* Institute of Cell Biology, Zentrum für die Molekularbiologie der Entzündung, University of Münster, Germany; and Istituto di
Ricerche Farmacologiche Mario Negri, Milano, Italy
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Abstract |
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It has been recently proposed that adhesion
of polymorphonuclear cells (PMNs) to human umbilical vein endothelial cells leads to the disorganization of
the vascular endothelial cadherin-dependent endothelial adherens junctions. Combined immunofluorescence
and biochemical data suggested that after adhesion of
PMNs to the endothelial cell surface, -catenin, as well
as plakoglobin was lost from the cadherin/catenin complex and from total cell lysates. In this study we present
data that strongly suggest that the adhesion-dependent disappearance of endothelial catenins is not mediated
by a leukocyte to endothelium signaling event, but is
due to the activity of a neutrophil protease that is released upon detergent lysis of the cells.
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Introduction |
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THE endothelium forms the main barrier that, under
homeostatic conditions, regulates the diffusion and
transport of both macromolecules and whole cells
from the blood stream to the underlying tissues. In response to an inflammatory stimulus, polymorphonuclear
leukocytes (PMNs)1 are the first cells that are recruited
from the blood to the site of an acute inflammatory reaction. This extravasation process is initiated by a cascade of
cell adhesion molecules and leukocyte-activating mediators, which control the adhesion of leukocytes to the apical surface of endothelial cells (EC) (Carlos and Harlan, 1994;
Springer, 1994
).
Whereas these initial interactions have been intensively
studied, the ensuing transmigration event is poorly understood. Transendothelial migration requires mechanisms
that open the endothelial cell layer and allow the passage
of leukocytes. Endothelial monolayer integrity and permeability, on the other hand, are largely controlled by intercellular junctions (Rubin, 1992; Dejana et al., 1995
). With respect to leukocyte extravasation, the so-called adherens
junctions appear to be of particular interest. These junctions are formed by the cadherins, transmembranous cell-
cell adhesion molecules that undergo homophilic interactions and that bind to each other in a Ca2+-dependent
manner. To perform their adhesive functions, these cadherins interact with the actin cytoskeleton through their
cytoplasmic tails, an association that is mediated by the intracellular catenins
-catenin,
-catenin, and plakoglobin
(Takeichi, 1991
; Kemler, 1993
; Aberle et al., 1996
). In the
endothelium, several cadherins have been described, of
which only vascular endothelial (VE)-cadherin (cadherin-5) is specific for endothelial cells (Liaw et al., 1990
; Suzuki
et al., 1991
; Lampugnani et al., 1992
). VE-cadherin is concentrated at sites of cell-cell contacts, and functions in the
maintenance of cell layer integrity of cultured human endothelial cells (Lampugnani et al., 1992
; Navarro et al.,
1995
). A monoclonal antibody against mouse VE-cadherin
accelerates the extravasation of neutrophils in a mouse
peritonitis model in vivo (Gotsch et al., 1997
), suggesting
that the opening of VE-cadherin-mediated cell contacts
may be a relevant step during neutrophil extravasation. Whereas the mechanisms that would lead to such an opening of adherens junctions have not been defined, it has
nevertheless been demonstrated that adhesion of PMNs
leads to an increase in endothelial cytosolic Ca2+ levels. In
addition, intracellular Ca2+ scavengers were shown to
block PMN transmigration (Huang et al., 1993
).
Based on this, Del Maschio et al. (1996) have recently
presented evidence that suggested that PMN adhesion
would trigger the disorganization of endothelial adherens
junctions. By using immunofluorescence as well as immunoprecipitation and Western blotting techniques, the authors found that the VE-cadherin/catenin constituents of adherens junctions disappeared from the endothelial cell-
cell contacts. In addition, and even more surprising,
-catenin as well as plakoglobin completely disappeared from total
cell extracts, suggesting that PMN adhesion would lead to
the activation of a catenin-degrading proteolytic activity
(Del Maschio et al., 1996
). Similar results were recently
also described by Allport et al. (1997)
, who extend the
above observations by showing that the endothelial proteasome is not involved in catenin degradation. Here, we
present evidence that strongly suggests that this catenin-degrading activity is not an endothelial enzyme but leukocyte encoded. Our data lead us to conclude that the disappearance of catenins after the adhesion of PMNs to EC
(and seen in immunofluorescence as well as by Western
blotting of total cell lysates), is because of a nonspecific
proteolytic event.
