* Division of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide,
South Australia, 5000 Australia; Vascular Biology Laboratory, Baker Medical Research Institute, Prahran Victoria 3181; and § Director of Lung Research, Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania
Medical Center, Philadelphia, Pennsylvania 19104-4283
Tumor necrosis factor-, interleukin-1, and
endotoxin stimulate the expression of vascular endothelial cell (EC) adhesion molecules. Here we describe
a novel pathway of adhesion molecule induction that is
independent of exogenous factors, but which is dependent on integrin signaling and cell-cell interactions. Cells plated onto gelatin, fibronectin, collagen or fibrinogen, or anti-integrin antibodies, expressed increased
amounts of E-selectin, vascular cell adhesion molecule-1,
and intercellular adhesion molecule-1. In contrast, ECs
failed to express E-selectin when plated on poly-L-lysine
or when plated on fibrinogen in the presence of attachment-inhibiting, cyclic Arg-Gly-Asp peptides. The duration and magnitude of adhesion molecule expression
was dependent on EC density. Induction of E-selectin
on ECs plated at confluent density was transient and returned to basal levels by 15 h after plating when only 7 ± 2% (n = 5) of cells were positive. In contrast, cells
plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 ± 4%
(n = 5) positive for E-selectin expression 15 h after
plating, and significant expression still evident 72 h after plating. The confluency-dependent inhibition of expression of E-selectin was at least partly mediated through the cell junctional protein, platelet/endothelial
cell adhesion molecule-1 (PECAM-1). Antibodies
against PECAM-1, but not against VE-cadherin, increased E-selectin expression on confluent ECs. Co-
culture of subconfluent ECs with PECAM-1- coated
beads or with L cells transfected with full-length PECAM-1 or with a cytoplasmic truncation PECAM-1
mutant, inhibited E-selectin expression. In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant
failed to regulate E-selectin expression. In an in vitro
model of wounding the wound front displayed an increase in the number of E-selectin-expressing cells, and
also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer. Thus we propose that the EC junction, and in
particular, the junctional molecule PECAM-1, is a powerful regulator of endothelial adhesiveness.
THE endothelial lining of the vascular system normally displays a nonactivated, nonadhesive phenotype. Stimulation with agents such as tumor necrosis
factor- The induction of E-selectin expression on endothelial
cells (ECs) in vitro after cytokine stimulation is transient
and independent of the continued presence of the stimulant (Pober et al., 1986a In contrast to the transiency of E-selectin and VCAM
expression demonstrated by the in vitro data, these antigens have been detected on venular endothelium in chronic
inflammatory lesions, such as the synovium in rheumatoid
arthritis (Koch et al., 1991 Since sites of inflammation are often associated with
morphological changes including cell retraction of the endothelium (Schumacher, 1973 Cell Culture
ECs were extracted from human umbilical veins by collagenase treatment,
according to a modified method of Wall et al. (1978) Antibodies
Mouse mAbs directed against E-selectin (49-1B11), VCAM-1 (51-10C9),
PECAM-1 (51-9H6, 51-6F6, 55-3D2), VE-cadherin (55-7H1), integrin Flow Cytometry
Flow cytometric analysis of in situ endothelial monolayers was performed
as previously described (Gamble et al., 1993 A minimum of 1,000 events per test was analyzed using an EPICS Profile II (Coulter Immunology, Hialeah, FL). Results of individual EC lines
are expressed either as a plot of frequency versus log fluorescence, or as
the mean fluorescence channel number, subtracting the accompanying
value for the negative control Ig. When results from multiple EC lines
have been pooled, the mean fluorescence intensity (MFI) represents n cell
lines.
In cocultures of ECs and L cells prepared for flow cytometric analysis,
the cells were stained with anti-VE-cadherin, detected with phycoerythrin
(PE)-conjugated anti-mouse F(ab Immunofluorescence Confocal Microscopy
Confocal microscopy was performed on ECs cultured on fibronectin-coated glass or Permanox® chamber slides. A staining three layer method
was used in wounding assays and involved initial fixation in ice-cold methanol for 5 min and then acetone 1 min and washing in M199-containing
2.5% FCS. Cells were stained with saturating amounts of mAb for 30 min
at 25°C. After two washes, cells were incubated with biotin-conjugated, affinity-purified anti-mouse Ig (Vector Labs, Burlingame, CA), washed
twice, and incubated with avidin-FITC (Dako Corp., Carpinteria, CA).
Slides were mounted using 2% propylgallate in glycerol as an anti-fade
agent. Images were captured with a laser scanning confocal microscope
(MRC600; Bio Rad Laboratories, Hercules, CA). Comparison images
were subjected to equivalent amounts of contrast enhancement.
