By
From * The Vascular Research Division, Departments of Pathology, Brigham and Women's Hospital
and Harvard Medical School, Boston, Massachusetts 02115; and Bayer Corporation, West Haven,
Connecticut 06516
Although several adhesion molecules expressed on leukocytes (1 and
2 integrins, platelet endothelial cell adhesion molecule 1 [PECAM-1], and CD47) and on endothelium (intercellular
adhesion molecule 1, PECAM-1) have been implicated in leukocyte transendothelial migration, less is known about the role of endothelial lateral junctions during this process. We have
shown previously (Read, M.A., A.S. Neish, F.W. Luscinskas, V.J. Palambella, T. Maniatis, and
T. Collins. 1995. Immunity. 2:493-506) that inhibitors of the proteasome reduce lymphocyte and
neutrophil adhesion and transmigration across TNF-
-activated human umbilical vein endothelial
cell (EC) monolayers in an in vitro flow model. The current study examined EC lateral junction
proteins, principally the vascular endothelial (VE)-cadherin complex and the effects of proteasome inhibitors (MG132 and lactacystin) on lateral junctions during leukocyte adhesion, to gain a
better understanding of the role of EC junctions in leukocyte transmigration. Both biochemical
and indirect immunofluorescence analyses of the adherens junction zone of EC monolayers revealed that neutrophil adhesion, not transmigration, induced disruption of the VE-cadherin
complex and loss of its lateral junction localization. In contrast, PECAM-1, which is located at
lateral junctions and is implicated in neutrophil transmigration, was not altered. These findings
identify new and interrelated endothelial-dependent mechanisms for leukocyte transmigration
that involve alterations in lateral junction structure and a proteasome-dependent event(s).
Localized leukocyte accumulation is the cellular hallmark of inflammation. Although this has been recognized for more than a century, it is only in the past decade
that the role of the endothelium has been appreciated. The
notion that the vascular endothelium actively participates
in leukocyte recruitment initially gained support from in
vitro studies demonstrating that treatment of cultured endothelium with inflammatory cytokines TNF- Recent reports have shown that the proteasome pathway
is involved in activation of NF- In light of our previous observations that proteasome inhibitors prevent firmly adherent neutrophils from penetrating between endothelial cell-cell lateral junctions, we reasoned that the function of endothelial cell-to-cell lateral
junctions may be critical during the process of leukocyte
transmigration. The molecular structure and organization of
endothelial cell-cell lateral junctions has been reviewed (5).
We focused our attention on the adherens type junctions
which appear to serve as a focal point for the connections
between the EC plasma membrane and its underlying actin-cytoskeleton complex. The adherens type junctions
contain cadherins (for review see reference 7), a family of
single-span transmembrane glycoproteins which directly associate with structural components of the cytoskeleton and
mediate Ca2+-dependent cell-cell adhesion in a homotypic
fashion. Cadherin-5, also termed vascular endothelial (VE)-
cadherin, is specific to vascular endothelium and localizes
exclusively to lateral junctions of intact, confluent endothelium (8, 9). A recent study (8) has revealed that VE-cadherin
associates with the cytosolic proteins To better understand the molecular basis of inhibition of
neutrophil transmigration by proteasome inhibitors and the
potential role of the components of the EC adherens junctions, we have evaluated the effects of two structurally different proteasome inhibitors, MG132 and lactacystin (13), on the
association of VE-cadherin with Materials
Dulbecco's phosphate-buffered saline (DPBS) with Ca2+ and
Mg2+, DPBS, M199, DMEM, RPMI-1640 with 25 mM Hepes,
and 1 M solution of Hepes were purchased from BioWhittaker
Bioproducts (Walkersville, MD). Human rTNF- mAbs
The following murine mAbs have been reported previously:
anti-E-selectin (H4/18 or 7A9, each an IgG1; reference 16), anti- ICAM-1 (Hu5/3, IgG1; reference 17), anti-VCAM-1 mAb E1/6
(IgG1; reference 18), anti-PECAM-1 (obtained from Iowa Hybridoma Bank, Iowa City, IA, or from Immunotech, Inc., Westbrook, ME), and anti-HLA-A,B (W6/32, IgG2a) mAb (19).
