From the Laboratoire d'Ingénierie des
Macromolécules and ¶ Laboratoire de Spectrométrie de
Masse des Protéines, Institut de Biologie Structurale Jean-Pierre
Ebel (Commissariat à l'Energie Atomique/CNRS,
Université Joseph Fourier), 41 rue Jules Horowitz,
38027 Grenoble Cedex, France
Received for publication, January 13, 2003, and in revised form, February 7, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transmigration of neutrophils across the
endothelium occurs at the cell-cell junctions where the vascular
endothelium cadherin (VE cadherin) is expressed. This adhesive receptor
was previously demonstrated to be involved in the maintenance of
endothelium integrity. We propose that neutrophil transmigration across
the vascular endothelium goes in parallel with cleavage of VE cadherin by elastase and cathepsin G present on the surface of neutrophils. This
hypothesis is supported by the following lines of evidence. 1)
Proteolytic fragments of VE cadherin are released into the culture
medium upon adhesion of neutrophils to endothelial cell monolayers; 2)
conditioned culture medium, obtained after neutrophil adhesion to
endothelial monolayers, cleaves the recombinantly expressed VE cadherin
extracellular domain; 3) these cleavages are inhibited by inhibitors of
elastase; 4) VE cadherin fragments produced by conditioned culture
medium or by exogenously added elastase are identical as shown by
N-terminal sequencing and mass spectrometry analysis; 5) both elastase-
and cathepsin G-specific VE cadherin cleavage patterns are produced
upon incubation with tumor necrosis factor The endothelium constitutes a physical barrier between blood and
the underlying tissues and is the point of entry of leukocytes into
inflamed tissues. The mechanism of leukocyte entrance into tissues is a
complex, multistep event involving leukocyte adhesion to endothelium
and subsequent migration across the blood vessel wall (1). Whereas the
early steps leading to leukocyte adhesion are well understood (2, 3),
it is still unclear how leukocytes crawl through the endothelial wall.
Many studies demonstrated that leukocytes exit blood vessels by
squeezing between adjacent endothelial cells (4), but a recent
study suggested that leukocytes could pass across the body of
endothelial cells using a transcellular route (5).
Two adhesive receptors, platelet-endothelial cell adhesion molecule-1
(PECAM-1)1 (6, 8, 9) and CD99
(7), were clearly demonstrated to be involved in the leukocyte
transmigration process. They are both expressed at the surface of
leukocytes and at endothelial cell-cell junctions and elaborate
homophilic interactions between the two cellular types. In fact,
PECAM-1-mediated homotypic interactions are relayed by CD99-mediated
ones to regulate the transmigration of leukocytes across the endothelium.
Additionally, a third adhesive receptor, VE cadherin (10), expressed at
endothelial cell-cell junctions, can modulate leukocyte transmigration. VE cadherin participates in the elaboration of inter-endothelial adherens junctions as its extracellular part establishes homophilic interactions resulting in cell-cell attachment (11, 12). These ectodomain-based interactions are reinforced by
interactions involving the cytoplasmic domain of VE cadherin, which
binds, in a mutually exclusive manner, the armadillo proteins VE cadherin also possesses a critical role in the transmigration of
leukocytes. Indeed, treatment with anti-VE cadherin antibodies, able to
disrupt interendothelial junctions, results in an increase of
neutrophil transmigration in vitro (20) and in
vivo (19, 44). Moreover, immunofluorescence staining reveals that
VE cadherin and The exact role played by VE cadherin in the transmigration
process is still controversial. It was first proposed that leukocyte adhesion might trigger intracellular signals, leading to the
destabilization of VE cadherin-based complexes and thus allowing
leukocyte transmigration to occur through the formed gaps (22, 21).
Indeed, disruption of the VE cadherin/catenin complexes (23) generally
parallels with the tyrosine phosphorylation of the different partners
within the cadherin complex. These phosphorylation events may be
modulated by the capacity of VE cadherin to associate with kinases such as the receptor tyrosine kinase VEGFR-2 (24, 25) and with phosphatases
such as vascular endothelial protein tyrosine phosphatase (VE-PTP) (26)
and SH 2 domain-containing tyrosine phosphatase 2 (SHP-2) (27, 28).
A second and more recent hypothesis concerning the role of VE cadherin
in transmigration proposes that migrating leukocytes locally push VE
cadherin in the plane of the junction thus allowing their passage
through the intercellular gaps (4). In this study, transmigration of
fluorescently labeled leukocytes was followed in real time by two-color
fluorescence microscopy using HUVECs expressing endogenous VE cadherin
and a VE cadherin protein fused in its C-terminal part with green
fluorescent protein. This study shows that transmigration occurs
through both preexisting gaps and through de novo gap
formation. Gap widening accommodates to the size of the transmigrating
leukocyte allowing a narrow contact between endothelial cell and
leukocyte. This widening of the gaps seems to be accompanied by a
clustering of the VE cadherin protein fused in its C-terminal part with
green fluorescent protein molecules on the edge of the forming clefts.
After transmigration, the displaced molecules diffuse back to
reconstitute the junctions. These results were recently confirmed by a
real-time study following the differential movements of VE cadherin in
living endothelial cells during transmigration of neutrophils using
antibodies which do not interfere with transendothelial migration (29).
The lateral movement of VE cadherin was hypothesized to be a
consequence of its decoupling from the cytoskeleton, probably initiated
by an intracellular signal as a result of leukocyte adhesion to the endothelium.
A possible role for neutrophil proteases is also suspected in
transmigration (30). These proteases could cleave proteins participating in the elaboration of intercellular contacts, thus facilitating the progression of neutrophils across the endothelial junctions. Among the proteases susceptible to involvement in
transmigration are serine proteases (including elastase, cathepsin G,
and proteinase 3) and matrix metalloproteases (such as MMP-8 and
MMP-9), all packed in the azurophil granules of neutrophils (31).
Recently, elastase was suspected to be able to degrade components of VE cadherin complexes during contact between neutrophils and endothelial cells (22, 21), but this result remains controversial. Indeed, the
adhesion-dependent degradation of endothelial catenins
described by Del Maschio et al. (22) and Allport et
al. (21) probably does not reflect transmigration events but
rather a release of neutrophil proteases upon cell lysis (32).
Moreover, the fact that, in MMP-9- and neutrophil elastase-deficient
mice, neutrophils transmigrate as efficiently as in wild-type mice
suggests that elastase and MMP-9 are not essential for the leukocyte
transmigration process (33). This is in conflict with the results of
Cepinskas et al. (30), who found that, on migrating
neutrophils, membrane-bound elastase, which localizes to the migrating
front, facilitates transendothelial migration. Moreover, in some
pathological situations, administration of an elastase inhibitor
attenuates the transmigration of neutrophils (34).
In the present report, we study the role of proteases and VE cadherin
in neutrophil transmigration. We demonstrate that, following adhesion
of neutrophils to endothelial monolayers, VE cadherin fragments are
released from the surface of endothelial cells, suggesting that
proteases are involved in the transmigration process. We present
evidence that purified elastase and cathepsin G can cleave
in vitro the extracellular part of VE cadherin. The exact positioning of the cleavage sites for each protease allows us to
clearly identify elastase and cathepsin G as the surface-bound proteases able to cleave VE cadherin. Moreover, we also demonstrate that specific blockage of both elastase and cathepsin G at the neutrophil surface significantly reduces their transmigration in an
in vitro assay. This strongly suggests that proteolytic events such as the cleavage of VE cadherin occurring at cell-cell junctions are required for neutrophil transmigration.
Cells--
Neutrophils were isolated from the peripheral blood
of healthy volunteers by using dextran-Ficoll (35) and resuspended in M199 medium (Biowhittaker, Verviers, Belgium). Human endothelial cells
were isolated from umbilical veins and cultured in M199 supplemented
with 20% fetal bovine serum (Biowhittaker), 50 µg/ml endothelial
cell growth supplement (Biowhittaker) and kept at 37 °C in a 5%
CO2 humidified atmosphere, as previously described (10).
Reagents--
Elastase was purchased from Athens Research
(Athens, GA); cathepsin G was from Elastin Products Co., Inc.
(Owensville, MI); human Secretory leukocyte protease inhibitor (SLPI)
was from R&D Systems Europe (Abingdon, UK); the specific human
leukocyte elastase inhibitor
N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone (MeOSuc-AAPV) was from Sigma-Aldrich Chimie (St Quentin
Fallavier, France). Human tumor necrosis factor
The bacterially expressed VE-EC1-4 fragment was produced as previously
described (11, 12).