Neutrophil-Endothelial Adhesion
Peripheral blood from normal volunteers was sedimented on dextran, followed by density-gradient centrifugation on Lymphoprep (Nycomed,
Oslo, Norway) at 450 g. Contaminating erythrocytes were then lysed by
hypotonic 0.2% sodium chloride. Cells were resuspended in RPMI-1640
with 2.5% FCS and yielded a purity of >98%. 5 × 105 neutrophils were
added in 125 µl medium to human umbilical vein endothelial cells (HUVECs), which had been plated 16 h earlier onto fibronectin-coated chamber slides at cobblestone and subconfluent densities. After 25 min at 37°C
in a humidified 5% CO2 in air atmosphere, the wells were washed three
times removing unattached neutrophils. The slides were fixed in 0.5% glutaraldehyde, examined by confocal microscopy, and the number of neutrophils attached per EC counted. At least 160 ECs were assessed.
Preparation of Protein-coupled Beads
Tosyl-activated paramagnetic beads (Dynabeads M-450; DYNAL A.S.,
Oslo, Norway) were coated with purified platelet PECAM-1 as previously
described (Plopper and Ingber, 1993 Endotoxin Assay
A quantitative, photometric assay (Coatest; Kabi Diagnostica, Stockholm,
Sweden) based upon activation of a proenzyme in limulus amoebocyte lysate was used, which detected endotoxin at 0.1-1.2 EU/ml.
Cytokines and Cytokine Antagonists
TNF- Reagents and Peptides
Polymyxin B sulfate (Sigma Chemical Co., St. Louis, MO) was used at 10 µg/ml. When added at plating, it effectively abolished induction of E-selectin
by LPS on ECs. Soluble PECAM-1 protein was purified from platelet and
was used at 0.01-100 µg/ml. Cyclic RGD and RAD peptides (EMD66203,
67679, 69601) were kindly supplied by A. Jonczyk from Merck KGaA
(Darmstadt, Germany). These peptides were identical to those used by
Brooks et al. (1994) Enumeration of EC Contacts
Multiple photomicrographs of low power, phase contrast fields (see Fig. 1)
were obtained of ECs plated at 0.25 and 105 cells per cm2. The number of
cell contacts made with adjoining ECs were counted for 10 ECs per field.
Wounding EC Monolayers
ECs plated at cobblestone density on fibronectin-coated chamber slides
were wounded by scraping with the tip of a 1,000-µl pipette. The wells
were washed three times with medium. All wounds consisted of a clearly
demarcated cross in the center of a monolayer and healed as an advancing
front of elongated, flattened cells. At specified times after wounding, cells
were stained for immunofluorescence confocal microscopy as described
above. Phase contrast microscopy with ×100 magnification was used to select fields at wound fronts and on areas of cellular monolayer, at least two
fields were removed from a wound. Selected fields were then examined
with the laser confocal microscope at ×200 magnification and the images
stored. Images were retrieved in SETCOL format (COMOS 7.0; Bio Rad
Laboratories, Hercules, CA), which displays fluorescence intensity on a
color scale (green being minimal, red maximal), and were then examined for the number of ECs positive and negative for E-selectin per ×200 field.
An EC was determined to be positive if any part of its surface, >5 mm in
diam was red. At least 1,700 ECs were counted at wounds or monolayers
per well and positivity was expressed as a percentage of all cells counted.
The proportion of ECs expressing E-selectin in the top 50% range of expression intensity was assessed by analysis of pixel intensity in areas of
monolayer and wound front and calculated using the histogram format.
Statistics
The statistical significance of results was assessed using the two-tailed Student's t test with either paired or unpaired groups of data as indicated.
The frequency histograms of neutrophil adherence to ECs were compared
by the Kolmogorov-Smirnov test and the effects of PECAM-1-coated
beads on E-selectin expression by the analysis of variance (ANOVA) test.
Confluency-dependent Expression of Endothelial
Adhesion Molecules
HUVECs were seeded onto gelatin-coated tissue culture
plates at varying numbers (Fig. 1). 15 h after plating, the
EC surface expression of E-selectin was measured by flow
cytometry. Confluent, cobblestone cultures as seen in Fig.
1 a, showed negligible E-selectin expression (Fig. 2 a). This
is in agreement with published work demonstrating a lack
of E-selectin on unstimulated ECs (Pober et al., 1986b
Time Course of E-selectin Expression
The expression of E-selectin was measured on ECs at
varying times after plating. Fig. 3 shows that cells plated at
high and low densities both displayed significant levels of
E-selectin early after plating that is within 4 to 12 h. However, expression of E-selectin on high density ECs was
fourfold less at its maximum 8 h after plating, was transient and returned to basal levels by 24 h. In contrast,
E-selectin expression on ECs plated at low density peaked
at 12 h after plating and was still evident at 32 h. ECs at
very sparse densities, such that single cells were maintained over the course of the experiment, displayed a persistent and significant expression of E-selectin even 72 h
after plating (data not shown). There was considerable
variation in the absolute levels of E-selectin between different EC preparations, the reason for which is not known.
