cell
adhesion event, these signaling molecules in lymphocytes were required
for migration. The inhibition of lymphocyte function during migration
also indicates that the inhibitors were active. Although the inhibitors
had no effect on endothelial cells for promotion of lymphocyte
migration, the inhibitors were used at concentrations reported to
modulate other endothelial cell functions (29, 30, 31, 32).
Furthermore, at these concentrations, the inhibitors had no effect on
lymphocyte or mHEV cell viability as determined by trypan blue
exclusion (data not shown). Thus, these results are consistent with a
requirement for activation of lymphocyte, but not endothelial cell,
tyrosine kinase, calmodulin, and PI3-K during lymphocyte migration
across the endothelial cell lines.
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). In contrast, pretreatment of
lymphocytes with DPI had no effect on lymphocyte migration across the
mHEV cells (data not shown). Because VCAM-1 is required for migration,
it was determined whether DPI affected VCAM-1 expression. Pretreatment
of mHEV cells with DPI for 30 min did not affect VCAM-1 expression
24 h later as determined by immunofluorescent labeling and flow
cytometry (98 ± 1% of the mHEVa cells expressed VCAM-1 with a
mean fluorescence intensity of 318 ± 33; 80 ±
4% of the mHEVc cells expressed VCAM-1 with a mean
fluorescence intensity of 53 ± 8). To determine which enzyme was
important for migration, reversible inhibitors that were specific for
NOS and NADPH oxidase were used. Coincubation with reversible
inhibitors was used, because DPI had no effect on the lymphocytes. The
arginine analogues L-NMMA (5–1000 µM) and
N5-(1-iminoethyl)-L-ornithine
(0.5–100 µM), which inhibit NOS (39), did not reduce
lymphocyte migration (data not shown). Apocynin, which inhibits NADPH
oxidase (40), exhibited dose-dependent (0.5–4 mM)
inhibition of lymphocyte migration across the mHEVa and mHEVc cells
(Fig. 3), suggesting that endothelial cell NADPH oxidase activity is
required for lymphocyte migration. Apocynin had no effect on mHEV cell
expression of VCAM-1 after 24 h as determined by immunofluorescent
labeling and flow cytometry (95 ± 5% of the mHEVa cells
expressed VCAM-1 with a mean fluorescence intensity of 271 ± 70;
75 ± 12% of the mHEVc cells expressed VCAM-1 with a
mean fluorescence intensity of 46 ± 9). Inhibitors of other
enzymes (xanthine oxidase and cytochrome P450) that catalyze the
production of ROS were also examined. These inhibitors were used at
concentrations reported to modulate endothelial cell functions other
than promotion of lymphocyte migration (41, 42, 43, 44, 45).
Inhibitors of xanthine oxidase (100–300 µM allopurinol) (46, 47) and cytochrome P450 (0.03–0.3 µg/ml methoxsalen or 10–20
µM troleandomycin) (48, 49, 50) did not affect lymphocyte
migration or cell viability (data not shown).
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). Therefore,
NADPH oxidase production of ROS by mHEV cells was required for
lymphocyte migration. Lymphocyte adhesion stimulated the production of ROS by endothelial cells
To demonstrate whether ROS were generated by lymphocyte binding to
mHEV cells, the production of low levels of intracellular ROS in the
mHEV cells was examined using the reactive oxygen-sensitive fluorescent
indicator DHR and confocal microscopy. The mHEV cell monolayers were
preloaded with DHR for 15 min. Lymphocytes were added in the presence
and the absence of the NADPH oxidase inhibitor apocynin. At 5, 10, 20,
30, and 40 min, rhodamine 123 fluorescence was examined by confocal
microscopy. Lymphocytes stimulated detectable accumulation of
endothelial cell rhodamine 123 fluorescence at 20–30 min after
addition of lymphocytes compared with nonstimulated endothelial cells
incubated for the same length of time (Fig. 4). Furthermore, apocynin inhibited this
generation of rhodamine 123 fluorescence (Fig. 4), indicating that mHEV
cell NADPH oxidase was required for lymphocyte-stimulated production of
ROS. Therefore, lymphocyte adhesion stimulated endothelial cell
production of ROS.