These mAb were used as hybridoma culture supernatant fluid for
surface immunofluorescence assays and as pure IgG for immunofluorescence microscopy, immunoprecipitations, and Western blotting. Murine mAb directed to VE-cadherin (clone TEA1/31,
IgG1) was purchased from Immunotech Inc. or from BIODESIGN Intl. (Kennebunk, ME). Murine mAb directed to plakoglobin (clone PG5.1, IgG2b) was purchased from BIODESIGN
Intl. Murine mAb to Cell Culture
ECs were isolated from two to five umbilical cord veins,
pooled, and established as detailed (19). Primary human umbilical vein endothelial cell (HUVEC) cultures were serially passaged (1:
3 split ratio) and maintained in M199 containing 10% FCS, EC
growth factor, porcine intestinal heparin, and antibiotics.
Experimental Protocols.
For immunofluorescence staining assays, ECs (passage 1-2) were plated at confluent density on 4-well
chamber glass slides (Lab-Tek; Nunc, Inc., Naperville, IL) until 2 d
after confluent. For flow assays, ECs were plated at confluence on
human fibronectin (2 µg/cm2)-coated 25-mm glass coverslips
(20), and used 3 d later. For quantitative surface immunofluorescence assays, ECs were plated on fibronectin-coated microtiter
plates (C96; Costar, Cambridge, MA), and used 2-3 d after confluence. For immunoprecipitation assays, EC were plated on 0.1%
gelatin-coated 100-mm diameter plastic petri dishes (Costar), and
used 3 d after attaining confluence. EC monolayers were not manipulated or fed for 48 h before use.
Leukocyte Isolation
Human neutrophils were purified from whole blood of volunteer donors as previously detailed (19). Isolated neutrophils (94% pure) were resuspended in cold DPBS containing 0.75 mM Ca2+,
0.75 mM Mg2+, and 0.2% HSA (assay buffer).
Endothelial-Leukocyte Interactions in a Parallel-plate
Flow Chamber
The parallel-plate flow chamber used in this study has been described in detail (20). A wall shear stress of 1.8 dynes/cm2 was
achieved with a flow rate of 0.85 ml/min (20). EC monolayers were assayed for leukocyte adhesion and transmigration under
flow conditions as before (16, 20).
Immunofluorescence Assays
Quantitative surface immunofluorescence assays for adhesion
molecule expression on EC were performed in triplicate wells as reported previously (17) using appropriate primary mAb-detected FITC-conjugated goat anti-mouse (F(ab Indirect Immunofluorescence Staining.
Indirect immunofluorescence staining of endothelial surface molecules was performed using the protocol of Gerritsen et al. (23). In brief, confluent EC
in 4-well chambers were activated as detailed above and then
washed with assay buffer. Neutrophils (0.3 × 106) or assay buffer
were added to wells and incubated at 37°C for 10 min. Wells
were washed twice with DPBS, and then fixed with methanol
( Immunoprecipitation of Endothelial Proteins
and Immunoblotting
Immunoprecipitation of Junctional Proteins.
VE-cadherin complex proteins were immunoprecipitated using a modification of
the method of Lampugnani et al. (8). In brief, EC were incubated
with inhibitors, ALLM, or carrier (0.02% DMSO) and/or TNF- or IL-1, and certain Gram-negative bacterial endotoxin could "activate" the endothelium to become adhesive for blood leukocytes and cell lines (1). Subsequently, the work of
many investigators has identified and molecularly cloned
several such endothelial cell (EC)1 adhesion molecules and
their cognate ligands on leukocytes, which support leukocyte adhesion to endothelium.
B, which is a transcription
factor necessary for activation of EC gene transcription
of E-selectin (CD62E), intercellular adhesion molecule 1 (ICAM-1) (CD54), and vascular cell adhesion molecule 1 (VCAM-1) (CD106) (4). Small peptide aldehyde inhibitors
(MG132, MG115) of the proteasome can dramatically reduce TNF-
-induced expression of E-selectin, VCAM-1, and ICAM-1 in human umbilical vein ECs (4). Functionally, neutrophil adhesion was reduced by 50%, and transmigration was reduced by >60%. Live-time video microscopy showed that many adherent neutrophils had flattened
and extended pseudopods into the EC junctions, but were
unable to transmigrate. This result raises the possibility that
the proteasome regulates an essential endothelial-dependent component(s) during transendothelial passage.
-and
-catenins to
form a complex and organize at nascent endothelial cell-to-cell contacts. Plakoglobin (also named
-catenin) associated
with VE-cadherin and
- and
-catenins at cell-to-cell contacts, through an undefined mechanism(s), only as EC approached confluence. p120 (p120cas), initially identified as
one of several substrates of the tyrosine kinase pp60src (10,
11), and a closely associated molecule termed p100 also have been reported to associate with
- and
-catenins and
VE-cadherin in umbilical vein endothelium (12). That the
VE-cadherin complex is dynamic and involved in regulating cell-to-cell contact is suggested by wounding (8) or
Ca2+ depletion (5) experiments where VE-cadherin and
plakoglobin rapidly and reversibly retract from the endothelial lateral junctions.
