Department of Physiology and Cell Biology, Neil Hellman Medical Research Building, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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Tumor necrosis factor- (TNF-
) causes
an increase in transendothelial protein permeability of confluent
monolayers of calf pulmonary artery endothelial (CPAE) cells, and the
addition of plasma fibronectin (pFn) to the culture medium can
attenuate this increase in permeability. We determined if reduced
integrin function had a role in decreased endothelial cell adhesion to
immobilized Fn after exposure of the endothelial monolayers to TNF-
.
TNF-
also causes a reorganization of the subendothelial Fn rich
matrix and a significant loss in RGD-dependent adhesion of TNF-
treated CPAE cells to pFn coated surfaces. However, flow cytometry
revealed no decrease in
5
1 or total
1 integrin expression on the surface of the CPAE
cells after TNF-
. Reduced CPAE adhesion to immobilized Fn was,
in part, due to a loss of
1-integrin function
since the
1-integrin blocking antibody mAb 13 significantly (P < 0.05) prevented the adhesion of
normal control CPAE cells but did not further reduce the adhesion of
TNF-
-treated cells. In addition, antibodies which activate
1 integrins restored (P < 0.05)
adhesion of TNF-
-treated cells to immobilized pFn but did not alter
the adhesion of control cells. Despite reduced ability to adhere to immobilized Fn, TNF-
-treated CPAE monolayers demonstrated increased binding and incorporation of fluid-phase pFn into the subendothelial extracellular matrix (ECM) as measured by the analysis of the deoxycholate (DOC) detergent insoluble pool of 125I-Fn in
the cell layer. In contrast to the RGD-mediated adhesion of CPAE cells
to matrix Fn, the increased binding of soluble pFn after TNF-
was
not inhibited by RGD peptides or mAb 13. Thus reduced
integrin-dependent adhesion of the CPAE cells to matrix Fn as well as
disruption of the Fn matrix may contribute to the increased protein
permeability of previously confluent endothelial monolayer after
TNF-
. In addition, increased ability for the monolayer to incorporate fluid-phase Fn into the ECM after TNF-
via
a non-
1- integrin dependent mechanism may be a
compensatory response to stabilize the Fn matrix and the endothelial barrier.
fibronectin; vascular permeability; integrins; lung permeability
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INTRODUCTION |
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ADULT RESPIRATORY
DISTRESS syndrome as a consequence of increased lung endothelial
protein permeability is often observed in critically ill surgical,
trauma, or burn patients with gram-negative sepsis (43).
Tumor necrosis factor- (TNF-
), an inflammatory cytokine released
from endotoxin-activated monocytes and macrophages sequestered in the
lung microcirculation and interstitium during sepsis or lung
inflammation, is believed to contribute to disruption of the lung
vascular barrier leading to the increase in protein permeability
(46, 47). Intravenous injection of recombinant human
TNF-
has been shown to elicit cardiovascular and pulmonary disturbances similar to those observed during endotoxemia or
gram-negative septic shock (38, 45), whereas the
intravenous infusion of neutralizing antibodies to TNF-
reduces
lethality in both endotoxemia and bacteremia in subhuman primates
(46). Moreover, mice devoid of the 55-kDa TNF-
receptor
are more resistant to the development of septic shock after endotoxin
or bacterial challenge (34).
Endothelial monolayer barrier integrity is influenced by both cell-cell
and cell-matrix interactions (13, 15, 24). The concept
that TNF- can alter vascular integrity is directly supported by
various in vitro studies measuring protein permeability of cultured
confluent endothelial monolayers after TNF-
exposure (10, 41,
48, 49). Defilipi et al. (12-14) showed that
exposure of endothelial cells to a combination of TNF-
and
interferon-
(IFN-
) reduced endothelial cell adhesion to specific
matrix proteins. For example, exposure of human umbilical vein
endothelial cells (HUVECs) to TNF-
decreased the expression of
6
1-integrins, resulting in reduced
adhesion to substrate laminin, whereas combined treatment with both
TNF-
and IFN-
decreased
v
3-integrin
surface expression, leading to reduced adhesion to vitronectin
(12-14). Changes in integrin activity have also been
implicated in a loss of endothelial cell adhesion, since parallel
exposure of human endothelial cells with both TNF-
and IFN-
reduced the ligand-binding activity of
v
3-integrins, resulting in a loss of
adhesion to either denatured collagen, vitronectin, or fibrinogen
(42).