Antibodies--
Four fragments, designated as EC1, EC4, Cad1,
and Cad3, encompassing various regions of the VE cadherin protein were
used as antigens to raise the anti-VE cadherin polyclonal antibodies named anti-EC1, anti-EC4, anti-Cad1, and anti-Cad3, respectively. The
shorter fragments VE-EC1 and VE-EC4 were produced as described (11).
The cDNA constructs encoding Cad1 and Cad3 were elaborated as
described previously for VE cadherin (18). The resulting plasmids
expressed the VE cadherin fragments Cad1 and Cad3 (amino acid stretches
1-439 for Cad1 and 259-439 for Cad3) fused at their N termini with a
16-amino acid fusion peptide (18).
Production of the polyclonal anti-VE cadherin antisera was performed as
previously described (18). Each polyclonal antibody was directly
purified by applying the corresponding antiserum on an affinity column
coupled to the recombinant fragment used as an antigen. The
characterization of these polyclonal antibodies was performed by
fluorescence-activated cell sorter analysis on Chinese hamster ovary
cells expressing human VE cadherin, by immunofluorescence staining and
Western blotting as previously described (18).
The anti-fragment antibodies were biotinylated with
D-biotinoyl- Capture Immunoassays--
Polyvinyl microtiter wells were coated
overnight at 4 °C with 100 µl of either anti-EC1 or anti-EC4 or
anti-Cad3 antibodies diluted at 10 µg/ml in 0.1 M sodium
carbonate buffer, pH 9.6. The plates were postcoated with
phosphate-buffered saline containing 1% bovine serum albumin, washed
with 0.05 M phosphate-buffered saline plus 0.05% Tween 20, and incubated with 100 µl of cell supernatants overnight at 4 °C.
Following incubation with supernatants, extensive washing was performed
with 0.05 M phosphate-buffered saline, 0.05% Tween 20. Bound VE cadherin-derived fragments were detected by adding 100 µl of
either biotinylated anti-Cad 1 or biotinylated anti-Cad 3 diluted in
phosphate-buffered saline containing 0.5% bovine serum albumin and
incubation was performed for 2 h at room temperature. After
washing with phosphate-buffered saline and 0.05% Tween 20, alkaline
phosphatase-conjugated streptavidin was added for 1 h at room
temperature. After washing, binding was quantified using
p-nitrophenyl phosphate as a substrate for alkaline
phosphatase and measuring the absorbance at 405 nm.
Western Blotting--
Following its proteolysis, VE-EC1-4 was
subjected to 12.5% polyacrylamide gel electrophoresis. Separated
fragments were then electroblotted onto pure nitrocellulose membranes
(Bio-Rad, Marnes-la-Coquette, France) and blocked with 3%
gelatin-containing PBS. The blots were incubated overnight with the
polyclonal anti-Cad3 antibody in PBS containing 1% gelatin, and
protein bands were then detected by a peroxidase-labeled goat
anti-rabbit antibody using the ECL kit (Amersham Biosciences).
Neutrophil Adhesion to Endothelial Cells--
Prior to the
addition of neutrophils, confluent endothelial cell monolayers were
treated with 100 units/ml TNF Digestion of the Recombinant Fragment VE-EC1-4 by Elastase,
Cathepsin G, or Cell Medium Supernatant--
VE-EC1-4 (final
concentration, 3 µM) was mixed with either elastase
(final concentration, 0.02 unit/ml) or cathepsin G (final concentration, 0.02 unit/ml) or the supernatant (dilution 1/3, see
above). The mixtures were incubated for different periods of time at
37 °C prior to Western blot or matrix-assisted laser desorption
(MALDI) mass spectrometry analyses. In some assays, the purified
proteases or the supernatants were preincubated with the protease
inhibitors MeOSuc-AAPV or with SLPI at 37 °C during 20 min before
addition of the fragment VE-EC1-4.
Degradation of the Recombinant Fragment VE-EC1-4 by Cell
Surface-bound Enzymes--
Neutrophils were stimulated for 30 min at
37 °C both with fMLP (10 Transendothelial Permeability Assay--
Cells were cultured
until confluence on polycarbonate membranes of Transwell inserts
(0.4-µm pore size, Costar, Cambridge, MA) as previously described
(18). Following verification of cell confluence by crystal blue
staining, the culture medium in the upper compartment was substituted
with medium containing purified elastase (2 × 10 MALDI Mass Spectrometry--
Mass spectra of the
proteolytic fragments derived from VE-EC1-4 were obtained with a
Perspective Biosystems Voyager Elite Xl time of flight mass
spectrometer with delayed extraction, operating with a pulsed nitrogen
laser at 337 nm (Framingham, MA). Positive-ion mass spectra were
acquired using linear, delayed extraction mode with an accelerating
potential of 25 kV, a 90% grid potential, a 0.2% guide wire voltage,
and a delay time of 200 ns. Each spectrum is the result of 200 averaged
laser pulses.
Samples were mixed with an equal volume of 1% trifluoroacetic acid and
concentrated on a ZipTipTM C4 (Millipore) as specified by
the manufacturer. The material eluted from the ZipTipTM C4
was partially evaporated and mixed with an equal volume of a saturated
solution of sinapinic acid prepared in 50% (v/v) aqueous acetonitrile,
0.1% trifluoroacetic acid. Aliquots of 2 µl of this mixture were
spotted on the stainless steel sample plate and dried in the air prior
to analysis. External calibration was performed with enolase from
bakers' yeast using the m/z values of 46,672 and
23,336 for the mono- and di-charged molecules, respectively. The
accuracy of MALDI molecular weight determinations comprises between
0.01 and 0.2%.
Blotting for N-terminal Sequencing--
Following
electrophoresis, proteolytic products were transferred to
polyvinylidene difluoride ProblottTM membranes (Applied
Biosystems, Foster City, CA) using a 10 mM CAPS, 10% (v/v)
methanol buffer at pH 11. Transfer was carried out during 1 h at
room temperature using a constant voltage of 50 V. After transfer, the
membranes were rinsed with distilled water, saturated with 100%
methanol for a few seconds, stained for 5 min with a solution of 0.1%
Coomassie Blue R250 in acetic acid/methanol/H2O (10:40:50,
v/v/v), and then destained with 50% methanol and air-dried. Protein
bands were excised and submitted to N-terminal sequencing. Amino acid
sequence determination was performed using a gas-phase Sequencer model
477A (Applied Biosystems).
Neutrophil Transmigration Assay--
HUVECs were grown to
confluence on fibronectin-coated porous membranes of Transwell units
(3-µm pore size, Falcon, Dutscher, Issy-les-Moulineaux, France) for 3 days at 37 °C. To study the influence of proteases on
transmigration, freshly purified neutrophils were activated at 37 °C
during 30 min in M199 containing 20% fetal bovine serum, 100 units/ml
TNF
In parallel, the presence of active proteases at the surface of
activated neutrophils or their inactivation following treatment with
inhibitors was verified by incubating differently treated neutrophils
with the fragment VE-EC1-4 as described above. As indicated by blue
trypan staining, the various treatments applied to neutrophils did not
induce cell death.
Cleavage of VE Cadherin Induced by Adhesion of Neutrophils onto
Endothelial Cell Monolayers--
To investigate the fate of VE
cadherin during transmigration of leukocytes across endothelium, human
neutrophils were added, in the presence of the chemoattractant reagent
fMLP, to confluent TNF
To understand the mechanism underlying the production of soluble VE
cadherin fragments, an ELISA using the antibodies anti-EC1 and
anti-Cad3 (Fig. 2A) was
performed on cell culture supernatants obtained from differentially
treated endothelial cells (Fig. 2B, ELISA
1). First, to analyze the role of TNF
In the ELISA 1, depicted in Fig. 2B, only
fragments overlapping at least the amino acid stretch Phe-104 to
Phe-259 and extending maximally from amino acid Asp-1 to Lys-439 of the
VE cadherin sequence could be detected. To improve the detection of VE
cadherin-derived fragments, several ELISAs were performed using
different pairs of polyclonal antibodies directed against various
regions of VE cadherin (Fig. 2A). In each case, adhesion of
neutrophils onto endothelial cells resulted in a significant increase
of the level of soluble fragments into cell supernatants (Fig.