-,
-, and
-catenin (plakoglobin), and p120/p100 in 6-h TNF-
-activated EC using
both indirect immunofluorescence microscopy and immunoprecipitation followed by immunoblotting. Second, we have
coincubated human neutrophils with control or proteasome inhibitor-treated TNF-
-activated endothelium, and
evaluated the staining patterns and biochemical association
of members of the VE-cadherin complex, and as a control,
platelet endothelial cell adhesion molecule 1 (PECAM-1;
CD31), which has been shown previously to colocalize to
EC lateral junctions (14) and has been implicated in leukocyte transmigration in in vivo and in vitro studies (15). The
results of these experiments suggest that novel endothelial-dependent mechanisms regulate neutrophil transmigration
which involves structural alterations of the VE-cadherin
complex, as well as a proteasomal-dependent step(s).
(produced in
Escherichia coli) was the gift of Dr. Baker (Genentech Inc., South
San Francisco, CA), and a concentration of 25 ng/ml gave maximal response and contained <10 pg/ml of endotoxin as reported
previously (16). The proteasome inhibitors, MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal-H) and lactacystin were the gift
of Dr. J. Adams (ProScript, Inc., Cambridge, MA). Each inhibitor was dissolved in DMSO at 40 mM and stored at
80°C. For
use in experiments, aliquots of inhibitors were thawed at 37°C
and diluted directly into appropriate culture media, or as otherwise noted in the text. Calpain inhibitor II (also abbreviated as
ALLM [N-acetyl-leucinyl-leucinyl-methional]) and aprotinin were
purchased from Calbiochem Corp. (La Jolla, CA). EDTA, Protein-G
Sepharose, PMSF, leupeptin, DMSO, BSA, and Hepes were purchased from Sigma Chemical Co. (St. Louis, MO). Human serum albumin (HSA) was obtained from Baxter Healthcare Corp.
(Glendale, CA).
- and
-catenins and p120 (clone 5, 14, and 98, respectively; all IgG1) were purchased from Transduction
Labs. (Lexington, KY). A second mAb to
-catenin (Zymed, S. San Francisco, CA) was used for immunofluorescence studies.
)2, 1:50 dilution; Caltag Labs., S. San Francisco, CA).
20°C) on ice for 5 min, followed by three washes. Immunofluorescence staining was performed as detailed (23). Fields of FITC
fluorescence-stained EC were visualized on a fluorescence microscope (Microfot FXA; Nikon, Inc., Melville, NY) equipped
with a ×20 objective and the images were captured using a
cooled charged-coupled device video camera. Exposure times
were matched in each instance (typically 1-3 s, final magnification of all images was 320, except where noted).
,
and then washed three times with assay buffer. EC were incubated at 37°C for 10 min under static conditions with either 107
human neutrophils (5 ml, ratio of neutrophils to EC was 2.5:1) or
assay buffer alone. Nonadherent neutrophils were removed by washing (2 times) the monolayers with DPBS alone. Plates were placed on ice and the Triton X-100 soluble fraction extracted for
30 min in lysis buffer (10 mM Tris, 150 mM NaCl, 1 mM PMSF, 40 U/ml aprotinin, 15 µg/ml leupeptin, 0.36 mM 1,10-phenanthroline, 2 mM CaCl2 (Tris-buffered saline [TBS] and protease
inhibitors), 1% NP-40, and 1% Triton X-100), mixing every few
minutes. For total lysates, monolayers were lysed in lysis buffer
containing 0.5% SDS. The supernatant was collected, microcentrifuged at 14,000 g for 5 min, and stored at
80°C. The lysed
monolayers were washed 3 times with TBS and protease inhibitors,
and the Triton X-100 insoluble fraction was extracted as detailed
(8) and stored at
80°C.
Immunoblot Analysis.
Immunoblots were performed using a
modification of the method of Donnelly et al. (25). The membrane
was blocked for 1 h with 5% dried milk protein in PBS (blocking
buffer) and then incubated for 2 h with primary mAb (either 5 µg/ml anti-VE-cadherin, 1 µg/ml anti--catenin, anti-
-catenin or anti-p120, or 5 µg/ml anti-plakoglobin or anti-PECAM-1
diluted in block). The membrane was washed six times at 5 min
intervals with PBS containing 0.05% (vol/vol) Tween 20 and
0.05% (wt/vol) BSA Tween 20, and then the primary mAbs were
detected with anti-mouse IgG conjugated to horseradish peroxidase (Sigma Chemical Co., 1/10,000 dilution, 1 h at room temperature). The immunoreactive bands were visualized using enhanced chemiluminescence (Amersham Corp.).