Integrin ligation is important in transducing signals from the
extracellular matrix (ECM) which in turn can influence
cytoskeleton-mediated cell motility and gene expression (1, 15,
23). Endothelial cells express several integrins on their
surface, including 5
1- and
v
3-integrins, which interact with a
three-amino acid cell attachment sequence, Arg-Gly-Asp (RGD), found in
fibronectin (Fn), fibrinogen, and vitronectin (13, 24,
35). Both
5
1- and
v
3-integrins recognize an RGD site
accessible in the III-10 module of immobilized or matrix-incorporated
Fn (35, 36). However, Dejana et al. (16)
demonstrated that
5
1 is the only integrin
found clustered in focal contacts when HUVEC cells are plated on Fn. In
addition to influencing cell adhesion, several studies have also
suggested that
5
1-integrins may
facilitate cell-mediated assembly of soluble Fn in the ECM (1,
18, 51), since the binding of the NH2-terminal end
of Fn to cell-associated matrix assembly sites on adherent cells
(22) may require the participation of
5
1-integrins (1, 18, 23, 27,
51).
We previously reported that the addition of TNF- to confluent calf
pulmonary artery endothelial (CPAE) monolayers causes disruption of the
underlying fine fibrillar Fn matrix and an increase in the endothelial
protein permeability (10, 11, 48, 49). This increase in
endothelial protein permeability can be attenuated by the addition of
purified human plasma Fn (pFn), presumably through its incorporation in
the ECM (10, 48, 49). The current study was designed to
determine if exposure of lung pulmonary artery endothelial monolayers
to TNF-
would alter their adhesion to immobilized Fn and to
determine if such reduced CPAE adhesion to Fn was related to either
reduced surface expression of the
5
1-integrins or a change in their
activity or functional state.
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METHODS |
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Endothelial cell culture.
CPAE cells (CCL-209; American Type Culture Collection, Rockville, MD)
were grown in MEM (GIBCO, Grand Island, NY) supplemented with 20% FBS
(Hyclone, Logan, UT), nonessential amino acids (10 mM; GIBCO),
penicillin (100 U/ml; GIBCO), and streptomycin (100 µg/ml; GIBCO).
Experiments were performed between passages 17 and
23. MEM containing 5% FBS was used during the experimental treatments with and without 200 U/ml (~9 ng/ml) of recombinant human
TNF- (Cellular Products, Buffalo, NY). The specific activity of
TNF-
is 2.1 × 107 AU/mg. All chemicals were
purchased from Sigma Chemical (St. Louis, MO) unless otherwise specified.
Determination of endothelial protein permeability using the
dual-chamber monolayer system.
A dual-chamber monolayer assay was used to evaluate transendothelial
125I-labeled albumin clearance as a measure of endothelial
protein permeability, as previously described (9, 10, 48,
49). CPAE cells were seeded (75,000 cells/well) in the luminal
chamber of tissue culture transwell filters (6.5 mm diameter, 0.4 µM
pore size; Nucleopore Polycarbonate Membrane; Costar, Cambridge, MA), grown to confluence, and exposed to 200 U/ml TNF- for 2, 4, 6, or
18 h. The BSA tracer was prepared by iodination with Na
125I (ICN, Irvine, CA) using chloramine-T, as previously
described (10, 48, 49).
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Dual-label immunofluorescence and differential interference
contrast microscopy.