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). As controls, rhodamine 123 fluorescence of
anti-VCAM-1-treated mHEV cell monolayers was examined without the
addition of lymphocytes. Soluble anti-VCAM-1 did not
stimulate accumulation of rhodamine 123 fluorescence compared with that
in nonstimulated endothelial cells (Fig. 4). Therefore, VCAM-1-mediated
adhesion was required for lymphocyte stimulation of endothelial cell
NADPH oxidase-dependent synthesis of ROS. Cross-linking VCAM-1 stimulates the production of ROS by endothelial cells
VCAM-1 as well as fibronectin are constitutively expressed on the
surface of the mHEV cell lines (11). To determine whether
VCAM-1 signaling could induce the activation of NADPH oxidase,
anti-VCAM-1-coated 10.4-µm diameter beads were used to cross-link
VCAM-1 on the endothelial cell surface. The mHEV cell monolayers were
preloaded for 15 min with DHR. Anti-VCAM-1-coated beads or
anti-CD44-coated control beads were added to the mHEV cell
monolayers, and rhodamine 123 fluorescence was examined by confocal
microscopy at 5, 15, 30, and 60 min. The beads bound to the endothelial
cells and were not phagocytosed by the endothelial cells (data not
shown). Cross-linking of VCAM-1 stimulated detectable accumulation of
rhodamine 123 fluorescence by 15–30 min compared with that in
nonstimulated endothelial cells (Fig. 5).
Anti-CD44-coated control beads did not stimulate rhodamine 123
fluorescence (Fig. 5). The anti-VCAM-1 bead-stimulated fluorescence
was inhibited by the addition of the NADPH oxidase inhibitor, apocynin
(Fig. 5), indicating that signals initiated by VCAM-1 stimulated NADPH
oxidase activity in the mHEV cells for the production of ROS.
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overnight to induce
VCAM-1 expression. The TNF--treated HUVECs expressed VCAM-1, as
determined by immunofluorescent labeling and fluorescent microscopy
(data not shown). Cross-linking VCAM-1 on TNF--treated
HUVECs with anti-human VCAM-1-coated beads stimulated
accumulation of rhodamine 123 fluorescence by 30 min (Fig. 6) as examined by confocal microscopy at
5, 15, 30, and 45 min. The anti-VCAM-1 bead-stimulated fluorescence
was blocked by the NADPH oxidase inhibitor, apocynin (Fig. 6),
indicating that VCAM-1 cross-linking activates endothelial cell NADPH
oxidase. Anti-PECAM-1-coated control beads did not stimulate rhodamine
123 fluorescence (Fig. 6). In summary, VCAM-1 cross-linking activated
endothelial cell NADPH oxidase.
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We have shown by time lapse confocal microscopy that during
lymphocyte migration across mHEV cells, the mHEV cell changes shape by
retracting its membrane, the lymphocyte migrates between adjacent mHEV
cells within a few minutes, and the mHEV cells reform their cell-cell
junctions (11). Cell shape changes are known to involve
rearrangement of the cytoskeletal structure of the cell
(51). Furthermore, membrane ruffling as well as NADPH
oxidase activation have been shown to be regulated by the G protein
Rac-1 (52, 53). Therefore, we hypothesized that VCAM-1
activates NADPH oxidase to produce low concentrations of localized ROS
that modulate actin structure and therefore mHEV cell shape. To examine
this, it was determined whether cross-linking VCAM-1 activated changes
in mHEV cell actin structure and whether these changes could be blocked
by the NADPH oxidase inhibitor apocynin. Lymphocytes or
anti-VCAM-1-coated beads were added to mHEV monolayers and allowed
to adhere for 5 min. The cells were fixed with paraformaldehyde and
permeabilized. The actin in these cells was then labeled with
TRITC-phalloidin and examined by confocal microscopy. The mHEV cell
actin locally coalesced around the bound bead or lymphocyte (Fig. 7), whereas the actin structure in the
center of the mHEV cells did not change (Fig. 7). The induction of
actin coalescence was inhibited by apocynin or cytochalasin D (Fig. 7).
Therefore, adhesion to VCAM-1 caused changes in the endothelial cell
cytoskeleton, and these changes were dependent on NADPH oxidase
activity. In summary, the data indicate that lymphocyte binding to
VCAM-1 on the endothelial cell lines stimulated endothelial cell NADPH
oxidase for the generation of ROS. This NADPH oxidase activity was
required for lymphocyte migration and for endothelial cell shape
changes that occur during the migration of lymphocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-treated HUVECs. In the past, NADPH oxidase has been primarily examined in leukocytes such as neutrophils and macrophages, where it functions to produce large amounts of superoxide for the destruction of pathogens (55). More recently, the production of low subtoxic concentrations of oxygen metabolites has been recognized as a potential cell signaling mechanism (33). For example, superoxide activates mitogenic signaling in Ras-transformed fibroblasts (56), mediates chemotactic responses by fibronectin- or platelet-derived growth factor-stimulated fibroblasts (57, 58), and inhibits phosphatases in phorbol ester-treated macrophages (59). Oxygen metabolites also play a role in signaling in endothelial cells. For example, in endothelial cells, exogenous superoxide stimulates a transient calcium release (60, 61). Furthermore, it has been shown that endothelial cells have NADPH oxidase components that produce low levels of ROS (35, 62, 63, 64). ROS also mediate signals required for induction of endothelial cell gene expression of VCAM-1 and M-CSF (62, 63). However, it is unlikely that ROS modulated VCAM-1 expression in the mHEV cell lines during lymphocyte migration, because mHEV cells constitutively express VCAM-1, and the migration of a lymphocyte across a particular mHEV cell occurs within minutes (11).