Statistics
Adhesion data was collected by analyses of variance and Student's two sample t test was used to calculate statistical significance (Minitab Software, State College, PA).
We have found previously that pretreatment of endothelial
monolayers with the aldehyde peptide 20S proteasome inhibitor MG132 before TNF- activation of EC dramatically reduces the induction of E-selectin, ICAM-1, and
VCAM-1 gene transcription (4). Functionally, this results
in reduced neutrophil and lymphocyte adhesion and inhibition of transendothelial migration under flow conditions. Lactacystin (13) is a Streptomyces metabolite that potently inhibits the 20S proteasome, and is structurally distinct from MG132 (4). Pretreatment of EC with lactacystin (20 µM)
before TNF-
also significantly inhibited surface expression
of adhesion molecules (Table 1), reduced adhesion by 88%,
and essentially ablated neutrophil transmigration (>95% inhibition).
|
To distinguish between the effects of the proteasome inhibitors on expression of endothelial leukocyte adhesion
molecules and the process of neutrophil transmigration,
MG132, lactacystin, or carrier control were added to 4 h
TNF--activated EC, and the monolayers further incubated. Under such conditions, both inhibitors have no effect on neutrophil adhesion (Fig. 1) or expression of endothelial adhesion molecules (Table 1 and data not shown), but reduce neutrophil transmigration by >50% (Fig. 1).
The effect on transmigration was observed by 60 min, and
by 120 min the level of blockade was >70% for MG132
and >60% for lactacystin. A direct effect of the inhibitors
on neutrophils is not likely because a 5-min pretreatment
of neutrophils with 5 µM MG132 before perfusion did not
alter neutrophil adhesion or transmigration (vehicle, 51.5 ± 5.9% transmigrated versus MG132 treated, 51.5 ± 6.3%; n = 3 experiments). We conclude that MG132 and lactacystin,
two structurally distinct inhibitors of the proteasome, act
on the endothelium to block transmigration, separately from
their effect on adhesion molecule expression.
The Endothelial VE-Cadherin Complex Is Not Altered by Treatment with TNF-
Inhibition of neutrophil penetration suggests
that the function of lateral junctions is an important endothelial-dependent component(s) for transendothelial passage
of the leukocyte. To examine the effects of TNF-, proteasomal inhibitors or both on the VE-cadherin complex,
confluent EC monolayers were incubated with or without
inhibitors and then with or without TNF-
for a total of
6 h. The effect of these treatments on the VE-cadherin complex was determined in Triton X-100-soluble lysates by
immunoprecipitation with anti-VE-cadherin mAb and immunoblot analysis using specific mAb, and by a second independent analysis using immunofluorescence photomicroscopy. VE-cadherin,
- and
-catenins, and plakoglobin were clearly identified (Fig. 2, lane C), and their migration in SDS-PAGE under reduced conditions was consistent with
previous reports (8, 12). Similarly, immunoprecipitation with
anti-VE-cadherin mAb, and subsequent immunoblotting
analysis with an anti-p120 mAb, detected a single band migrating at 120 kD (data not shown), but the results were not
consistently found in every EC culture examined. However, analysis of EC lysates with anti-p120 mAb followed by blotting with anti-p120 consistently revealed a band at
120 kD and a second band at 100 kD, consistent with a recent analysis in endothelium derived from brain (12). The
endothelial VE-cadherin complex was not altered by 6 h of
incubation with TNF-
, MG132, ALLM, lactacystin, or
carrier control (Fig. 2, compare lane C with lanes T, MG,
and ALLM; data with lactacystin not shown). Similarly,
treatment with TNF-
for 4 h followed by 2 h of coincubation with inhibitors, ALLM, or carrier control did not alter the VE-cadherin complex (data not shown).
Analysis of Triton X-100 cell lysates also revealed that the majority of VE-cadherin and other members of the cadherin complex remained in the Triton X-100-soluble fraction, not in the cytoskeleton-associated Triton X-100- insoluble lysate (data not shown), and this distribution was not altered by any of the above treatments.