CPAE cells (280,000 cells/well) were seeded on glass coverslips in
12-well tissue culture dishes and grown to confluence. After treatment
with TNF-, the cells were fixed in 3% formaldehyde in PBS (GIBCO)
for 15 min, permeabilized with 0.5% Triton X-100 at 4°C for 5 min,
blocked with 2% BSA + 50 mM glycine + 0.2% Tween 20 in PBS for
1 h, incubated with primary antibody (Ab) for 1 h at room
temperature, and washed with PBS before being incubated with secondary
Ab for 1 h. Primary Abs included rabbit polyclonal Ab against
bovine Fn (1:100 dilution; Calbiochem, La Jolla, CA) and mouse
monoclonal antibody (mAb) against the human
5
1-integrin complex (mAb HA5; 1:500
dilution; Chemicon, Temecula, CA). Secondary Abs included
rhodamine-isothiocyanate conjugated goat anti-rabbit IgG (Cappel
Organon Teknika, Durham, NC) and FITC goat anti-mouse IgG (Boehringer
Mannheim, Indianapolis, IN). Coverslips (Fisher Scientific, Pittsburgh,
PA) were mounted on slides using AntiFade (Molecular Probes, Eugene,
OR) and were viewed using a Nikon Microphot SA fluorescent microscope.
Immunoprecipitation of total cell surface
5
1-integrins.
Confluent CPAE cell layers were trypsinized, washed with trypsin
inhibitor (1 mg/ml) in PBS, and prelabeled with 0.5 mg/ml Sulfo-NHS-biotin (Pierce, Rockford, IL) for 30 min at 4°C, and 0.05 mM Tris · HCl (pH 7.4), 0.15 mM NaCl, 0.1 mM MgCl2,
and 0.1 mM CaCl2 (TBS) was added to stop the reaction. The
mAb 9EG7 or mAb HA5 against the
5
1-complex (4 µg/ml) was added to
106 suspended cells in 1 ml of TBS. Pellets were treated
with extraction buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris (pH
7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 0.2 mM phenylmethylsulfonyl
fluoride, and 0.5% Nonidet P-40], and protein-G agarose was added to
the supernatants for precipitation. Pelleted samples were washed with extraction buffer, resuspended with gel sample buffer, and boiled for 5 min. Total cell lysates, immunoprecipitated proteins, and IgG controls
were separated using 7.5% SDS-PAGE and transferred to nitrocellulose
(Schleider and Schuell, Keene, NH). Blots stained with
streptavidin-horseradish peroxidase (HRP; Amersham Pharmacia Biotech,
Piscataway, NJ) or
5- or
1-polyclonal Abs
(Chemicon, Temecula, CA) and goat anti-rabbit IgG-HRP were then
developed using chemiluminescence (Amersham Pharmacia Biotech). Equal
loading was determined by cell number before extraction.
Flow cytometry.
Confluent CPAE monolayers treated with or without TNF- for 18 h
were trypsinized, washed, and resuspended in PBS (1 × 106 cells/ml). Cells were incubated at 4°C with either
mAb clones 9EG7, HA5, or AIIB2 (a gift from Caroline Damsky at the
University of California-San Francisco) for 30 min, washed with PBS,
and labeled for 30 min at 4°C with either FITC-conjugated goat
anti-rat IgG (Boehringer Mannheim Biochemical, Indianapolis, IN) or
FITC-conjugated goat anti-mouse IgG, respectively. Samples were fixed
with 3% paraformaldehyde, and 104 cells were analyzed
using a Becton-Dickinson FACScan.
Binding of soluble pFn to CPAE cells.