However, the fold induction calculated between high and
low density cells was relatively consistent (21.1 ± 3.6%,
n = 30) when measured 16-20 h after plating.
VCAM-1 and ICAM-1 (as well as E-selectin) demonstrated EC density-dependent expression, but the expression of PECAM-1 (or CD31), a non-inducible adhesion
protein was not altered (Fig. 4). Increased expression of
VCAM-1, ICAM-1, and E-selectin was independent of
cell size, as identical forward scatter gates of high and low
density cells were always used in the FACS® analysis. Furthermore, the change in adhesion molecule expression was
observed, whether the cells were stained in situ before detachment, or after extraction from matrix (data not shown).
E-selectin Expression by Subconfluent
ECs Is Independent of Cytokines and Supports
Neutrophil Adhesion
The time course of expression seen in Fig. 2 suggested two
phases in the regulation of E-selectin expression: an induction phase, and a maintenance phase. Induction of the expression of adhesion molecules by subconfluent ECs occurred in the absence of exogenous cytokines. As shown in
Fig. 5 a, IL-1 receptor antagonist (IL-1ra) or blocking
anti-TNF-
Table I.
E-selectin Expression Is Not Stimulated by
Supernatant from Migrating Endothelial Cells
(TNF-
)1, interleukin-1 (IL-1), or lipopolysaccharide (LPS) are known to induce the expression of proteins
on the endothelial surface that mediate coagulation (Bevilacqua et al., 1986
), leukocyte adhesion (Bevilacqua et al.,
1985
; Gamble et al., 1985
; Pober et al., 1986b
; Doherty et
al., 1989
), and leukocyte transendothelial migration (Furie
et al., 1989; Moser et al., 1989
). The endothelial antigens
that are important for the adhesion of leukocytes are members of the selectin family, E- and P-selectin, and the
immunoglobulin gene superfamily, vascular cell adhesion
molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) (Carlos and Harlan, 1994
; Litwin et al.,
1995
).
). Previous studies have shown that
E-selectin mRNA and protein levels peak between 2 and 4 h,
respectively, after treatment with an agonist, returning to
near basal levels by 24 h (Bevilacqua et al., 1989
; Read et
al., 1994
). VCAM-1 (Osborn et al., 1989
) and ICAM-1
(Pober et al., 1986b
) are maximal 6 and 12 h, respectively, after stimulation.
), and the skin in psoriasis (Petzelbauer et al., 1994
). E-selectin expression is also detected on angiogenic vessels in human hemangiomas, a
noninflammatory angiogenic disease (Kraling et al., 1996
).
Moreover, the architecture and anatomic localization of
capillary loops influence the pattern of endothelial expression of E-selectin and VCAM-1, independently of the
availability of cytokines (Petzelbauer et al., 1994
). Thus it
is likely that alternate control mechanisms exist to allow
prolonged, locality-based expression of adhesion molecules on the endothelium. At least one of these alternate mechanisms may be flow, since increased shear stress has
been shown to selectively modulate adhesion molecule expression, upregulating ICAM-1 but not E-selectin or
VCAM-1 (Nagel et al., 1994
).
), we hypothesized that cell
contacts may be important in the regulation of endothelial
phenotype. We describe here the central role of the junctional protein, platelet/endothelial cell adhesion molecule-1 (PECAM-1), through the formation of cell-cell interactions, in the maintenance of the functional integrity
of the endothelial monolayer. Furthermore, we demonstrate a novel pathway for the induction of adhesion molecules on endothelial cells that is independent of exogenous
addition of cytokines, but is related to integrin- and cell
shape-associated signaling events.