Results of the immunoprecipitation and immunoblots
were supported by data obtained from indirect immunofluorescence photomicroscopy (Fig. 3). TNF-, lactacystin,
MG132, or ALLM treatments alone, or incubations for 4 h
with TNF-
followed by 2 h incubations with either inhibitors or ALLM, had no effect on the localization of the
components of VE-cadherin complex to the lateral junctions. The results suggest that treatment of confluent EC
monolayers with TNF-
or inhibitors, alone or in combination, does not alter the biochemical composition or lateral junction localization of the VE-cadherin complex.
Neutrophil Adhesion/Transmigration Dramatically Disrupts the VE-Cadherin Complex.
To examine whether neutrophil transmigration correlated with any alterations in the
VE-cadherin complex, neutrophils were incubated with
TNF--activated EC monolayers for 10 min, and then the
nonadherent neutrophils were removed by washing. Visual inspection of TNF-
-treated EC cultures before lysis
showed intact and tightly confluent monolayers with many
adherent and transmigrated neutrophils. The ratio of adherent neutrophils to individual EC was 1:2. In contrast, few
neutrophils adhered to unactivated EC, and no neutrophils transmigrated. When the levels of VE-cadherin complex
were determined in Triton X-100-soluble lysates by immunoprecipitation with anti-VE-cadherin mAb and immunoblotting with specific mAb to each component, dramatic alterations were observed. Neutrophil adhesion and/or
migration across TNF-
-activated EC monolayers induced
loss of VE-cadherin,
-catenin, and plakoglobin, whereas
the level of
-catenin was not decreased (Fig. 4 A, compare lane 3 to 4). Over the course of our experiments, we
noted that the native immunoreactive species of
-catenin,
plakoglobin, and p120/p100 were always below detectable levels, whereas there was often retention of a small amount
of VE-cadherin. Coincubation of control unactivated EC
monolayers with unactivated neutrophils consistently had no
significant effect on the VE-cadherin complex (compare
lane 1 and 2). Since equal numbers of EC were used for
each sample, and this ECL detection system is very sensitive, we infer the losses are not due to unequal sample
loading.
Neutrophil Adhesion Induces Rapid Disruption and Degradation of VE-Cadherin Complex.
The disruption of the VE-
Cadherin complex by addition of neutrophils was accompanied by significant loss and proteolytic cleavage of each
component, except -catenin. This was addressed by using
total EC monolayer lysates (soluble in 1% Triton X-100, 1% NP-40, and 0.5% SDS) and performing immunoprecipitation with specific mAb directed against each component. Immunoprecipitated proteins were detected subsequently by immunoblotting with the same mAb. Addition
of SDS dissociates the complex from the cytoskeletal components (8), and thus allows for determination of the total cellular content (soluble and cytoskeletal-associated) of each component of the complex. Total lysates were prepared
from EC treated with TNF-
and incubated with media
alone or media with neutrophils for 0, 3, or 10 min (Fig. 4
B, lanes 0, 3, and 10, respectively, for each mAb listed). In
parallel, adhesion and transmigration was assessed by phase
contrast microscopy. The levels of native
-catenin, VE-
cadherin, plakoglobin, and p120/p100 were decreased dramatically in TNF-
-activated EC coincubated with neutrophils for 3 min, a time point when neutrophil adhesion, but no transmigration, had occurred. By 10 min, when many
neutrophils had adhered and transmigrated, the levels of each
component, except
-catenin, was reduced dramatically or
undetectable (Fig. 4 B, lanes labeled 10). The level and apparent Mr of
-catenin was stable at each time point. These
results are consistent with the immunoblots of the VE-cadherin complex shown in Fig. 4 A. In contrast, PECAM-1,
which has been demonstrated to localize to endothelial cell-to-cell lateral junctions and is involved in neutrophil
migration (15), remained at similar levels at both 0 and 10 min.
Inspection of immunoblots of total lysates (Fig. 4 B) from
TNF--activated EC coincubated with neutrophils using
anti-VE-cadherin mAb revealed the native VE-cadherin
species (140 kD, Fig. 4 B, large arrow, VE-cadherin, lane 0),
and two immunoreactive bands with an apparent Mr of
~100 kD (small arrow) after 3 or 10 min. That
-catenin remains associated with VE-cadherin, as demonstrated in Fig.
4 A, may be due to its continued association with these 100-kD VE-cadherin immunoreactive degradation products
identified in Fig. 4 B. Previous studies have reported that
-catenins can associate directly with VE-cadherin in some
cells types (26), but not in others (27). Immunoblots of total
lysates with mAb to
-catenin revealed an immunoreactive
degradation product at ~75 kD, and essentially total loss of
immunoreactive bands for p120/p100 and plakoglobin.