Confluent CPAE monolayers grown in 12-well culture plates were treated
for 18 h with or without TNF-. pFn used for binding and
incorporation experiments was iodinated using the chloramine-T method,
as previously described (37). The cell layers were washed, and 1 ml of MEM containing 1% BSA and 1 µg/ml of human
125I-pFn [2.4 × 106
counts · min
1 (cpm) · 1 µg
1] was added to each well at 4°C. The cell layers
were incubated with 125I-pFn, washed with cold PBS,
dissolved with 1 N NaOH, and counted. Unlabeled pFn (300 µg/ml) was
added to control and TNF-
-treated cells with 125I-pFn to
quantify nonspecific binding, which was subtracted from experimental
values. Samples were quantitated using a Wallac 1470 Wizard gamma counter.
Assay of 125I-pFn incorporation in ECM of CPAE
monolayers.
CPAE monolayers grown to confluence in 12-well culture plates were
exposed for 18 h to TNF- or diluent medium alone, washed with
PBS, and supplemented with 1 ml of MEM containing 5% FBS (Fn depleted)
and 1 µg/ml of human 125I-pFn (2.4 × 106 cpm/µg). The cell layers were incubated at 37°C for
1, 3, 6, or 24 h. To evaluate the incorporation of
125I-Fn in the ECM, cells were detergent extracted with
deoxycholate (DOC), and the DOC insoluble pool of 125I-pFn
(covalently incorporated Fn) was quantitated as previously described
(37). Fn-depleted FBS was obtained by passing the serum
over a gelatin-Sepharose column.
Microscopic analysis of cell adhesion to substrate Fn.
Fn (0.5-2 µg/ml) diluted in PBS was adsorbed on 24-well cell
culture plates and blocked with 1% BSA at 4°C. Confluent CPAE cell
layers treated with TNF- for 18 h were trypsinized, and 5 × 104 CPAE cells suspended in 0.5 ml of MEM were added to
each well for 30 min at 37°C. Thereafter, wells were washed three
times with PBS, and the number of adherent cells per 1 mm2
was counted using an inverted microscope. Depending on the protocol, cell layers were preincubated with either a RGD-containing peptide (GRGDSP; GIBCO), a control RGE-containing peptide (GRGESP; GIBCO), mAb
13 (Becton-Dickinson, Franklin Lakes, NJ), mAb 12G10 (Chemicon), or mAb
9EG7 for 30 min at 4°C before analysis in the adhesion assay.
Electrical cell impedance sensor analysis of cell adhesion to
substrate Fn.
Cell adhesion was also studied with an electrical cell impedance sensor
(ECIS) from Applied Biophysics (Troy, NY). This system can analyze the
dynamic behavior of cells in culture by continuously recording changes
in electrical resistance resulting from altered cell attachment and/or
spreading on gold-plated sensors fixed within the ECIS culture wells
(19). Confluent CPAE monolayers were pretreated with
TNF- for 18 h, lifted by trypsinization, suspended (5 × 104 cells) in 400 µl of MEM supplemented with 1% BSA,
and added to eight-well ECIS plates precoated with human pFn (25 µg/ml) or 1% BSA. The electrical resistance in ohms was recorded
over a 60-min interval. To verify RGD-dependent adhesion to the
Fn-coated surface, 250 µg/ml of the RGD peptide or the control RGE
peptide was added.
Statistical methods. Data are presented as means ± SE. Data were analyzed by a Student's t-test or a two-way ANOVA, with significance from controls determined by a Tukey test. A confidence level of 95% (P < 0.05) was used to establish statistical significance.
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RESULTS |
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Protein permeability increase, Fn rearrangement, and intercellular
gap formation after TNF- exposure to CPAE monolayers.
Figure 1 presents the endothelial protein
permeability of the CPAE monolayers after TNF-
treatment. Protein
clearance across control lung endothelial monolayers was 0.031 µl/min. This was not significantly elevated during the initial 6 h but did increase significantly (P < 0.05) by 18 h post-TNF-
. We used immunofluorescence microscopy to study the
endogenous bovine Fn-rich subendothelial matrix (Fig.