2B, ELISA 2-5). This suggested that
adhesion of neutrophils to TNF In Vitro Cleavage of the VE Cadherin Extracellular Region by Cell
Culture Supernatants--
We hypothesized that adhesion of neutrophils
to TNF
In an attempt to detect the proteases able to cleave VE cadherin, the
cell culture supernatants were mixed with a recombinant protein
encompassing the majority of the extracellular part of VE cadherin,
designated as VE-EC1-4, which was previously demonstrated to be able
to self-assemble as a stable hexamer (11, 12).
Treatment of endothelial cells with either TNF
To observe the potential intermediate proteolytic fragments, the
neutrophil:endothelial cell ratio was diminished from 10:1 to 4:1 and
cell culture supernatants were diluted (1/3) prior to their addition to
the recombinant fragment VE-EC1-4. The time course of the proteolysis
was followed by Western blot using the polyclonal anti-VE cadherin
anti-Cad3 antibody directed against the C-terminal part of VE-EC1-4.
In these conditions, two distinct cleavage products with apparent
molecular masses of 38 and 27 kDa were detected 2 or 4 h after
addition of the culture supernatant to VE-EC1-4 (Fig.
4A). Re-probing the Western
blot membranes with the anti-VE cadherin antibody directed against the
N-terminal module EC1 (Fig. 2A) allowed the detection of an
additional fragment of 22 kDa (data not shown). These results were
systematically observed using neutrophils from 10 different donors
(data not shown).
Identification of the Neutrophil Proteases Involved in the Cleavage
of VE Cadherin--
To identify the supernatant-containing proteases
involved in the VE cadherin cleavage, we used a physiological
inhibitor, the human SLPI, in our cleavage assays. SLPI is known to
specifically block the neutrophil granule-containing proteases elastase
and cathepsin G (37). At a final concentration of 0.5 µg/ml, SLPI effectively blocked the supernatant-induced degradation of VE-EC1-4 (Fig. 4B, lanes 7-9). This suggested
strongly that elastase and/or cathepsin G were the active proteases
present in the supernatants.
We then verified that purified elastase, at concentrations as low as
0.02 unit/ml, was able to significantly cleave VE-EC1-4. The pattern
of digestion of this enzyme (Fig. 4C, lanes
5 and 6) appeared very similar to those obtained
with the supernatants (Fig. 4B, lanes
5 and 6). Indeed, both the 38- and the 27-kDa proteolytic products were clearly detected by Western blot following 2 or 4 h of treatment. This strongly indicated that one of the proteases secreted by neutrophils, following their adhesion on endothelial cells, is elastase. Similarly, purified cathepsin G was
also able to cleave VE-EC1-4. After a 2-h incubation time, the
digestion pattern (Fig. 4D, lane 5)
was very similar to that observed with purified elastase (Fig.
4C, lane 5). In contrast, 4 h
after addition of cathepsin G, the 38-kDa band disappeared, whereas the
27-kDa band appeared slightly less abundant (Fig. 4D,
lane 6). Furthermore, addition of SLPI to each
purified enzyme blocked their proteolytic activity. Indeed, as shown in
Fig. 4 (C and D, lanes 8 and 9), SLPI inhibited the elastase activity totally and
that of cathepsin G partially.
To confirm that elastase was one of the proteases responsible for the
proteolytic cleavage of fragment VE-EC1-4, the chemical neutrophil
elastase-specific inhibitor MeOSuc-AAPV (37) was incubated with
supernatant before its addition to VE-EC1-4. In these conditions,
intensities of both the 38- and 27-kDa proteolytic bands were strongly
decreased (Fig. 5, lanes
7-10). Concomitantly, we also verified that this
elastase-specific inhibitor only affects the elastase activity (Fig. 5,
lanes 3-6) and not the cathepsin G activity
(Fig. 5, lanes 11-14).
Identification of the Elastase- and Cathepsin G-induced Cleavage
Sites by N-terminal Sequencing and MALDI Mass
Spectrometry--
N-terminal sequencing of the 27-kDa electroblotted
bands observed in Fig. 4 (panels C and
D, lanes 5) showed that they
corresponded to C-terminal peptides beginning at positions Thr-201 or
Gln-203 when VE-EC1-4 was digested either by elastase or cathepsin G, respectively (Table I). The 38-kDa band
generated by elastase digestion corresponded to a single C-terminal
peptide starting at the amino acid Lys-94. In contrast, the 38-kDa
cathepsin G-generated band contained two peptides starting at amino
acids Thr-92 and Val-95 (Table I).
MALDI mass spectrometry analyses were also performed to confirm and
complete the N-terminal sequencing data (Fig.
6). Mass spectra indicated that treatment
of VE-EC1-4 with purified elastase gave a major proteolytic product
possessing a molecular mass of 26,611 Da (Fig. 6A and Table
I), whereas purified cathepsin G released a product of 26,424 Da (Fig.
6B and Table I). The masses of the proteolytic fragments
were determined with sufficient accuracy to establish that the
26,611-Da product corresponds to the amino acid stretch Thr-201 to
Glu-432 (calculated mass, 26,651 Da) (Fig. 6A), whereas the
26,424-Da fragment can be assigned to the fragment Gln-203 to Glu-432
(calculated mass, 26,402 Da) (Fig. 6B). Moreover, with both
elastase and supernatant, the initial fragment VE-EC1-4 (calculated
mass, 48,941 Da) could still be detected (Fig. 6, A and
C).
The 38-kDa fragments (Figs. 4 and 5) were not detected by mass
spectrometry. This was probably a result of the method used for
treating the samples before mass spectrometry analysis. Indeed, to
increase their concentration, samples were passed through a ZipTip
column. Analysis by Western blot of the samples prior to and after
elution on ZipTip columns indicated that large amounts of the 38-kDa
fragments remained fixed on the columns. This explains the discrepancy
of protein fragment quantities observed between Western blot and mass
spectrometry analyses. In contrast, MALDI mass spectrometry analyses
detected additional fragments. These fragments generated by elastase or
cathepsin G, respectively, possess molecular masses of 22,300 Da (Fig.
6A) or 22,669 Da (Fig. 6B) and correspond to the
N-terminal peptide fragments MD1-V200 (calculated mass, 22,300 Da) or
MD1-Q203 (calculated mass, 22,669 Da). These bands are absent from the
Western blots revealed with the antibody anti-Cad3 (Figs. 4 and 5) but
are detected with the antibody anti-EC1 as previously mentioned.
Altogether, mass spectrometry and N-terminal sequencing analyses
indicate that elastase cleaves the VE-EC1-4 fragment after residues
Ile-93 and Val-200, whereas cathepsin G cleaves after residues Phe-91,
Lys-94, and Gln-203, as illustrated in Table I.
MALDI mass spectrometry combined to N-terminal sequencing allowed us to
clearly identify the nature of the protease secreted by neutrophils
following their adhesion on endothelial cell monolayers. Indeed, the
supernatant contained a protease that generated a 26,625-Da fragment,
similarly to elastase but different from the 26,424-Da cathepsin
G-generated fragment (Fig. 6C). This result strongly
suggests that elastase is probably the only protease released from
neutrophils that is capable to digest VE cadherin. To verify this
hypothesis, we added purified cathepsin G to the supernatant prior to
its addition to the VE-EC1-4 fragment (Fig. 6D). MALDI
analysis exhibited two major cleavage products of 26,604 and 26,395 Da,
corresponding, as previously established, to fragments Thr-201 to
Glu-432 and Gln-203 to Glu-432, respectively. This result indicated
that, if cathepsin G would have been released into the culture
medium, it could easily be detected by mass spectrometry.
The selective secretion of elastase in the supernatant ruled out the
possibility that VE cadherin cleavage resulted from neutrophil crushing
during the cell purification step. In this case, both elastase and
cathepsin G would have been detected in cell supernatants.
Cleavage of the Extracellular Domain of VE Cadherin by Elastase and
Cathepsin G Bound to the Leukocyte Cell Surface--
Several papers
mention that exposure of neutrophils to cytokines or chemoattractants
such as fMLP and TNF
It thus remains to be shown whether, following adhesion of neutrophils
on endothelial cell monolayers, the cleavage of VE cadherin is induced
by proteases secreted into the extracellular milieu or bound to the
neutrophil surface membrane. To answer this question, neutrophils,
stimulated both by fMLP and TNF
This Western blot analysis did not allow to discriminate whether bound
elastase or bound cathepsin G or both were involved in the cleavage of
VE cadherin. In contrast, MALDI mass spectrometry analysis revealed two
distinct major products having molecular masses of 26,615 and 26,395 Da
(Fig. 7B). They were identical to those generated by
elastase and by cathepsin G as previously demonstrated. To exclude the
possibility that this proteolysis was caused by the release of enzymes
from intracellular granules, postwashing supernatants from fixed
neutrophils were also incubated with VE-EC1-4. In these conditions, no
proteolytic product was detected by mass spectrometry (data not shown).