These data rule out the possibility that components of the
VE-cadherin complex shift their distribution from Triton
X-100-soluble to -insoluble fraction (i.e., cytoskeletal associated) after neutrophil adhesion. From these findings,
we infer that neutrophil adhesion alone is sufficient to induce rapid endothelial-dependent disruption and partial
degradation of members of the VE-cadherin complex.
We then performed several experiments to show that
neutrophils trigger an endothelial-dependent signal that
leads to degradation of specific members of the complex.
First, the members of the VE-cadherin complex were clearly
disrupted after a 10 min coincubation of TNF--activated
EC with neutrophil membranes (representing 107 neutrophils, Fig. 4 C, lane 3), which are devoid of granule contents and had only residual elastase activity (0.38% of total
elastase activity when compared to intact neutrophils). In
addition, disruption of the VE-cadherin complex by incubation of EC monolayers with neutrophil membranes was
demonstrated by indirect immunofluorescence studies (Fig.
4 D). Thus, the components that triggered disruption of
the VE-cadherin complex are present in neutrophil plasma
membranes. This suggests that the degradation is not due to
nonspecific degradation via neutrophil granule proteolytic
enzymes. Second, transfer of conditioned medium from
coincubation of neutrophils with TNF-activated EC to
TNF-activated EC did not alter the VE-cadherin complex
(Fig. 4 C, lane 4), suggesting degradation is not induced by
a soluble factor. Lastly, neutrophil interactions specifically
trigger both the disruption and partial degradation of VE-
cadherin complex since incubation of EC monolayers with
EDTA, which released VE-cadherin from the lateral junctions (Fig. 5 A), did not trigger degradation of VE-cadherin complex (Fig. 5 B). Taken together, these findings suggest that the dissociation and cleavage of VE-cadherin
and its associated proteins are carefully controlled endothelial-specific event(s) that are triggered by leukocyte contact.
Indirect immunofluorescence photomicroscopy supported
the findings that neutrophil adhesion induced loss of the
VE-cadherin complex at EC lateral junctions (Fig. 6; neutrophils are identified with arrows). TNF--activated EC
monolayers were incubated with or without human neutrophils for 5 min, fixed, and then stained with mAb to the
VE-cadherin complex. The loss is clearly demonstrated for
monolayers stained to detect VE-cadherin,
-catenin, p120/ p100, and plakoglobin, whereas the level and junctional
colocalization of PECAM-1 remained constant. A significant overall reduction in immunoreactive staining of
-catenin, p120/p100, and plakoglobin was observed in the presence of neutrophils, whereas VE-cadherin was lost specifically
from regions of neutrophil adhesion/transmigration (small
arrows). This is consistent with the immunoprecipitation data where, in the majority of experiments, a small amount
of native VE-cadherin is retained, whereas other components of the complex are not detectable.
To investigate whether the degradation of the VE-cadherin complex involved the cellular proteasome system, the
effects of proteasome inhibitors were evaluated. As shown
in Fig. 7, disruption of the VE-cadherin complex was not
prevented by 2 h of incubation with 20 µM lactacystin or
MG132, even though such treatments significantly reduced
transmigration (Fig. 1). For comparison, the components of
the VE-cadherin complex in TNF--activated EC remained constant in the absence of neutrophils. In addition,
shorter incubations (5 or 30 min) with 50 µM MG132 did
not prevent neutrophil-dependent dissociation of the VE-
cadherin complex (data not shown). These findings establish that neutrophil adhesion triggers rapid EC-dependent degradation of the VE-cadherin complex through an enzymatic pathway that appears to be independent of the proteasome.
The current work demonstrates that neutrophil adhesion to TNF--activated endothelial monolayers dramatically alters the molecular composition and organization of
the endothelial cell VE-cadherin complex, which has been
implicated in maintenance of endothelial cell-to-cell adhesion and cell-to-cytoskeletal integrity. Neutrophil adhesion
to confluent TNF-
-activated endothelial monolayers induced rapid and near complete dissociation of
-catenin, p120/p100, and plakoglobin from VE-cadherin and loss of
their lateral junction colocalization. Biochemical analysis of
TNF-
-activated EC monolayers using immunoprecipitation and immunoblotting revealed that these proteins are
degraded rapidly through a proteolytic mechanism that is
dependent on the endothelium and does not involve granule components of the neutrophil. In contrast, PECAM-1,
which is not associated directly with the VE-cadherin complex, but does colocalize to the lateral EC junctions and is
involved in leukocyte transmigration (28), remained at constant levels and retained lateral junction localization. Given
the rapid and specific cleavage of the components of VE-
cadherin complex by the endothelial cells, we suspected
that the cellular proteasome was involved. Surprisingly, two
different proteasome inhibitors, MG132 or lactacystin, did not prevent dissociation or degradation of components of
the VE-cadherin complex, however, both inhibitors did
prevent >60% of neutrophil transmigration, specifically by
preventing neutrophil penetration of the lateral junctions.