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Materials and Methods |
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Cell Culture
Human umbilical vein endothelial cells (HUVEC) were isolated as described (Warren, 1990), and cultured in M199, 20% FCS, 50 µg/ml endothelial cell growth supplement (Sigma Chemical Co., St. Louis, MO), 100 µg/ml Heparin (Sigma Chemical Co.). Alternatively, HUVEC were purchased commercially (Promocell, Heidelberg, Germany) and cultivated in
MCDB 131 (GIBCO BRL, Gaithersburg, MD), 20% FCS, 50 µg/ml
ECGS, 100 µg/ml Heparin. Cells were used up to passage number 8. Mouse bEnd.3 endothelioma cells (Williams et al., 1989
) were cultured as
described (Hahne et al., 1993
). Activation of the EC was done with 400 U/ml
tumor necrosis factor (TNF)-
for 4 h or 24 h as indicated.
Leukocyte Adhesion to EC
Human PMN were isolated from fresh buffy coats on Histopaque 1077 and Histopaque 1119 density gradients according to the manufacturer's instructions (Sigma Chemical Co.). The recovered cells were washed twice
in HBSS without Ca2+ and Mg2+ (GIBCO BRL, Gaithersburg, MD) and
kept in this buffer at room temperature until use. The leukocytes were
then resuspended into M199, 20% FCS (for HUVEC) or DME, 10% FCS
(for bEnd.3), and then added to the EC at a 10:1 or 2:1 ratio of leukocytes
to EC as indicated. The cells were coincubated for 5 min at 37°C in a humidified atmosphere. Nonadherent cells were washed off with PBS without Ca2+ and Mg2+, and the remaining cells were lysed. Lysis was carried
out in either lysis buffer containing 1% Triton X-100, 150 mM NaCl, 20 mM
Tris/HCl, pH 8.0, 1 mM CaCl2, 10 µg/ml leupeptin, 1 mM PMSF, 2 µg/ml
pepstatin, 40 U/ml aprotinin, 30 µg/ml eglin C, or in boiling (95°C) Laemmli SDS-PAGE sample buffer (2% SDS, 10% glycerol, 65 mM Tris/HCl,
pH 6.8, 50 mM DTT, [Laemmli, 1970]). For Triton X-100 lysates, lysis was
carried out on the tissue culture plates for 25 min on ice with occasional
gentle agitation. Cell extracts were then centrifuged at 14,000 rpm in a table
top microfuge for 5 min at 4°C, and the supernatants used (Triton X-100-
soluble fraction). The residual material on the tissue culture plate was extracted with 0.2% SDS, briefly sonicated, and then analyzed (Triton
X-100-insoluble fraction; data not shown). In some instances, after the
rinse in PBS, the adherent PMNs were removed by incubation with PBS
without Ca2+ and Mg2+, 5 mM EDTA for about 30 s. Subsequently, the
PMNs were washed off once more with PBS before extraction with Triton
X-100 lysis buffer. For the lysate mixing experiment, Triton X-100 lysates of 4 × 105 HUVEC each were prepared as described above. To such lysates, 2 × 106 lysed PMNs, or mononuclear cells (without centrifugation),
or the respective cleared PMN lysates (with nuclei and Triton X-100- insoluble material pelleted) were added as described in the text. Under
the experimental conditions used, i.e., PMN/EC ratio of 10:1, we found
that approximately five PMNs adhere per endothelial cell. The final volume (300 µl) was the same as for lysates prepared on cell culture plates.
Western Blot Analysis
Cell extracts corresponding to 6 × 104 EC/lane were used and the Western
blots performed essentially as described (Zöllner and Vestweber, 1996).
To load approximately the same amount of PMN lysates in the lanes
where PMNs were analyzed for their respective catenin and
-actinin expression levels, lysates corresponding to 3 × 105 cells were used (see
above). For detection of the human antigens monoclonal antibodies
against
-catenin (No. C19220) and plakoglobin (No. C26220; all from
Transduction Laboratories, Lexington, KY), and against
-actinin (BM-75.2; Sigma Chemical Co.) were used according to the manufacturer's instructions.
Immunofluorescence Microscopy
Immunofluorescence analysis was performed essentially as described
(Lampugnani et al., 1992; Del Maschio et al., 1996
), except that endothelial cells were grown on coverslips coated with 0.2% gelatine. The removal
of adherent PMN with EDTA was carried out as described above. Cells
were fixed in 3% paraformaldehyde (PFA) in PBS; 0.5% Triton X-100 for
3 min at room temperature, followed by 3% PFA in PBS for 15 min at
room temperature. Identical results were obtained when cells were first
fixed with 3% PFA for 10 min at room temperature, and subsequently
permeabilized with 0.5% Triton X-100 in HBSS for an additional 5 min.