This indicated that the proteolytic activity was cell-associated and
not a result of the release of intracellular leukocyte-derived enzymes.
Altogether, data indicated that both elastase and cathepsin G bound to
neutrophil cell membranes could cleave the extracellular part of VE cadherin.
Elastase- and Cathepsin G-mediated Increase of Endothelial
Monolayer Permeability--
To test whether elastase or cathepsin G
could be responsible for the opening of inter-endothelial cell
junctions during transmigration of leukocytes, transendothelial
permeability was measured in the presence of these purified proteases.
The modification of permeability was quantified by establishing the
transmigration time course of a peroxidase-conjugated anti-mouse IgG
across endothelial cell monolayers seeded on porous Transwell chambers
(Fig. 8). A clear accumulation of the
marker with time was observed in the lower compartments for
elastase-treated monolayers compared with untreated ones (Fig.
8A). Similar results were observed when elastase was replaced by either cathepsin G (Fig. 8B) or by supernatants
collected from endothelial cells on which neutrophils had adhered (Fig. 8C). This last observation was consistent with the fact that
these cell supernatants, as previously demonstrated, contained
elastase. In fact, permeability increased with time and reached a
plateau 90 min after the addition of proteases. It can be deduced that resealing of adherent junctions takes place after 90 min of incubation, thus preventing additional accumulation of the marker in the lower compartment. This confirmed our previous finding obtained with an
anti-VE cadherin antibody, which was able to transiently increase transendothelial permeability by destabilizing endothelial cell-cell junctions (18). Altogether, these results suggest both elastase and
cathepsin G are able to perturb the integrity of the endothelial cell
monolayer likely by damaging VE cadherin at cell-cell junctions.
Neutrophil Transmigration Blockage by Protease Inhibitors--
To
assess the role of elastase and cathepsin G in neutrophil
transmigration, an in vitro assay was performed using
Transwell units. Differentially treated neutrophils were added to
confluent endothelial cell monolayers seeded on porous membranes and
pre-activated with TNF
To detect the presence or the absence of protease activity at the
neutrophil cell surface, differentially treated neutrophils were fixed
before being incubated with fragment VE-EC1-4. The typical fragments
of 38 and 27 kDa previously described in Fig. 7 are detected when
VE-EC1-4 was incubated with untreated neutrophils attesting that some
proteases are bound to the surface of these neutrophils (Fig.
9A,
lane 2). Treatment of neutrophils
with both TNF
TNF The mechanism by which neutrophils transmigrate across the
vascular endothelium is controversial. Some studies suggest that adherent neutrophils trigger intracellular events in the endothelium, which lead to the disorganization of the junctional complexes involved
in the maintenance of endothelial integrity (4, 21, 22). Other studies
indicate that these junctional complexes disrupt under the action of
neutrophil-derived proteases, thus facilitating neutrophil migration
(30, 34).
Herein, we show that VE cadherin, an adhesive receptor participating at
the maintenance of endothelial integrity (18), is cleaved following
adhesion of neutrophils to endothelial cell monolayers.
Indeed, small but detectable amounts of soluble fragments of VE
cadherin accumulated in the cell culture supernatants after adhesion of
neutrophils. We can exclude an artifactual degradation of VE cadherin
by released proteases caused by neutrophil lysis in our experiments.
Therefore, the low level of soluble VE cadherin fragments probably
reflects a physiologically relevant, local proteolysis activity close
to the very sites of neutrophil adhesion.
Using specific inhibitors of neutrophil proteases, we were able to
identify elastase and cathepsin G as the major proteases involved in
the cleavage of VE cadherin. There is a redundancy between these two
proteases to cleave VE cadherin. To abolish neutrophil transmigration,
these cells must at least be deficient in both elastase and cathepsin
G. This can explain why neutrophils still transmigrate across
endothelial monolayers in the presence of specific inhibitors of
elastase and also why neutrophils from elastase-deficient mice show no
defect in transendothelial migration (33). Indeed, cathepsin G can
replace elastase, thus allowing the neutrophil transmigration to occur
despite elastase inhibition or deficiency. Redundancy is a commonly
observed feature of complex biological processes allowing for
flexibility under different physiological conditions.
Using MALDI-time of flight mass spectrometry and N-terminal sequencing,
we demonstrate that purified elastase can cleave the recombinant
fragment VE-EC1-4 overlapping the four-N-terminal extracellular
modules of VE cadherin after the amino acids Ile-93 and Val-200.
Similarly, purified cathepsin G produces three cleavage sites on
VE-EC1-4 located after amino acids Phe-91, Lys-94, and Gln-203,
i.e. shifted by only two or three amino acids when compared with those of elastase. All of these cleavage sites map within the
EC1-EC2 and the EC2-EC3 interdomain regions of VE cadherin, which are
structurally more prone to be exposed than the compact domains they
connect. The recombinant fragment VE-EC1-5, extended by the C-terminal
module EC5 and corresponding to the complete extracellular domain of VE
cadherin, was also used in these proteolysis experiments (data not
shown). Because of the heterogeneity of its glycosylation, it cannot be
used for determining the position of the cleavage sites. Nevertheless,
when digested by elastase and cathepsin G, its digestion pattern
appears more complex compared with that of VE-EC1-4. This suggests
that these two proteases possess additional cleavage sites localized
between the ends of modules EC4 and EC5 of VE cadherin.
Both fragments VE-EC1-4 and VE-EC1-5 elaborate a hexameric structure
in solution (12),2 probably
reflecting the self-association of VE cadherin at the surface of
endothelial cells. Based on our previous studies (11, 12), we know that
the extracellular module EC1 and the intact EC3-EC4 tandem-module
(amino acid stretch T212-E431) are both required for VE cadherin
self-assembly. Consequently, at the endothelial cell surface, elastase
and cathepsin G may cleave the VE cadherin molecule at positions
identical to those observed for the recombinant fragment VE-EC1-5,
thus probably destroying its hexameric assembly. Based on our previous
knowledge on the self-assembly of VE cadherin, the short fragments left
at the endothelial cell surface membrane are probably unable to self
associate. We know that destabilization of VE cadherin homophilic
interactions by an anti-VE cadherin antibody alters the integrity of
endothelial cell monolayers by creating numerous gaps at cell-cell
junctions (18). Similarly, cleavage of VE cadherin by elastase or
cathepsin G could induce formation of gaps between endothelial cells
through which neutrophils could migrate from the vasculature into the
underlying tissues.
We demonstrate that adhesion of neutrophils on endothelial cell
monolayers induces the release of elastase from neutrophils into the
supernatant of the cell culture. In contrast, no cathepsin G was
extracellularly detected, although its intracellular content within
azurophil granules is as abundant as that of elastase (38). Because of
a different surface charge, elastase does not remain bound to the
neutrophil outer membrane and therefore is released into the
extracellular milieu (36, 38-40). Our results also confirm that,
following neutrophil stimulation by TNF Furthermore, we prove that purified elastase and cathepsin G are able
to increase endothelial monolayer permeability in vivo using
Transwell units. It can therefore be deduced that treatment of
endothelial cell monolayers with elastase or cathepsin G disrupt cell-cell junctions, probably by proteolysing VE cadherin molecules involved in endothelium monolayer integrity.
Altogether, our data suggest that neutrophil transmigration is
facilitated by elastase secreted into the extracellular milieu and by
membrane-bound elastase or cathepsin G despite specific inhibitors such
as The leukocyte transmigration through the endothelium is a multistep
process. First, the leukocyte must firmly adhere to the endothelium
involving dynamic interactions between the leukocyte integrin
-stimulated and fixed
neutrophils; 6) transendothelial permeability increases in
vitro upon addition of either elastase or cathepsin G; and 7)
neutrophil transmigration is reduced in vitro in the
presence of elastase and cathepsin G inhibitors. Our results suggest
that cleavage of VE cadherin by neutrophil surface-bound proteases
induces formation of gaps through which neutrophils transmigrate.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
or
catenin (13, 14). By mediating
catenin binding (15), F-actin-bundling proteins,
or
catenins, promote connections between the actin cytoskeleton and the VE cadherin-based intercellular junction (16). An other member of the armadillo family,
p120ctn or
catenin, binds to the membrane
proximal region of the VE cadherin cytoplasmic part and modulates the
adhesive strength of VE cadherin (17). By establishing these multiple
interactions, VE cadherin plays an important role in the maintenance of
the endothelial integrity (18). Indeed, anti-VE cadherin antibodies, when administered to mice, induce an increase in vascular permeability (19) and, when added to endothelial cell monolayers, provoke the
appearance of gaps at cell-cell junctions (18).