2, left) and differential
interference contrast (DIC) microscopy to visualize intercellular gap
formation (Fig. 2, right). Rearrangement or disruption of
the Fn matrix was readily apparent at 18 h, with a loss of the
fibrillar Fn network from the ECM. Parallel DIC analysis
demonstrated a tight and uniform monolayer with slight changes in
morphology by 6 h. Gap formation was evident at 18 h after
exposure to TNF-
, consistent with the significant increase in
protein permeability. These results suggest a temporal relationship
between the structural rearrangement of the Fn matrix, increased
protein permeability, and gap formation of the endothelial monolayer,
all which seemed to occur in parallel after TNF-
treatment. Based on
these data, we used an 18-h TNF-
treatment period for the remainder
of this study.
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Loss of 5
1-integrin association with
fine Fn fibers of the ECM after TNF-
exposure to CPAE monolayers.
We next examined the effect of TNF-
exposure on the presence of the
Fn
5
1-integrins in relation to the fine
Fn fibers from the ECM by costaining for both the
5
1-integrins and endogenous Fn fibers and
visualizing them by fluorescence microscopy. In confluent control
monolayers (Fig. 3, top left),
5
1-integrins are seen to align (Fig. 3,
top right) with fine Fn fibers at the periphery of the
cells. With higher magnification, cells could be seen within the center
of these dense bundles (data not shown). In contrast, after treatment
with TNF-
the presence of both fine Fn fibers (Fig. 3, bottom
left) and the
5
1-integrins (Fig. 3, bottom right) within the bundles was lost although cells
were still present. Another integrin found on the surface of
endothelial cells that is capable of interacting with the RGD site of
Fn is the
v
3-integrin (13).
Costaining for both the
v
3-integrins with
mAb LM609 and the bovine Fn fibers did not show colocalization within
circular Fn bundles, but rather diffuse staining of the
v
3-integrins around the periphery of
cells (data not shown). The reduced colocalization of the fine Fn
fibers with
5
1-integrins after TNF-
suggested that the increase in protein permeability may reflect
decreased
5
1-mediated cell adhesion to Fn
in the matrix.
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Effect of TNF- on adhesion of endothelial cells to immobilized
Fn.
We confirmed a decrease in adhesion of TNF-
-treated CPAE cells to
pFn-coated wells by direct cell counting via microscopic analysis. No
significant differences occurred between control and TNF-
-treated
cells plated on wells coated with 0.5 µg/ml pFn or BSA alone (Fig.
4A). However, a significant
(P < 0.05) decrease of 35 and 50% in adhesion of
TNF-
-treated CPAE cells to wells coated with 1.0 and 2.0 µg/ml
pFn, respectively, was observed compared with control cells. The assay
was repeated with the addition of RGD- and RGE-containing peptides to
evaluate the role of integrins in the observed adhesion to the
immobilized Fn surface. Control and TNF-
-treated CPAE cells were
plated on wells coated with pFn (2 µg/ml) after preincubation with
RGD or RGE peptide (500 µg/ml). RGD peptide caused a significant
(P < 0.05) loss of adhesion to Fn in both the control
and TNF-
-treated groups (Fig. 4B).
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Temporal effect of TNF- on adhesion of endothelial cells to
immobilized Fn as measured by ECIS.
ECIS (19) was used to examine the temporal aspects of cell
adhesion to wells coated with 25 µg/ml pFn over 60 min after addition
of the cells to the culture medium. Resistance began to increase in
both control and TNF-
-treated groups by 20-25 min after plating
(Fig. 5), with significance
(P < 0.05) observed by 30 min. Thereafter, the
TNF-
-treated group remained significantly lower for the remainder of
the 1-h incubation. The presence of cells on the unwashed electrodes
from all groups was confirmed by visualization using an inverted
microscope after the ECIS recordings were completed. As measured by
ECIS (Fig. 5), the addition of RGD peptides inhibited both control and
TNF-
-treated CPAE cell attachment and spreading on the Fn surface,
whereas RGE peptides had no effect (data not shown). Using an inverted
microscope, we observed that control cells were spread on the
electrode, whereas those exposed to the RGD peptide were found to be
rounded in appearance (data not shown). Thus exposure of CPAE
cells to TNF-
caused a reduction in their RGD-dependent adhesion to
Fn.