Mouse VE-cadherin and
-catenin were detected using rabbit polyclonal
antipeptide antibodies (5 µg/ml in HBSS, 3% FCS) and have been described (Breier et al., 1996
; Gotsch et al., 1997
).
-catenin was detected using a monoclonal antibody (1:100 in HBSS, 3% FCS; No. C19220) followed by a TRITC-conjugated, goat anti-mouse secondary antibody (1:50 in HBSS, 3% FCS; Dianova, Hamburg, Germany).
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Results and Discussion |
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Neutrophil-induced Catenin Disappearance Depends on Cell Lysis Conditions
Recent work by Del Maschio et al. (1996) suggested that
PMN adhesion to EC leads to the complete loss of
-catenin and plakoglobin from total cell extracts, as well as to
an at least partial disappearance of VE-cadherin and
-catenin. We could reproduce this effect by adding human
PMNs for 5 min to monolayers of 4 h TNF-
-stimulated HUVEC, washing unbound cells away, lysing endothelial
cells and bound neutrophils with Triton X-100 (in the
presence of a cocktail of protease inhibitors), and immunoblotting the extracts with antibodies to
-catenin and
plakoglobin (Fig. 1 A, lanes 3 and 4). The same effect was
seen with HUVEC activated for 24 h (data not shown).
-catenin and plakoglobin also disappeared from the Triton X-100-insoluble fraction, which was solubilized by
SDS (data not shown).
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Since neutrophils are known to produce a large number
of proteases, we were concerned as to whether the colysis
of EC and bound PMNs in a Triton X-100-containing
buffer could possibly lead to the nonspecific liberation of
neutrophil proteasessufficiently active even in the presence of protease inhibitors. Therefore, we changed the lysis
conditions and extracted HUVEC and bound neutrophils
with boiling (95°C) SDS-PAGE sample buffer. Subsequent immunoblot analysis revealed that
-catenin as well
as plakoglobin were not degraded if cells were lysed under
these conditions (Fig. 1 A, lanes 1 and 2). These results
strongly suggest that degradation of catenins does not occur before cells are lysed and depends on the lysis conditions.
To rule out a potential contribution of neutrophil proteases in the degradation of the catenins Del Maschio et al.
(1996) separately extracted HUVEC and PMNs with Triton X-100, pelleted the insoluble material, and then coincubated the detergent extracts on ice. This treatment did
not result in any degradation of either VE-cadherin or the
catenins. Since the preparation of Triton X-100 lysates and
the centrifugation of insoluble material takes about 30 min, we analyzed whether the suspected proteolytic activity in the PMN lysates would be unstable. We prepared Triton X-100 extracts from HUVEC, cleared from insoluble
material, and then added PMN lysates prepared in three
different ways. First, PMNs were lysed and the total lysate
was immediately added to the HUVEC lysate without
prior pelleting (Fig. 1 B, lane 2). Second, the total PMN lysate was incubated for 30 min on ice before adding it to the
HUVEC lysate, again without prior precipitation of insoluble material (Fig. 1 B, lane 3). Third, PMN lysates were
incubated for 30 min on ice, cleared from insoluble material by centrifugation, and then added to the HUVEC lysate (Fig. 1 B, lane 4). The two lysates were coincubated
for another 20 min on ice, and then analyzed by immunoblotting. As shown in Fig. 1 B, fresh PMN lysates contained a proteolytic activity that degraded
-catenin and
plakoglobin (lane 2). This activity was strongly reduced after a 30-min incubation on ice. Plakoglobin appeared to be
more protease sensitive than
-catenin. Interestingly, we
found that the neutrophil protease activity was even lost
within 30 min if no external protease inhibitors were
added (data not shown). Thus, neutrophil lysates degrade endothelial catenins only when immediately added to endothelial lysates.
Removal of Bound Neutrophils with EDTA Before Analysis Prevents Catenin Degradation
We examined whether the disappearance of the endothelial catenins would still be observed if neutrophils that had
attached to the endothelial surface were removed by washing them off with EDTA before the analysis. As shown in
Fig. 2 the PMN-induced -catenin and plakoglobin degradation (lane 4) was largely prevented by washing off the
PMNs before extracting the EC with Triton X-100 (lane
6). Thus, the binding of neutrophils to the endothelial cells
was not responsible for the degradation of the catenins, but rather the presence of neutrophils during the lysis procedure.
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In addition to the biochemical techniques, immunofluorescence analysis had been used to study the effects of
neutrophil binding on the distribution of the catenins at
endothelial cell contact sites (Del Maschio et al., 1996).