MATERIALS AND METHODS
. Cells were grown in
25-cm2, gelatin-coated Costar flasks (Costar Corp., Cambridge, MA), maintained with endotoxin-free medium 199 (Cytosystems, Sydney, Australia), 20% FCS (Commonwealth Serum Laboratories, Melbourne, Australia), 20 mM Hepes, 2 mM glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids (Cytosystems), 0.225% sodium bicarbonate and
antibiotics in the absence of exogenously added heparin or basic fibroblast
growth factor, at 37°C in a humidified, 5% CO2 (in air) atmosphere. After
2-5 d of culture, the cells were harvested by trypsin-EDTA treatment
(Cytosystems) and replated at specified cell densities on 1.9-cm2, gelatin-
and fibronectin-coated multiwell dishes (Nunc, Roskilde, Denmark) for
flow cytometry experiments, or on fibronectin-coated glass or Permanox®
chamber slides (Labtek, Nunc, Roskilde, Denmark) for immunofluorescence and adhesion assays. The preparation of PECAM-1-transfected L cells has been reported previously (DeLisser et al., 1993
). The cells were
maintained in RPMI 1640 (GIBCO BRL, Gaithersburg, MD) 10% FCS
with 0.5 mg/ml G418.
1
(61-2C4), colony-stimulating factor (CSF), common
c chain (3D7, supplied by Q. Sun, Hanson Centre for Cancer Research [HCCR], Adelaide,
Australia), and Keyhole Limpet Hemacyanin (23-1F11) were raised at the
HCCR. The anti-PECAM-1 mAbs were functional in an assay of neutrophil transendothelial migration and anti-VE-cadherin antibody was functional in an assay of EC aggregation. For coating of wells with antibody,
plates were coated with rabbit anti-mouse Ig (50 µg/ml) for 18 h, blocked
with 1% BSA, and then anti-integrin or anti-CSF
c chain-purified antibodies added for 1 h at 37°C. Wells were then washed and further blocked
before addition of cells.
). EC monolayers were
blocked in 5% sheep serum, and then were stained with primary antibody
for 30 min at 37°C, washed twice with RPMI 1640 containing 2.5% FCS,
and then stained with FITC-conjugated, anti-mouse Ig (Fab2, DAF; Silenus Laboratories, Hawthorn, Australia) for 30 min at room temperature.
Cells were washed twice, removed by trypsin-EDTA treatment, and then
fixed in 1% formaldehyde, 0.02% azide, and 0.02% glucose. In experiments involving endothelial pretreatment with mAbs, E-selectin was detected using a single layer, FITC-conjugated, anti-E-selectin mAb (49-1B11).
)2 (DDAPE; Silenus Laboratories,
Hawthorn, Australia) and simultaneously stained with goat anti-E-selectin detected with FITC-conjugated, anti-goat antibody (Silenus Laboratories). L cells were negative for VE-cadherin, EC were 100% positive for
VE-cadherin and were selected for analysis of E-selectin (FITC staining)
using a second fluorescence detector. The flow cytometer was calibrated
using single PE- or FITC-stained cells.
). Essentially 99% of beads were
coated with PECAM-1 as assessed by flow cytometry using polyclonal
anti-PECAM-1 antibody staining.
(lot S9010AX; sp act 6.27 × 107 U/mg), TGF-
(lot 8987-53), and a
monoclonal anti-TNF were gifts from Genentech, Inc. (South San Francisco, CA) IL-1
(108 thymocyte mitogenesis U/mg) was kindly supplied
by Immunex (Seattle, WA). IL-1ra was a gift from Synergen (Boulder,
CO). All cytokines contained <3 U/ml of LPS.
in inhibiting
v
3-dependent angiogenesis.
Fig. 1.
Phase contrast photomicrographs of EC monolayers 15 h
after seeding at confluent 1.0 (a), subconfluent 0.25 (b), and
sparse 0.05 × 105 cells per cm2 densities (c). Bar, 40 µm.
[View Larger Version of this Image (101K GIF file)]
RESULTS
;
Bevilacqua et al., 1989
). However, subconfluent ECs (Fig.
1 b) displayed substantial expression of E-selectin (Fig. 2
a). A comparison between subconfluent and confluent
density cells using five separate EC lines showed a 17-fold
greater induction in subconfluent ECs, (6.4 ± 1.0 vs. 0.37 ± 0.19 mean fluorescence intensity units ± SEM, P = 0.0003, unpaired t test). In these five experiments, 57 ± 4% of ECs at subconfluent density were positive for
E-selectin, as opposed to 7 ± 2% of cells in a cobblestone monolayer. The confluency-dependent expression of E-selectin was evident over a range of EC densities (Fig. 2 b) and
was seen on HUVECs extracted from their original monolayer culture by treatment with trypsin-EDTA or EDTA
alone (data not shown).
Fig. 2.
E-selectin expression varies with EC density.
(a) Flow cytometry profile
of a representative EC line
stained for E-selectin 15 h after plating. Cells were plated
at cobblestone (, 105
cells per cm2) or subconfluent densities (thick line; 0.25 × 105 cells per cm2). (thin line)
Nonbinding control immunoglobulin, which gave a similar
profile for confluent or subconfluent cells. (b) ECs were cultured for 15 h after plating
at cell densities ranging from sparse to confluent (0.125-
2.0 × 105 cells per cm2). The
MFI ± SEM of E-selectin expression is shown for five EC lines, except for values at densities 0.5 and 2.0 × 105 cells per cm2,
which are triplicates. Asterisks indicate values significantly different (P < 0.003) from confluent density ECs (105 cells per cm2) by
unpaired t test.