,
, and
catenins disappear from cell-to-cell
contacts following neutrophil adhesion to endothelial monolayers. This effect appears only where neutrophils firmly adhere, whereas VE cadherin-based complexes remain intact in areas devoid of adherent neutrophils (21, 22). By contrast, PECAM-1 distribution remained unaffected (22).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(TNF
) and
N-formyl-Met-Leu-Phe (fMLP) were from Roche Molecular
Biochemicals (Meylan, France) and Sigma, respectively.
-aminocaproic acid
N-hydroxysuccimide ester according to the instructions from
the supplier (Roche Molecular Biochemicals).
for 24 h and then washed with
PBS. Neutrophils, suspended in M199 medium containing 10 µM fMLP, were then added to monolayers at final
leukocyte:endothelial cell ratios of either 10:1 (Fig. 3) or 4:1 (Figs.
4 and 5) and incubated for 1 h at 37 °C. Under this condition,
the integrity of endothelial cell monolayers was preserved. In some
experiments, neutrophils were layered on resting endothelial cells.
Following an incubation of 1 h, cell culture media containing
nonadherent neutrophils were collected and gently centrifuged (300 × g, 10 min) to avoid neutrophil crushing. Supernatants
were separated from neutrophil cells and then tested for their capacity
to cleave the recombinant fragment VE-EC1-4.
8 M) and TNF
(100 units/ml) as previously described (36). Following stimulation,
neutrophils were fixed for 3 min at 4 °C with PBS containing 3%
paraformaldehyde (w/v) and 1% glutaraldehyde (v/v). Neutrophils were
then centrifuged at 300 × g for 5 min, washed three
times in PBS, and resuspended in cell culture medium M199 without
serum. Either fixed neutrophils (105 cells) or purified
human leukocyte elastase (7 × 10
4 units) or
purified human leukocyte cathepsin G (7 × 10
4
units) were added to the recombinant fragment VE-EC1-4 (5 µg) in the
presence or absence of either SLPI (10
10 mol) or
MeOSuc-AAPV (10
10 mol) and incubated at 37 °C for
3 h. Following centrifugation at 300 × g for 5 min, the cell-free supernatants were mixed with
-mercaptoethanol
containing Laemmli buffer and subjected to SDS-polyacrylamide electrophoresis. Western blots were then performed as described above.
2
units/ml), purified cathepsin G (2 × 10
2 units/ml),
or cell culture supernatant (see above). At the same time, horseradish
peroxidase-linked goat IgG (Rockland, Gilberville, PA) was added to the
upper compartment of each Transwell unit. After various incubation
times, the medium in the lower compartments was assayed photometrically
for the presence of peroxidase with o-phenyldiamine
dihydrochloride (Dako, Glostrup, Denmark) as a substrate according to
the instructions from the supplier. Three individual Transwell units
were used for each incubation time. To verify whether protease
inhibitors modify endothelial permeability, SLPI (4 × 10
10 mol/Transwell insert) or MeOSuc-AAPV (2 × 10
6 mol/Transwell insert) were added to the upper
compartments of Transwell units and the kinetic of marker migration
across endothelial monolayers was established as described above.
, and 10
8 M fMLP. In these conditions,
proteases are translocated from azurophil granules to the external
surface of neutrophils (36). In some assays, to inhibit surface
proteases, activated neutrophils were incubated with either SLPI
(2.5 × 10
10 mol/assay) or MeOSuc-AAPV (2 × 10
6 mol/assay) during 2 h at 37 °C. Then,
activated neutrophils, treated or not with inhibitors, were rinsed,
washed twice in PBS, and diluted in M199 medium containing 20% fetal
bovine serum before their addition to TNF
-activated (5 × 104 cells/insert). To create a chemotactic gradient for
neutrophils, 2 × 10
8 M fMLP in M199 was
added to the lower compartments of the Transwell units. To evaluate the
number of transmigrated neutrophils, the Transwell units devoid of
their upper compartments were centrifuged at 4 °C for 10 min at
300 × g. The supernatants were discarded and the cell
pellet lysed in experimental conditions allowing the extraction of
neutrophil DNA using the high pure template kit (Roche Molecular
Biochemicals). The number of transmigrated neutrophils was expressed as
a function of the amount of neutrophil DNA, quantified by measuring
optical density at 260 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-activated endothelial cell monolayers. In
these conditions, neutrophils could adhere onto endothelial cells.
Following this adhesion, we observe VE cadherin fragments in the cell
culture supernatants using an immunocapture assay (Fig.
1). As indicated in Fig. 1A,
the amount of soluble VE cadherin fragments increased as contact time
between endothelial cells and neutrophils increased. Moreover, the
level of VE cadherin accumulated in the cell culture supernatant could
be correlated with the neutrophil/HUVEC ratio (Fig. 1B).
View larger version (15K):
[in a new window]
Fig. 1.
Detection of soluble VE cadherin-derived
fragments in cell culture supernatants following adhesion of
neutrophils on endothelial cell monolayers. A, increase of
the amount of VE cadherin fragments with contact time between
endothelial cells and neutrophils. Neutrophils were incubated on
TNF -activated HUVEC monolayers during 0, 1, or 2 h at 37 °C
using a neutrophil/HUVEC ratio of 10. The immunocapture assay was
performed using the antibodies anti-EC1 for coating and biotinylated
anti-Cad3 for detection of soluble VE cadherin. B, increase
of the amount of soluble VE cadherin with increasing neutrophil/HUVEC
ratio. Neutrophils were incubated on TNF
-activated HUVEC monolayers
during 1 h at 37 °C using neutrophil/HUVEC ratios of 0, 10, and
15. Soluble VE cadherin fragments were detected using the immunocapture
assay described in panel A. In panels
A and B, bars at the left
correspond to the background absorption.
in this process, the content of soluble VE cadherin in supernatants derived from endothelial cells either activated or not by this cytokine was quantified. Slightly more soluble VE cadherin fragments were detected in TNF
-activated supernatants when compared with supernatants from
untreated cells. To test the impact of neutrophil adhesion to
endothelial cells, the content of VE cadherin fragments in supernatants
from TNF
-activated endothelial cells on which neutrophils had
adhered was measured. Results showed that adhesion of neutrophils to
TNF
-activated cells significantly increased the amount of VE
cadherin detected in the supernatants. In contrast, a very low amount
of VE cadherin fragments was detected in supernatants derived from
cells on which neutrophils had not adhered (i.e. in assays
in which neutrophils were layered on resting endothelial cells).
View larger version (20K):
[in a new window]
Fig. 2.
Relationship between adhesion of neutrophils
on endothelial cell monolayers and accumulation of soluble fragments of
VE cadherin in cell culture supernatants. A, schematic
representation of VE cadherin and recombinant fragments used to raise
the polyclonal antibodies anti-Cad1, anti-EC1, anti-EC4, and anti-Cad3.
The black bar in Cad1 and
Cad3 represents an N-terminal fusion peptide. B,
detection of soluble fragments of VE cadherin using various ELISAs.
Neutrophils were added to either TNF -activated (black) or
non-activated (light gray) endothelial cell
monolayers. After a 1-h contact period at 37 °C, supernatants were
collected and their content in VE cadherin-derived fragments analyzed
using ELISA. For comparison, supernatants derived from TNF
-activated
(dark gray) and non-activated (white)
endothelial cell monolayers without neutrophil treatment were also
collected and analyzed in parallel. ELISAs were performed using five
different sets of polyclonal antibody pairs; capture of VE cadherin
fragments was obtained with different polyclonal anti-VE cadherin
antibodies (1 and 2, anti-EC1; 3 and
4, anti-Cad3; 5, anti-EC4); detection of the
bound fragments was then performed using various biotinylated anti-VE
cadherin antibodies (1, 3, and 5,
anti-Cad3; 2 and 4, anti-Cad1).