We used the same approach as decribed above (Fig. 2) to
control these experiments, i.e., previously bound neutrophils were removed from the endothelial cell monolayer with EDTA before fixation and staining for the endothelial
-catenin. Since the integrity of HUVEC monolayers
was very sensitive to the treatment with EDTA, we used
the mouse endothelioma cell line bEnd.3 for these experiments. bEnd.3 cells not only express VE-cadherin and
catenins, but also form functional adherens junctions
(Breier et al., 1996
; Gotsch et al., 1997
) (Fig. 3, A-C).
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We found that human PMNs bound well to TNF-stimulated bEnd.3 cells. After the removal of unbound neutrophils, bEnd.3 cells with the bound PMN were fixed with
PFA and stained after permeabilization for
-catenin.
As shown in Fig. 3 D,
-catenin staining was almost completely gone at intercellular contacts. However, removal of specifically bound cells by washing with EDTA before fixation and permeabilization of the monolayer in Triton X-100
completely prevented the disappearance of
-catenin (Fig.
3 F). Thus permeabilization of endothelial cells in the
presence of adhering PMNs can lead to a neutrophil protease-mediated degradation of
-catenin
despite fixation
of the cells with PFA.
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Concluding Remarks |
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In this study we have examined the mechanism by which
the binding of neutrophils to the monolayer of endothelial
cells causes the degradation of endothelial catenins. In contrast to previous interpretations (Del Maschio et al., 1996;
Allport et al., 1997
), we found that the catenin-degrading
protease in this experimental setting is not an endothelial
enzyme, but a leukocyte enzyme that is released upon detergent lysis of the cells. This is based on the following evidence. First, catenin degradation was only observed when
the endothelial cells and bound PMNs were detergent lysed under nondenaturing conditions. If the cells were lysed at
95°C in an SDS-containing buffer, degradation was not detected. Second, if PMNs were first allowed to bind to activated HUVEC for 5 min at 37°C, and were then removed
by EDTA before detergent lysis of the EC, degradation of
catenins was not observed. Third, mixing experiments with
detergent extracts of EC and PMNs revealed that catenin
disappearance could be observed after mixing extracts from quiescent cells on ice. PMN lysates lost this activity
upon incubation on ice for 30 min. This strongly suggests
that the PMN-induced endothelial catenin disappearance
is not mediated by a transmembrane signaling event, but is
because of a neutrophil protease that is released upon detergent lysis.
The nature of the neutrophil protease degrading the endothelial catenins remains obscure. Our data suggest that
the activity of this enzyme is not immediately destroyed by
3% PFA. Furthermore, a mixture of several protease inhibitors (see Materials and Methods) did not inactivate
the enzyme, and only boiling in 2% SDS efficiently and
rapidly destroyed this protease activity. Moreover, several proteins were unaffected by this protease, such as platelet/
endothelium (PE)CAM-1 and F-actin (Del Maschio et al.,
1996), as well as
-actinin (this study). This may explain
why this neutrophil protease activity was overlooked in
the former study.
It has been shown that the binding of PMNs to the apical surface of HUVEC increased the permeability of the
endothelial monolayer (Del Maschio et al., 1996), and leads
to an increase in endothelial Ca2+ levels (Huang et al.,
1993
). The VE-cadherin/catenin complex is important for
the junctional integrity of EC layers (Navarro et al., 1995
;
Gotsch et al., 1997
). Therefore, the concept of adhering
leukocytes having an effect on the VE-cadherin junctional complex still remains attractive, although evidence is still
lacking. To establish a potential signaling pathway in endothelial cells that connects the docking of PMN at the
apical surface to the regulation of VE-cadherin/catenin
function, one would have to efficiently inhibit the neutrophil proteolytic activity. Alternatively, one would have to
find means to manipulate such a signaling pathway independently of neutrophil adhesion.
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Footnotes |
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Received for publication 2 October 1997 and in revised form 26 November 1997.
Address all correspondence to D. Vestweber, Institute of Cell Biology, ZMBE, Technologiehof Münster, Mendelstrasse 11, D-48149 Münster, Germany. Tel.: 49-251-835-86-17; FAX: 49-251-835-86-16; E-mail:vestweb{at}uni-muenster.de ![]() |
Abbreviations used in this paper |
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EC, endothelial cells; HUVEC, human umbilical vein endothelial cells; PFA, paraformaldehyde; PMN, polymorphonuclear cell; VE, vascular endothelial.
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The E-selectin ligand-1 is selectively activated in Chinese hamster ovary cells by the ![]() |