[View Larger Version of this Image (20K GIF file)]
Fig. 3.
Time course of
E-selectin expression after
EC plating. ECs were plated
at confluent (, 105 cells per
cm2) and subconfluent (
,
0.25 × 105 cells per cm2) densities. The expression of
E-selectin was assayed by
flow cytometry at specified
times after plating. The MFI
(± SEM) of three to five cell
lines is shown but values at 2, 32, and 72 h after plating are singlicates. Asterisks denote values
significantly different between the two cell densities (P < 0.03)
by paired t test.
[View Larger Version of this Image (17K GIF file)]
Fig. 4.
Expression of
E-selectin, VCAM-1, and
ICAM-1 are confluency dependent. ECs plated at subconfluent density (stripes,
0.25 × 105 cells per cm2) and
confluent density (solid, 105
cells per cm2) were stained
20 h later for a, E-selectin, b,
VCAM-1, c, ICAM-1, or d,
PECAM-1. Expression is
shown as the MFI of 23, 11, 7, and 7 EC lines, respectively. Error bars represent
the SEM. Asterisks denote
values significantly different
from cells plated at cobblestone density (P < 0.03) by
paired t test.
[View Larger Version of this Image (45K GIF file)]
antibody potently and specifically inhibited
IL-1 or TNF-
-mediated induction of E-selectin, respectively, but these agents were ineffective on the induction
of E-selectin by subconfluent ECs (Fig. 5 b). Furthermore,
conditioned medium taken from subconfluent ECs or
from cells multiply wounded such that the majority of cells
were undergoing migration, did not induce E-selectin expression on confluent density ECs (Table I). Thus a role
for endogenous endothelial cytokine production, or the release of some other stimulant from the EC, appeared unlikely.
Fig. 5.
The induction of
E-selectin on subconfluent
ECs is not mediated through
TNF- or IL-1. (a) ECs
plated at subconfluent density (0.25 × 105 cells per cm2)
were stimulated with 10 U/ml
TNF-
(TNF) or 1 ng/ml
IL-1
(IL-1) in the presence
of anti-TNF-
(TNF + anti-TNF) (1:1,000), or IL-1ra (IL-1 + IL-1ra) (100 ng/ml), respectively. Inhibitors and agonists were added immediately after EC plating and E-selectin expression was assessed 12 h later. (b) ECs were plated at subconfluent
density (stripes, 0.25 × 105 cells per cm2) and confluent density
(solid, 105 cells per cm2). Anti-TNF-
or IL-1ra were added at plating. E-selectin expression was assessed 12 h later. Results shown
are of one representative experiment of four performed.
[View Larger Version of this Image (37K GIF file)]
Further, the induction of E-selectin is not related to cell
proliferation. Rapamycin, an inhibitor of the G1-S transition (Kato et al., 1994) showed no effects on E-selectin induction in subconfluent ECs (data not shown) although
TGF-
, another inhibitor of cell proliferation (Heimark et
al., 1986
), which we have shown previously to inhibit cytokine-induced E-selectin expression (Gamble et al., 1993
)
did inhibit the cytokine-independent induction of E-selectin (data not shown). Moreover, E-selectin induction was independent of cell cycling since no difference in the level
of E-selectin expression was seen in cells in G0/G1 with respect to S/G2/M at either cell density, both up to 20 h and
even at 72 h after EC plating (data not shown).
Low doses of TNF- were able to increase E-selectin expression on both low and high density ECs (Fig. 6 a) suggesting that the cytokine and non-cytokine pathways of induction are at least additive. Although the level of E-selectin
expression induced by the cytokine-independent mechanism is less than that induced by TNF or IL-1 (Figs. 5 and
6), it is however, functionally relevant. Assessment of the
number of neutrophils adherent per EC showed that there
was an increase in the number of neutrophils attached per
EC in cells plated at low versus high density EC (Fig. 6 b). In three experiments, the percentage of EC supporting the
adhesion of two or more neutrophils was 24.3% ± 14% for
low density EC and 1.4% ± 1.0% for high density EC
(mean ± SEM; P = 0.02).