-activated endothelial cells induces
the cleavage of the extracellular part of VE cadherin.
-activated endothelial monolayers activated neutrophil
proteases, which could release extracellular fragments of VE cadherin
from the cell surface into the culture medium.
or fMLP or both gave
supernatants that did not induce a significant degradation of the
recombinant fragment (Fig. 3,
lanes 2-4). Moreover, supernatants obtained
after addition of neutrophils to non-activated endothelial cells left
the recombinant fragment intact, indicating that no active protease was
present in this condition (Fig. 3, lane 5). In
contrast, a complete degradation of the fragment was observed with
supernatants derived from endothelial cells on which neutrophils had
adhered (Fig. 3, lane 6). This suggested that
adhesion of neutrophils on endothelial cells caused a secretion and/or
an activation of proteases in the cell culture supernatant.
View larger version (41K):
[in a new window]
Fig. 3.
Supernatant-induced proteolysis of the
recombinant fragment VE-EC1-4. The recombinant fragment VE-EC1-4
(10 µM) was mixed with endothelial cell supernatants and
incubated at 37 °C for 1 h. The mixtures were electrophoresed
on a 7.5% polyacrylamide gel and transferred on nitrocellulose
membranes before probing with the polyclonal antibody anti-Cad3 (Fig.
2A). Supernatants were obtained from TNF -treated
(lanes 2, 4, and 6) or
non-treated (lanes 1, 3, and
5) endothelial cells in the presence (lanes
3, 4, and 6) or in the absence
(lanes 1, 2, and 5) of
fMLP. Lanes 5 and 6 correspond to
supernatants derived from endothelial cells to which neutrophils had
been added (neutrophils/endothelial cell ratio = 10).
View larger version (50K):
[in a new window]
Fig. 4.
Nature of the supernatant-containing
proteases. Supernatants used in panels A and
B of this figure were obtained in conditions identical to
those depicted in Fig. 3 (lane 6). The molecular
masses of the markers are indicated at the left margin in
kDa. A, time course of supernatant-induced proteolysis of
VE-EC1-4. Supernatants were diluted to 1/3 and mixed with VE-EC1-4
(final concentration, 3 µM) (lanes
2, 4, and 6). In control experiments,
buffer was mixed with VE-EC1-4 (lanes 1,
3, and 5). The mixtures were then incubated at
37 °C during different periods of time (0 h, lanes
1 and 2; 2 h, lanes 3 and 4; 4 h, lanes 5 and
6). B, supernatant-induced proteolysis is blocked
by the protease inhibitor SLPI. VE-EC1-4 (3 µM) was
mixed either with supernatant (dilution 1/3) (lanes
4-9) or, in controls, with buffer (lanes
1-3). The various mixtures were incubated at 37 °C
during 0 h (lanes 1, 4, and
7), 2 h (lanes 2, 5,
and 8), and 4 h (lanes 3,
6, and 9). SLPI was added at 0.5 µg/ml to the
mixtures (lanes 7, 8, and 9).
C, VE-EC1-4 digestion by purified elastase. VE-EC1-4 (3 µM) was mixed either with purified elastase (0.02 unit/ml) (lanes 4-9) or, in controls, with
buffer (lanes 1-3). The various mixtures were
incubated at 37 °C during 0 h (lanes 1,
4, and 7), 2 h (lanes
2, 5, and 8), and 4 h
(lanes 3, 6, and 9). SLPI
was added at 0.5 µg/ml to the mixtures (lanes
7-9). D, VE-EC1-4 digestion by purified
cathepsin G, VE-EC1-4 (3 µM) was mixed either with
purified cathepsin G (0.02 unit/ml) (lanes 4-9)
or, in controls, with buffer (lanes 1-3). The
various mixtures were incubated at 37 °C during 0 h
(lanes 1, 4, and 7), 2 h (lanes 2, 5, and 8), and
4 h (lanes 3, 6, and
9). SLPI was added at 0.5 µg/ml to the mixtures
(lanes 7-9). Western blot analysis was then
performed as described in Fig. 3.
View larger version (33K):
[in a new window]
Fig. 5.
Presence of elastase within cell
supernatants. Supernatants used in this figure were obtained in
conditions identical to those depicted in Fig. 3 (lane 6).
VE-EC1-4 (3 µM) was mixed either with purified elastase
(0.02 unit/ml) (lanes 3-6) or with purified
cathepsin G (0.02 unit/ml) (lanes 11-14) or with
supernatant (dilution 1/3) (lanes 7-10) or, in
control assays, with buffer (lanes 1 and
2) and incubated at 37 °C during 0 h
(lanes 1, 3, 5,
7, 9, 11, and 13) or 2 h (lanes 2, 4, 6,
8, 10, 12, and 14). The
specific inhibitor of elastase MeOSuc-AAPV was added at a final
concentration of 0.5 mM (lanes 5,
6, 9, 10, 13, and
14). Detection of the proteolytic fragments was performed as
described in Fig. 4.
Identification by N-terminal sequencing and MALDI mass spectrometry of
the proteolytic peptides derived from VE-EC1-4 digested with either
purified elastase or purified cathepsin G or cell supernatant
indicates the position of the cleavage site.
The calculated molecular weights of the proteolytic fragments were
determined using the computer program Protparam Tools of the Expasy
server (www.expasy.ch/tools/protparam.html). N.D., not determined.
View larger version (20K):
[in a new window]
Fig. 6.
Identification of elastase by MALDI mass
spectrometry as the only protease released into the cell culture medium
following adhesion of neutrophils on endothelial monolayers.
Supernatants used in this figure were obtained in conditions identical
to those depicted in Fig. 3 (lane 6). VE-EC1-4 (3 µM) was mixed with purified elastase (0.02 unit/ml,
panel A), with purified cathepsin G (0.02 unit/ml, panel B), or with supernatant (dilution
1/3, panel C) and incubated at 37 °C during
2 h. Following proteolysis, the various mixtures were analyzed by
MALDI mass spectrometry. In panel B, based on
band intensity variations, the 19,666- and 16,476-Da products were
probably generated from the 22,606-Da fragment. D, prior to
the addition to VE-EC1-4, the diluted supernatant (1/3) was mixed with
purified cathepsin G (0.02 unit/ml). Incubation and mass spectrometry
were performed as previously described in panels
A-C. The inset presents an enlargement of the
mass spectrum corresponding to VE-EC1-4 digestions either by the
supernatant alone (dotted line) or by the
cathepsin G-treated supernatant (continuous
line). The arrow indicates an additional
proteolytic product resulting from the addition of cathepsin G to the
supernatant. The asterisk marks an artifact resulting from
the use of matrix-containing sinapinic acid.
induces translocation of elastase and cathepsin
G from the azurophil granules to the external surface plasma membrane
of neutrophils (36, 38, 39). These surface-bound proteases were
demonstrated to retain their enzymatic activity.
, were first fixed with
paraformaldehyde and glutaraldehyde and subsequently incubated with the
VE-EC1-4 fragment. Western blot analysis showed that the resulting
digestion pattern was comparable with that previously observed using
purified elastase or cathepsin G (Fig.
7A, lanes
2, 5, and 8). Indeed, the typical
fragments of 38 and 27 kDa generated by leukocyte enzymes were clearly
detected, indicating the presence of active elastase or cathepsin G at
the cell surface of neutrophils.
View larger version (38K):
[in a new window]
Fig. 7.
Proteolysis of VE-EC1-4 by elastase and
cathepsin G bound to the leukocyte cell surface. A, Western
blot analysis of VE-EC1-4 proteolysis. Neutrophils were incubated with
10 8 M fMLP and 100 units/ml TNF
for 30 min
and then fixed. Fragment VE-EC1-4 (5 µg) was incubated for 3 h
at 37 °C with purified elastase (7 × 10
4 units)
(lanes 2-4), purified cathepsin G (7 × 10
4 units) (lanes 5-7), or fixed
neutrophils (105 cells) (lane 8). The
leukocyte protease inhibitor SLPI (10
10 mol)
(lanes 3 and 6) or the
elastase-specific inhibitor MeOSuc-AAPV (10
10 mol)
(lanes 4 and 7) were added.