Oxygen metabolites have also been shown to modulate actin structure. For example, catalase scavenging of H2O2 blocks FCS-stimulated mesangial cell contraction and production of ROS (65). Furthermore, addition of oxygen to posthypoxic cultured aortic endothelial cells induces reorganization of actin into ruffles, and this actin change is blocked by overexpression of superoxide dismutase, which scavenges superoxide (66).
It is interesting to discuss potential mechanisms for ROS modulation of endothelial cell actin structure. ROS have been shown to inhibit phosphatases and activate metalloproteinases, both of which regulate cell shape and/or cell attachment. H2O2 can directly oxidize phosphatases (67) by oxidizing cysteine residues in the phosphatase catalytic site (68, 69). Phosphatase activity in neutrophils and Fao hepatoma cells is inhibited by exogenous H2O2 or activation of NADPH oxidase (67, 70). Phosphatases regulate many cell functions, including cytoskeletal structure, cell adhesion to extracellular matrix, and cell-cell junctions in fibroblasts, neutrophils, platelets, and endothelial cells (71, 72, 73, 74, 75, 76, 77, 78, 79). For example, cell constriction and reorganization of actin and microtubules in HUVECs are induced by inhibitors of phosphatases PTP1 and PTP2A (80), and cell-cell junction separation occurs when phosphatase PTP1B activity is blocked (81). In addition, the phosphatase inhibitor pervanadate initially induces an increase in endothelial cell phosphotyrosine labeling at cell junctions and an increase in levels of the cytoskeletal proteins vinculin, actin, and plakoglobulin, whereas a prolonged incubation with pervanadate induces dissociation of cell-cell junctions (71). Inhibition of protein phosphatases also decreases endothelial cell barrier function, increases protein phosphorylation, and induces localization of actin at the endothelial cell periphery (82). Therefore, the endothelial cell actin coalescence at the site of VCAM-1 binding may be mediated by reactive oxygen inhibition of phosphatases.
Another potential target of ROS is matrix metalloproteinases (MMPs), which degrade extracellular matrix (ECM), thus altering cell shape (83). MMP degradation of ECM modulates the shape of endothelial cells and endothelial cell growth, because the concentration of ECM regulates capillary endothelial cell growth (84) and endothelial cell spreading (85). Furthermore, T cell adhesion to VCAM-1 on the rat microvascular endothelial cell line RFC or T cell adhesion to recombinant VCAM-1 induces T cell MMP2 mRNA and MMP2 enzyme activity (86, 87). Furthermore, lymphocyte migration across these cells is inhibited by the MMP2 inhibitor TIMP2 (tissue inhibitor of metalloproteinase-2) (86, 87). However, the mechanism for activation of the MMPs during lymphocyte migration across endothelial cells is not known. Latent MMP2 is activated by low concentrations of H2O2 (4 µM), whereas higher concentrations of H2O2 (50 µM) inactivate MMP2 (83). We report that production of H2O2 by endothelial cell lines was required for lymphocyte migration across these endothelial cells that express ECM on their cell surface (11). Therefore, low localized production of H2O2 by endothelial cells may activate local MMP activity to degrade endothelial cell ECM at cell junctions. The endothelial cells would then retract at that site and allow the lymphocyte to migrate beneath the endothelial cells. Future studies will focus on whether ROS inhibit phosphatases and/or activate MMPs for the migration of lymphocytes across endothelial cells.
In summary, VCAM-1 is not simply a scaffold for lymphocyte adhesion, but activates endothelial cell functions that regulate lymphocyte migration. Specifically, VCAM-1 mediates outside-in signaling, and this signaling is via endothelial cell NADPH oxidase activity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Joan M. Cook-Mills, Department of Pathology and Laboratory Medicine, University of Cincinnati, P.O. Box 670529, Cincinnati, OH 45267-0529.
3 Abbreviations used in this paper: HEV, high endothelial venule; PECAM-1, platelet endothelial cell adhesion molecule-1; TRITC, tetramethylrhodamine isothiocyanate; DHR, dihydrorhodamine 123; DPI, diphenyleneiodonium; ECM, extracellular matrix; L-NMMA, NG-methyl-L-arginine; MMP, matrix metalloproteinases; NOS, NO synthase; ROS, reactive oxygen species; PI3-K, phosphatidylinositol 3-kinase; PTP, protein tyrosine phosphatase.
Received for publication August 2, 1999. Accepted for publication April 6, 2000.
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