E-selectin Induction Is Dependent on Integrin Engagement
ECs plated on collagen 1, gelatin, fibronectin, fibrinogen,
and laminin in BSA-containing media resulted in an upregulation of E-selectin when measured 6 h after plating
(Fig. 7 a). The level of induction on these matrices in the
absence of FCS in the media was never as high as in the
presence of FCS. E-selectin expression was not induced on
ECs plated on poly-L-lysine (Fig. 7 a) although they responded to TNF stimulation, suggesting that the poly-
L-lysine was not toxic (data not shown). The induction was
seen whether cells were plated at subconfluent density
(Fig. 7) or at high density (data not shown). Cells plated
on anti-1 integrin-coated surfaces, attached, spread, and
induced E-selectin. In contrast, cells plated on an antibody
to the common
chain (
c) of GM-CSF, IL-3, and IL-5 receptor, which is also expressed on EC (Korpelainen et al.,
1993
), attached but failed to spread and also did not result
in E-selectin induction (data not shown). Cells plated on
fibrinogen in the presence of cyclic RGD peptide also
failed to induce E-selectin (Fig. 7 b) although the peptide had no effect on E-selectin expression on cells plated on
collagen (data not shown). These results suggest integrin
engagement and possibly cell shape changes also are important in E-selectin induction.
PECAM-1 Regulates the Maintenance of Endothelial Adhesion Molecules
Although E-selectin is induced on all cells, only subconfluent EC maintain this expression. The chief difference between these density phenotypes lies in the number of cell- cell contacts and their establishment rate. ECs plated at confluent density (105 cells per cm2) were rapidly surrounded by other cells. Within 1 h they had established 5 ± 1 contacts with adjoining cells (mean ± SEM; n = 3). This number remained constant over the following 24 h. By contrast, subconfluent ECs (0.25 × 105 cells per cm2) had an average of only 1 ± 1 contact (n = 3) at 1 h after plating and continued to form new associations such that 24 h later they had 4 ± 1 contacts (n = 6).
Two molecules known to be concentrated in cell-cell
contacts and implicated in establishment of some of the
junctional properties of endothelial cell monolayers are
PECAM-1 (Albelda et al., 1991; DeLisser et al., 1994
) and
VE-cadherin (Lampugnani et al., 1992
; Ayalon et al.,
1994
). To determine whether PECAM-1 was involved in
the cytokine-independent regulation of E-selectin, three
independent methods were used. Firstly, confluent density
EC monolayers were exposed to functional antibodies directed to PECAM-1 or VE-cadherin. Monoclonal anti-
VE-cadherin antibody (antibody 7H1) had no effect. Polyclonal anti-PECAM-1 resulted in a twofold increase in
E-selectin expression (Fig. 8, a and b). Addition of both
anti-PECAM-1 and anti-VE-cadherin antibodies produced no further increase than with anti-PECAM-1 antibody alone (data not shown). Although two mAb directed
to domain one of PECAM-1 consistently and significantly enhanced E-selectin expression their activity was always
less than that seen with the polyclonal anti-PECAM-1 antibody (Fig. 8 b) suggesting the involvement of multiple
domains. 55-3D2, an mAb directed to domain two-thirds
of PECAM-1 was without function in these assays although it inhibits neutrophil transendothelial cell migration (Yan et al., 1995
). The anti-PECAM-1 antibody effect was dose dependent (maximal efficacy at 5 µg/ml) and
not due to contaminating endotoxin as polymyxin B did
not abrogate the enhancement, the antibodies did not
have detectable endotoxin and boiling the antibody abolished its potency (Fig. 8 a). VCAM-1 was also upregulated on cobblestone ECs by polyclonal anti-PECAM-1 antibody by 1.7 ± 0.19-fold (mean ± SEM, n = 3, P = 0.03, paired t test).
The second approach used platelet purified PECAM-1
immobilized on beads. E-selectin expression on EC plated
at low density in the presence of PECAM-1-coated beads
was inhibited when measured 18 h after plating (inhibition
was 61 ± 20%; n = 3). BSA-coated beads had no effect
(Fig. 9). Interestingly, neither purified, soluble PECAM-1,
nor its immobilization on plastic was able to regulate E-selectin expression (data not shown) suggesting that valency,
concentration, or microenvironment problems may be operating.
The third approach used PECAM-1 transfectants. EC
were plated at subconfluent density in the presence of L
cells expressing full-length PECAM-1, PECAM-1 lacking
domain 2, or lacking the cytoplasmic tail. Both the full
length and the truncation of the cytoplasmic tail inhibited
E-selectin expression, whereas the mutant lacking domain
2 had no effect (Fig. 10 a). Untransfected L cells (Fig. 10 b)
or L cells expressing the L1 adhesion glycoprotein (a
member of the Ig superfamily expressed by neural cells
and lymphocytes (Hubbe et al., 1993) failed to regulate
E-selectin expression.