Lane 1 corresponds to VE-EC1-4 alone. Note the
38- and 27-kDa immunoblotted bands in the presence of fixed
neutrophils. B, MALDI mass spectrometry analysis of the
VE-EC1-4-derived proteolytic products. Mixtures containing both fixed
neutrophils and VE-EC1-4 were centrifuged and the supernatants
collected. MALDI analysis performed on these supernatants showed a
double peak (*) representing proteolytic fragments with
molecular masses of 27 kDa and a 49-kDa peak corresponding to residual
uncleaved VE-EC1-4 (**). The inset shows an
enlargement of the 27-kDa doublet (*).
View larger version (13K):
[in a new window]
Fig. 8.
Time course of transendothelial permeability
induced by either elastase or cathepsin G or cell supernatants.
Supernatants used in this figure were obtained in conditions identical
to those depicted in Fig. 3 (lane 6). Cell permeability was
quantified by measuring the transmigration of peroxidase-conjugated
anti-mouse IgG across endothelial cell monolayers seeded on porous
Transwell filters. Practically, once cells reached confluence, mixtures
of either elastase (2 × 10 2 units/ml) and marker
(panel A, curve 1) or
cathepsin G (2 × 10
2 units/ml) and marker
(panel B, curve 1) or
supernatant and marker (panel C, curve
1) were added to the upper compartments of Transwell units.
In the controls (curves 2), only the marker was
added. Optical density represents the amount of marker accumulated for
the different incubation times in the lower compartments. They
correspond to mean values obtained from three similarly treated
independent Transwell units.
. Prior to their addition to endothelial cell
monolayers, freshly purified neutrophils underwent specific treatments.
First, they were treated with both TNF
and fMLP in such a way that
maximal amounts of elastase and cathepsin G were translocated at the
neutrophil external membrane (36); second, to block the surface-bound
proteases, TNF
- and fMLP-stimulated neutrophils were incubated with
either SLPI or MeOSuc-AAPV. This procedure was necessary to avoid
direct contact of SLPI with endothelial cells, an event that increases by itself the permeability of the endothelial monolayers (data not shown).
and fMLP increases the amount of these proteolytic
fragments slightly (Fig. 9A, lane 3).
By contrast, they disappear when VE-EC1-4 was incubated with either
SLPI- or MeOSuc-AAPV-treated neutrophils (Fig. 9A,
lanes 4 and 5). This indicates that
the addition of protease inhibitors fully hampers protease activity at
the cell surface of TNF
- and fMLP-stimulated neutrophils. However, a
subsequent 30-min incubation of the SLPI-treated neutrophils with
TNF
induced a new translocation of proteases at the neutrophil cell
surface. Indeed, after this second contact with TNF
, the 38- and
27-kDa fragments reappeared as illustrated in Fig. 9B
(lanes 1-3).
View larger version (21K):
[in a new window]
Fig. 9.
Inhibition of neutrophil transmigration by
protease inhibitors. A, inhibition of proteases at the
neutrophil cell surface. The neutrophils were stimulated with both
TNF (100 units/ml) and fMLP (10
8 M) during
30 min at 37 °C. They were then rinsed twice with PBS and incubated
with SLPI (2.5 × 10
10 mol) (lane
4), MeOSuc-AAPV (2 × 10
6 mol)
(lane 5), or PBS (lane 3)
for an additional 2 h. TNF
- and fMLP-stimulated
(lanes 3-5) or non-stimulated (lane
2) neutrophils (105 cells) were fixed with
paraformaldehyde and glutaraldehyde prior to incubation with VE-EC1-4
(5 µg) for 30 min. For comparison, undigested VE-EC1-4 is shown
(lane 1). Western blot analysis was then
performed on the digested products as described in Fig. 3. In the
presence of SLPI or MeOSuc-AAPV, the 38- and 27-kDa proteolytic
products disappeared. B, stimulation of cell surface
expression of neutrophil proteases. TNF
- and fMLP-stimulated
neutrophils were treated (lane 2) or not
(lane 1) with SLPI as previously described in
panel A of this figure. TNF
- and
fMLP-stimulated neutrophils that were also treated with SLPI
(lane 2) were stimulated a second time with
TNF
and fMLP during 30 min (lane 3). The
differently treated neutrophils were then fixed as previously described
prior to incubation with VE-EC1-4 for 30 min. Western blot analysis
was then performed on the digested products as described in Fig. 3. The
second TNF
and fMLP stimulation induced the apparition of the 38- and 27-kDa proteolytic products. C, transmigration assay of
neutrophils. TNF
- and fMLP- stimulated neutrophils treated with either SLPI (lane
3) or MeOSuc-AAPV (lane 4) or
untreated with inhibitor (lane 2) were added to
the upper compartments of the Transwell units at 5 × 104 cells/insert. For comparison, non-stimulated
neutrophils were added to inactivated endothelial cell monolayers
(lane 1). 30 min after the addition of
neutrophils, the lower compartments of the Transwell units were
centrifuged and the supernatants discarded. Extraction and
quantification of DNA from the transmigrated neutrophils enabled us to
count their number. Solid bars represent the means of four
experiments, and their calculated errors are shown.
- and fMLP-stimulated neutrophils easily transmigrate
across TNF
-activated endothelial cell monolayers (Fig.
9C, lane 2). Inhibition of proteases
by SLPI or MeOSuc-AAPV significantly reduces the neutrophil
transmigration rate (Fig. 9C, lanes 3 and 4). The partial inhibition of transmigration can be the
result of reappearance of proteases at the neutrophil cell surface
following their contact with TNF
-activated endothelial cell
monolayers. Furthermore, some untreated neutrophils transmigrate across
inactivated endothelial cells (Fig. 9C, lane
1) probably attracted by the presence of fMLP in the lower
compartments of Transwell units. These results indicate that elastase
and cathepsin G localized at the neutrophil cell surface participate in
the transmigration process probably by locally cleaving VE cadherin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and fMLP, elastase and
cathepsin G are present at the cell surface whereas they are mainly
stored within internal azurophil granules in resting cells (36). Our
data indicate that both cell surface-bound enzymes are active and
sufficiently accessible to be able to cleave, similarly to their
soluble forms, the VE-EC1-4 fragment. This result suggests that,
following close contact between neutrophils and endothelial cells, cell
surface proteases may locally cleave VE cadherin.
1-antitrypsin or SLPI within the blood. Indeed, in
zones of close contact between neutrophils and endothelial cells, the
concentration of soluble elastase is several orders of magnitude higher
than the serum concentration of inhibitors. Consequently, the soluble
enzymes may transiently overwhelm the local inhibitor concentration,
thus allowing VE cadherin proteolysis to take place (41). Moreover, it
is possible that membrane-bound elastase and cathepsin G may be more
resistant to inhibition when compared with secreted elastase,
inhibitors being unable to reach the zones of tight adhesion between
neutrophils and endothelial cells (31). The action of these
physiological inhibitors confines the activity of elastase and
cathepsin G to the very sites of neutrophil adhesion on endothelial
cells and thus limits their damaging activity.
L
2 and the endothelial immunoglobulin
superfamily proteins such as JAM-1 (42) and ICAM-1. Homophilic
interactions mediated by PECAM-1 (43) and CD99 (7) act in concert to
facilitate the progression of the leukocyte across the adherens
junctions. During this transendothelial migration, VE cadherin moves
away to different ends of the transmigration site (4, 29). The event
initiating the lateral movement of VE cadherin may correspond to the
cleavage of this adhesive receptor by surface-bound elastase or/and
cathepsin G. The subsequent disruption of its hexameric assembly might
open the way at the front of leukocyte migration.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to staff of Hôpital Sud (Grenoble, France) for kindly collecting umbilical cords for these experiments.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant 4447 from the Association pour la Recherche sur le Cancer and from Groupement des Entreprises Françaises dans la Lutte contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from Association de Recherche sur la Polyarthrite.
Current address: European Molecular Biology
Laboratory, 38 042, Grenoble, France.
** To whom correspondence should be addressed. E-mail: gulino@ibs.fr.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M300351200
2 S. Bibert, E. Concord, E. Hewat, B. Dublet, T. Vernet, and D. Gulino-Debrac, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PECAM, platelet-endothelial cell adhesion molecule;
VE cadherin, vascular
endothelium cadherin;
CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid;
ELISA, enzyme-linked immunosorbent assay;
HUVEC, human umbilical
vein endothelial cell;
fMLP, formyl-Met-Leu-Phe;
MALDI, matrix-assisted
laser desorption;
SLPI, secretory leukocyte protease inhibitor;
TNF, tumor necrosis factor
;
MeOSuc-AAPV, N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone;
PBS, phosphate-buffered saline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Worthylake, R. A., and Burridge, K. (2001) Curr. Opin. Cell Biol. 13, 569-577[CrossRef][Medline] [Order article via Infotrieve] |
2. | Dunon, D., Piali, L., and Imhof, B. A. (1996) Curr. Opin. Cell Biol. 8, 714-723[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Johnson-Leger, C.,
Aurrand-Lions, M.,
and Imhof, B. A.