Taken together, the results demonstrate that leukocyte adhesion and transmigration across the vascular endothelium
is more complex than previously appreciated, and that neutrophil adhesion induces substantial alterations in structural components of the adherens junctions that may allow for
subsequent transmigration of neutrophils.
The current finding that neutrophil coincubation with
TNF--activated EC disrupts the VE-cadherin complex as
early as 3 min demonstrates that neutrophil-EC adhesion,
rather than transmigration, is the initiating signal. As reported in the text, visual inspection of monolayers consistently showed that no transmigration had occurred at 3 min,
even though disruption and degradation of the VE-cadherin complex was easily detected (Fig. 4 B). These data
suggest engagement of adhesion molecules may be crucial
in triggering EC-dependent events, although signaling through
molecules not involved in adhesion is certainly possible.
This notion is further supported by the positive effects of
neutrophil membranes on the VE-cadherin complex. Additional studies are necessary to elucidate the intracellular signaling mechanisms underlying VE-cadherin complex alterations.
The observed changes in the association of VE-cadherin
with cytosolic -catenin,
-catenin, and plakoglobin after
leukocyte adhesion/transmigration is interesting in a few
respects. Using conditions to retain intact VE-cadherin complex,
-catenin remains associated with the ~100-kD fragments of VE-cadherin, whereas
-catenin and plakoglobin
do not. This is somewhat unexpected since
-catenin does
not appear to associate directly with members of the cadherin family in most cells examined (29, 30). One explanation is that the cleaved ~100-kD fragments of VE-cadherin can associate directly with
-catenin, even though
VE-cadherin is not localized at the lateral junction. This is
not unreasonable since
-catenin has been reported recently to stably associate with VE-cadherin in EC and in
Chinese hamster ovary cells transiently transfected with
VE-cadherin (26). Secondly, the immunoprecipitation and
blotting analyses strongly suggest that p120/p100 associates with the VE-cadherin complex in confluent EC monolayers. Although the association of p120/p100 with VE-cadherin complex was not observed in every EC culture, direct
immunoprecipitation from total EC extracts and immunoblotting with anti-p120 mAb consistently detected two
specific bands at 120 and 100 kD, which is in line with the
report of Staddon and co-workers (31). Moreover, both biochemical and indirect immunofluorescence evidence showed
that neutrophil coincubation with activated EC rapidly led
to the degradation and loss of lateral junctional staining of
p120/p100. One possible explanation for the inconsistency
of immunoprecipitation experiments using anti-VE-cadherin mAb is that only a small fraction of the cytosolic pool
of p120/p100 associates with VE-cadherin, or that the association fluctuates due to tissue culture conditions and thus the signal is low relative to the VE-cadherin complex
signal. Staddon et al. (12) have reported that in Mardin-Darby canine kidney epithelial cells, only 20% of the cytosolic pool of p120/p100 associates with
-catenin.
While this manuscript was being reviewed, Del Maschio
et al. (32) reported a similar effect of neutrophils on the
VE-cadherin complex in human EC monolayers. The current work confirms that neutrophils induce rapid adhesion-dependent alterations in the VE-cadherin complex, and extends these observations to show that (a) disruption and
degradation is endothelial-dependent and independent of neutrophil proteolysis and (b) independent of the endothelial proteasome system. Interestingly, Del Maschio et al. described significant retention of VE-cadherin while still detecting dramatic loss of -catenin and plakoglobin (essentially equivalent to the data reported in Fig. 4). The reason
for this difference in the level of VE-cadherin retained at
EC junctions is not clear. It cannot be explained by a difference in buffers because we still detect a dramatic loss of
VE-cadherin and its components when using their buffer
system for neutrophil-EC coincubations (M199 with or
without 20% FCS), lysis, and immunoprecipitation.