E-selectin Is Upregulated at Endothelial Wound Edges Whereas the Cell Junctional Molecule PECAM-1 Is Diminished
An in vitro wound assay was established as an in vivo correlate of EC migration (Schimmenti et al., 1992; Taylor
and Alexander, 1993
). ECs were plated and allowed to
come to confluence. At variable times thereafter wounds
were made and the cells stained 11 and 27 h later for
E-selectin and PECAM-1. As seen in Fig. 11 b, the cells at
the wound front displayed a spread, motile, morphology, and had advanced beyond the wound edge. These migrating cells had less PECAM-1 staining at the cell-to-cell borders. Significantly more ECs at the wound front and immediately behind the front expressed E-selectin in comparison
to the nonwounded areas (Fig. 11, c and d), and this was
substantiated by direct counts of the number of E-selectin
expressing cells (Table II). The proportion of ECs expressing E-selectin in the top 50% range of intensity of expression was 1.7 ± 0.7% in the cell monolayer compared to 16 ± 5.0% at the wound front (mean ± SEM of three separate experiments at 16 h after wounding; P = 0.05).
Table II. E-selectin Expression by ECs at Wound Edges |
In this article, we have demonstrated a novel mechanism for the induction and expression of adhesion molecules on ECs. The induction is initiated through an integrin-dependent process and is associated with cell spreading. The intensity and duration of expression of the adhesion molecules is inversely proportional to cell density, and is mediated at least in part through the junctional protein, PECAM-1. This phenomenon does not involve the known cytokines and differs from the cytokine-dependent pathways in a number of ways: (a) it is dependent on integrin engagement and cell shape; (b) it is regulated by cell junctions; and (c) it can result in prolonged cell surface expression of adhesion molecules (Table III).
Table III. Mechanisms Controlling Expression of Endothelial Adhesion Molecules |
Since the phenomena described in this paper represent a fundamentally new mechanism underlying important pathological processes, we suggest nomenclature where type I induction refers to the classic cytokine-dependent process, and type II induction refers to the new cytokine-independent pathway. Although the level of E-selectin induced in type II conditions is less than that seen for maximal doses of cytokine-induced E-selectin, it is functional as evidenced by increased neutrophil attachment, and is at least additive with low doses of TNF. Such situations of low cytokine concentrations may be relevant during early phases of inflammatory lesions, suggesting the potential importance of both these induction processes in determining endothelial phenotype.
Type II induction of E-selectin described herein is independent of cell size, cell proliferation, and cell cycle, but is initially integrin mediated and seen in cells plated at both high and low density. Cells plated onto ligands that facilitated integrin-mediated attachment induce E-selectin expression (Fig. 7). However if the initial attachment is blocked, as with the addition of cyclic RGD peptides to cells on fibrinogen, E-selectin induction is inhibited. Binding of RGD to cells per se is insufficient to inhibit E-selectin induction since RGD peptides have no effect on cells plated onto collagen.
The intensity and duration of induction of type II responses was related to the density of cell plating. Cells
plated at confluent density expressed less E-selectin when
measured 8-48 h after plating, compared to cells plated at
subconfluent density (Fig. 3). Morphologically, cells plated
at high density form rapid cell-cell interactions in contrast
to low density cells. Thus, junctional control of EC adhesion molecule expression was considered. Two molecules have been described that are known to be important in endothelial junctional integrity (Albelda et al., 1991; Lampugnani et al., 1992
; Ayalon et al., 1994
; DeLisser et al.,
1994
): PECAM-1, a transmembrane glycoprotein belonging to the Ig gene superfamily containing six extracellular,
Ig-like domains; and VE-cadherin, an endothelium-specific member of the cadherin family of cell junctional molecules. The involvement of PECAM-1 in the control of
E-selectin expression was shown by three independent
methods. Firstly, anti-PECAM-1 but not anti-VE-cadherin antibodies induced E-selectin expression on confluent ECs (Fig. 8). Although the increases achieved with the
antibodies were not to the level seen on low density cells,
they were consistent. This may suggest that molecules
other than PECAM-1 could exert secondary events that influence the level of expression of E-selectin. Secondly,
PECAM-coated beads, when added at plating, inhibited
the level of E-selectin expression on low density cells compared to BSA-coated beads (Fig. 9). Thirdly, transfectants
expressing full-length PECAM-1, or those expressing the
extracellular domains but lacking the cytoplasmic domain,
inhibited E-selectin expression on subconfluent ECs (Fig. 10). In contrast, L cells expressing the domain 2 deletion
mutant failed to effect E-selectin expression, suggesting a
critical involvement of domain 2 in this regulation. These
results are in agreement with other studies showing the
importance of domain 2 in cell aggregation (Sun et al.,
1996
). Together with our antibody studies showing that
antibodies to domain 1 increased the level of E-selectin
expression on high density cells, our results suggest that
both domains 1 and 2 of PECAM-1 are critical in regulation of E-selectin expression.