(2000)
J. Cell Sci.
113,
921-933 |
4. |
Shaw, S. K.,
Bamba, P. S.,
Perkins, B. N.,
and Luscinskas, F. W.
(2001)
J. Immunol.
167,
2323-2330 |
5. |
Feng, D.,
Nagy, J. A.,
Pyne, K.,
Dvorak, H. F.,
and Dvorak, A. M.
(1998)
J. Exp. Med.
187,
903-915 |
6. |
Newman, P. J.
(1999)
J. Clin. Invest.
103,
5-9 |
7. | Schenkel, A. R., Mamdouh, Z., Chen, X., Liebman, R. M., and Muller, W. A. (2002) Nat. Immunol. 3, 143-150[CrossRef][Medline] [Order article via Infotrieve] |
8. | Rival, Y., Del Maschio, A., Rabiet, M. J., Dejana, E., and Duperray, A. (1996) J. Immunol. 157, 1233-1241[Abstract] |
9. | Vaporciyan, A. A., DeLisser, H. M., Yan, H. C., Mendiguren, I. I., Thom, S. R., Jones, M. L., Ward, P. A., and Albelda, S. M. (1993) Science 262, 1580-1582[Medline] [Order article via Infotrieve] |
10. | Lampugnani, M. G., Resnati, M., Raiteri, M., Pigott, R., Pisacane, A., Houen, G., Ruco, L. P., and Dejana, E. (1992) J. Cell Biol. 118, 1511-1522[Abstract] |
11. |
Bibert, S.,
Jaquinod, M.,
Concord, E.,
Ebel, C.,
Hewat, E.,
Vanbelle, C.,
Legrand, P.,
Weidenhaupt, M.,
Vernet, T.,
and Gulino-Debrac, D.
(2002)
J. Biol. Chem.
277,
12790-12801 |
12. |
Legrand, P.,
Bibert, S.,
Jaquinod, M.,
Ebel, C.,
Hewat, E.,
Vincent, F.,
Vanbelle, C.,
Concord, E.,
Vernet, T.,
and Gulino, D.
(2001)
J. Biol. Chem.
276,
3581-3588 |
13. | Ozawa, M., Baribault, H., and Kemler, R. (1989) EMBO J. 8, 1711-1717[Abstract] |
14. | Butz, S., Stappert, J., Weissig, H., and Kemler, R. (1992) Science 257, 1142-1144[Medline] [Order article via Infotrieve] |
15. | Jou, T. S., Stewart, D. B., Stappert, J., Nelson, W. J., and Marrs, J. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5067-5071[Abstract] |
16. | Rimm, D. L., Koslov, E. R., Kebriaei, P., Cianci, C. D., and Morrow, J. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8813-8817[Abstract] |
17. | Ferber, A., Yaen, C., Sarmiento, E., and Martinez, J. (2002) Exp. Cell Res. 274, 35-44[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Gulino, D.,
Delachanal, E.,
Concord, E.,
Genoux, Y.,
Morand, B.,
Valiron, M. O.,
Sulpice, E.,
Scaife, R.,
Alemany, M.,
and Vernet, T.
(1998)
J. Biol. Chem.
273,
29786-29793 |
19. |
Corada, M.,
Mariotti, M.,
Thurston, G.,
Smith, K.,
Kunkel, R.,
Brockhaus, M.,
Lampugnani, M. G.,
Martin-Padura, I.,
Stoppacciaro, A.,
Ruco, L.,
McDonald, D. M.,
Ward, P. A.,
and Dejana, E.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
9815-9820 |
20. | Hordijk, P. L., Anthony, E., Mul, F. P., Rientsma, R., Oomen, L. C., and Roos, D. (1999) J. Cell Sci. 112 12, 1915-1923 |
21. |
Allport, J. R.,
Ding, H.,
Collins, T.,
Gerritsen, M. E.,
and Luscinskas, F. W.
(1997)
J. Exp. Med.
186,
517-527 |
22. | Del Maschio, A., Zanetti, A., Corada, M., Rival, Y., Ruco, L., Lampugnani, M. G., and Dejana, E. (1996) J. Cell Biol. 135, 497-510[Abstract] |
23. |
Rabiet, M. J.,
Plantier, J. L.,
Rival, Y.,
Genoux, Y.,
Lampugnani, M. G.,
and Dejana, E.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
488-496 |
24. | Carmeliet, P. (2000) Nat. Med. 6, 389-395[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Shay-Salit, A.,
Shushy, M.,
Wolfovitz, E.,
Yahav, H.,
Breviario, F.,
Dejana, E.,
and Resnick, N.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
9462-9467 |
26. |
Nawroth, R.,
Poell, G.,
Ranft, A.,
Kloep, S.,
Samulowitz, U.,
Fachinger, G.,
Golding, M.,
Shima, D. T.,
Deutsch, U.,
and Vestweber, D.
(2002)
EMBO J.
21,
4885-4895 |
27. |
Ukropec, J. A.,
Hollinger, M. K.,
Salva, S. M.,
and Woolkalis, M. J.
(2000)
J. Biol. Chem.
275,
5983-5986 |
28. | Ukropec, J. A., Hollinger, M. K., and Woolkalis, M. J. (2002) Exp. Cell Res. 273, 240-247[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Su, W. H.,
Chen, H. I.,
and Jen, C. J.
(2002)
Blood
100,
3597-3603 |
30. |
Cepinskas, G.,
Sandig, M.,
and Kvietys, P. R.
(1999)
J. Cell Sci.
112,
1937-1945 |
31. | Owen, C. A., and Campbell, E. J. (1995) Semin. Cell Biol. 6, 367-376[Medline] [Order article via Infotrieve] |
32. |
Moll, T.,
Dejana, E.,
and Vestweber, D.
(1998)
J. Cell Biol.
140,
403-407 |
33. |
Allport, J. R.,
Lim, Y. C.,
Shipley, J. M.,
Senior, R. M.,
Shapiro, S. D.,
Matsuyoshi, N.,
Vestweber, D.,
and Luscinskas, F. W.
(2002)
J. Leukocyte Biol.
71,
821-828 |
34. | Carden, D., Xiao, F., Moak, C., Willis, B. H., Robinson-Jackson, S., and Alexander, S. (1998) Am. J. Physiol. 275, H385-H392[Medline] [Order article via Infotrieve] |
35. | Weiss, J., Kao, L., Victor, M., and Elsbach, P. (1985) J. Clin. Invest. 76, 206-212[Medline] [Order article via Infotrieve] |
36. | Owen, C. A., Campbell, M. A., Sannes, P. L., Boukedes, S. S., and Campbell, E. J. (1995) J. Cell Biol. 131, 775-789[Abstract] |
37. | Weinrauch, Y., Drugan, D., Shapiro, S. D., Weiss, J., and Zychlinsky, A. (2002) Nature 417, 91-94[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Campbell, E. J.,
Campbell, M. A.,
and Owen, C. A.
(2000)
J. Immunol.
165,
3366-3374 |
39. | Owen, C. A., Campbell, M. A., Boukedes, S. S., and Campbell, E. J. (1995) J. Immunol. 155, 5803-5810[Abstract] |
40. | Owen, C. A., Campbell, M. A., Boukedes, S. S., and Campbell, E. J. (1997) Am. J. Physiol. 272, L385-L393[Medline] [Order article via Infotrieve] |
41. | Stockley, R. A. (2001) Novartis Found. Symp. Disc. 234, 189-204 |
42. | Ostermann, G., Weber, K. S., Zernecke, A., Schroder, A., and Weber, C. (2002) Nat. Immunol. 3, 151-158[CrossRef][Medline] [Order article via Infotrieve] |
43. | Vestweber, D. (2002) Curr. Opin. Cell Biol. 14, 587-593[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Gotsch, U.,
Borges, E.,
Bosse, R.,
Boggemeyer, E.,
Simon, M.,
Mossmann, H.,
and Vestweber, D.
(1997)
J. Cell Sci.
110,
583-588 |