The proteasome may play an important role in regulating endothelial-leukocyte interactions. If selective proteolysis lies at the center of a diverse array of cellular processes, then a question is what controls its timing. Resolving these issues may be important in understanding the role of the proteasome in leukocyte transmigration. How the proteasomal inhibitors interfere with neutrophil transmigration is not clear. Several possibilities can be considered. First, specific signal transduction events initiated by endothelial-leukocyte interactions may require the ubiquitin-proteasome proteolytic pathway. The paradigm for this is well established for several processes (13, 33). Second, the proteasome may degrade key architectural components of the junctional complex. The VE-cadherin complex is probably not the target of these events. Endothelial-leukocyte interactions could alter the activity of the endothelial proteasome by altering the composition or by increasing the activity of specific catalytic components of the proteasome facilitating transmigration. Third, the proteasome inhibitors may act indirectly by altering the activity of important processes other than the proteasome. The structure-function relationship between the inhibitors used in this study suggests that this possibility is unlikely. Two structurally distinct proteasome inhibitors, lactacystin and MG132, specifically blocked leukocyte transmigration. A structurally homologous peptide aldehyde (ALLM), which inhibits cathepsin B and calpain but is a much weaker inhibitor of the proteasome, did not block transmigration. Collectively, these findings distinguish the proteasome pathway rather than cathepsin/calpain-mediated protein degradation or processing as an important step in leukocyte transmigration.
The Process of Leukocyte Transendothelial Migration. The
current report, and several previous studies, further expand
the notion that EC-leukocyte adhesion triggers EC-dependent changes that correlate with leukocyte transmigration.
Huang et al. (34) showed that intracellular Ca2+ gradients
in EC were coupled to transendothelial migration. Neutrophil adhesion to EC induced rapid and transient several fold
increases in cytosolic Ca2+ concentrations ([Ca2+]i), which
paralleled the time course of neutrophil transmigration across
cytokine-activated EC grown on human amnion preparations. Pharmacological clamping of [Ca2+]i with an intracellular Ca2+ chelator, bis-(2-amino-5-methylphenoxylethane-N,N,N,N
-tetraacetic acid tetraacetoxymethyl ester,
inhibited >90% of migration, but had no effect on adhesion. Pfau et al., working with lymphocytes and EC (35),
and Zeigelstein et al., using both neutrophils and monocytes interacting with endothelial monolayers under flow conditions in vitro (36), also reported leukocyte adhesion- dependent changes of [Ca2+]i in EC and the leukocyte.
Neither of these studies report on the spatial resolution of
the Ca2+ flux, so it remains to be determined whether the
cationic flux relates to an adhesion event or to a migratory
event. Thus, the role of increases of [Ca2+]i in EC appear
compelling, whereas the relative role in leukocytes is less
clear. Interestingly, in a static assay system, pharmocologic clamping of [Ca2+]i in neutrophils did not dramatically alter
their migration across resting or cytokine-activated EC,
suggesting that changes in neutrophils [Ca2+]i are not a prerequisite (37). In addition, Yoshida et al. (31) have reported
that binding of leukocytes to 4-h IL-1-activated EC induce
E-selectin linkage at its cytoplasmic domain to the EC actin
cytoskeleton. This link, or similar mechanisms, may supply cues to the EC that influence downstream processes involved in stable adhesion or in transmigration.
The results presented here raise several issues concerning the function of lateral junctions during leukocyte trafficking at sites of inflammation. A basic understanding of these processes may lead to insight into therapies for prevention of tissue damage and abnormal wound healing that occur as a result of a pathological inflammatory response.
Address correspondence to Dr. Francis W. Luscinskas, Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115. Phone: 617-732-6004; FAX 617-732-5933; E-mail: fluscinska{at}bics.bwh.harvard.edu
Received for publication 6 November 1996 and in revised form 9 June 1997.
1 Abbreviations used in this paper: ALLM, N-acetyl-leucinyl-leucinyl- methional; DPBS, Dulbecco's phosphate-buffered saline; EC, endothelial cell; HUVEC, human umbilical vein endothelial cell; HSA, human serum albumin; ICAM, intercellular adhesion molecule; PECAM-1, platelet endothelial cell adhesion molecule 1; TBS, Tris-buffered saline; VCAM 1, vascular cell adhesion molecule 1.The authors wish to thank Ms. Kay Case and Mr. William Atkinson for providing cultured vascular endothelium, and Drs. Michael A. Gimbrone, Jr., James Madara, and Douglas Goetz for helpful discussions.
This work was supported by National Institutes of Health grants HL36028 (F.W. Luscinskas, T. Collins), HL47646 and HL53939 (F.W. Luscinskas, J.R. Allport) and a postdoctoral fellowship from The Crohn's and Colitis Foundation of America (J.R. Allport).
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