Cell-cell interactions are known to regulate a number of
events, including cell proliferation (Gradl et al., 1995), release of bFGF from astrocytes (Murphy et al., 1988
), responsiveness of fibroblasts to TGF-
(Paulsson et al.,
1988
), and the regulation of intracellular pH (Galkina et
al., 1995
). The mechanisms that underlie such regulation
however have not been elucidated, although autocrine release of growth inhibitory factors (Antonelli-Orlidge et al.,
1989
; Gradl et al., 1995
) and gap junction changes (Chen et
al., 1995
) have been proposed. As described here, a cell
surface junctional protein, PECAM-1, plays a central role
in the density-dependent regulation of endothelial E-selectin
expression. The possibility of a signaling role for PECAM-1
has been raised previously in a number of different systems. These include activation of integrins on T cells
(Tanaka et al., 1992
), natural killer cells (Berman et al.,
1996
), monocytes and neutrophils (Berman and Muller,
1995
), and on CD34+ hematopoietic progenitor cells
(Leavesley et al., 1994
), inhibition of EC proliferation
(Fawcett et al., 1995
), and platelet aggregation (Newman
et al., 1992
). The association of the tyrosine phosphatase-SHP2 with aggregated platelets (demonstrated recently by
Jackson et al. [1997]) has put credence to a signaling pathway associated with PECAM-1 itself. Of interest to our
study here is the recent observation by Lu et al. (1996)
that
integrin engagement and cell spreading results in PECAM-1
dephosphorylation in EC. Thus, the likely cross-talk between integrin and PECAM-1 is further strengthened.
Our in vitro model of cell wounding displays similar features to that described in the density-dependent regulation of adhesion molecules. Firstly, the induction of E-selectin expression is restricted to the migrating front and is associated with a change in cell shape and PECAM-1 redistribution away from the cell junction. Secondly, the E-selectin expression on these migrating cells is maintained at least up to 30 h after the wound signal. Thus we would suggest that this model may reflect identical signaling pathways as those operating in our plating experiments, and may therefore be appropriate as a model for in vivo endothelial regulation at wound sites.
Pathological tissue inflammation is characterized by increased and chronic expression of endothelial adhesion
molecules (Koch et al., 1991; Petzelbauer et al., 1994
; Kraling et al., 1996
), changes in endothelial morphology and
angiogenesis (Fitzgerald et al., 1991
). These morphological
changes include the formation of high endothelial venules
(Freemont et al., 1983
) and cell retraction with associated
intercellular gaps (Nagel et al., 1994
). In balloon angioplasty, expression of adhesion molecules at the wound
front associated with morphological changes has been observed in contrast to the lack of expression in the area behind the wound edge (Tanaka et al., 1993
). More recently,
a role for E-selectin in angiogenesis has been postulated
(Nguyen et al., 1993
; Koch et al., 1995
; Kraling et al.,
1996
). The results reported here, demonstrate: (a) a cytokine-independent mechanism of adhesion molecule expression, and (b) that cell-cell interactions influence the
duration and magnitude of this expression, may explain
some of these in vivo observations. Furthermore, the observation that restoration of PECAM-1 interactions can
downregulate adhesion molecule expression on ECs offers
the promise that manipulation of EC junctional molecules
may permit the development of novel therapeutics.
Received for publication 23 July 1996 and in revised form 6 December 1996.
M. Vadas and J. Gamble contributed equally to this paper.We thank Y. Khew-Goodall for helpful discussions and M. Walker for manuscript preparation. We express our gratitude to the staff at the delivery wards of the Women's and Children's Hospital (Adelaide, South Australia), and the Burnside War Memorial Hospital (Adelaide, South Australia) for collection of umbilical cords.
This work was supported by the National Health and Medical Research Council (Australia), the Anti-Cancer Foundation of the Universities of South Australia, and the National Heart Foundation of Australia.
CSF, colony stimulating factor;
EC, endothelial cell;
HUVEC, human umbilical vein endothelial cells;
ICAM-1, intercellular adhesion molecule-1;
IL-1 and IL-1ra, interleukin-1 and IL-1
receptor agonist;
LPS, lipopolysaccharide;
MFI, mean fluorescence intensity;
PECAM, platelet/endothelial cell adhesion molecule;
TNF-, tumor
necrosis factor-
;
VCAM-1, vascular cell adhesion molecule-1.
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