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Influence of integrin-blocking Abs on the adhesion of
TNF--treated CPAE cells to immobilized pFn.
We examined the role of integrins in adhesion of CPAE cells to
immobilized pFn using integrin-blocking Abs. Control and
TNF-
-treated cells were preincubated with 10 µg/ml mAb 13 (
1-integrin blocking), 10 µg/ml mAb LM609
(
v
3-integrin blocking), or no mAb at all and were applied to wells coated with 2.0 µg/ml pFn. Significant (P < 0.05) losses in adhesion to Fn were observed with
control cells preincubated with mAb 13 and mAb LM609 (Fig.
6). Each Ab alone reduced cell adhesion
by ~40%, down to the level of TNF-
treated cells. In contrast,
adhesion of TNF-
-treated cells to immobilized pFn was not inhibited
(P > 0.05) by mAb 13 or mAb LM609 (Fig. 6).
Interestingly, preincubation with both antibodies together did not
further reduce adhesion in either group (data not shown). These data
further suggested that a loss of integrin-mediated adhesion to surface
Fn occurs in CPAE cells treated with TNF-
.
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Surface expression of 5
1-integrin by
flow cytometry.
We then speculated that the loss of integrin-mediated adhesion to Fn by
CPAE cells treated with TNF-
may be the result of a loss of
5
1-surface expression. To measure surface
expression on CPAE cells, we performed flow cytometry using mAb HA5 to
detect the
5
1-complex. First, the
specificity of mAb HA5 and cross-reactivity for the bovine
5
1-complex was confirmed by Western blot
analysis of biotinylated surface proteins immunoprecipitated with mAb
HA5. Staining blots with streptavidin-HRP revealed two bands consistent with the molecular masses of the
5- and
1-integrin subunits (Fig.
7A), and reprobing with a
polyclonal Ab against
5-integrin confirmed the upper
band was
5.
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Surface expression of 9EG7-detectable epitope by flow cytometry.
The 1-integrin subunit can play a major role in the
activation of the integrin complex and exists in distinct conformations or activity states that influence their interactions with ligand (2, 32). Moreover, expression of the mAb 9EG7-dependent
epitope on the
1-integrin reflects a high affinity or
ligand-bound state that can be influenced by cations, chelating agents,
antibodies, inside-out signaling, or ligand occupation (2, 26,
32). Mastrangelo et al. (26) demonstrated a
decrease in mAb 9EG7-dependent expression associated with a loss of
Fn-dependent adhesion in MG-63 cells transfected with chimeric
receptors containing the
1-cytoplasmic domain. Since we
hypothesized that reduced adhesion of TNF-
-treated cells to
immobilized pFn might correlate with reduced surface expression of the
mAb 9EG7-dependent epitope, we next investigated the possibility that
TNF-
treatment had altered the activity state of
5
1-integrins. First, cross-reactivity to
bovine
1-subunits was confirmed by probing Western blots
of immunoprecipitated
5
1-integrin with
mAb 9EG7 (data not shown). Flow cytometry performed using mAb 9EG7 and
mAb AIIB2, an Ab against
1-integrins, revealed a slight
but nonsignificant (P > 0.05) increase in both
fluorescence intensity for the mAb 9EG7-dependent epitope and the
1-integrin on TNF-
-treated CPAE cells (Fig. 8). Thus the decreased adhesion of
TNF-
-treated cells to Fn was not caused by a significant decrease in
1-integrin surface expression.
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Effect of Mn2+ on the mAb
9EG7-detectable epitope in bovine endothelial cells.
Bazzoni et al. (3) demonstrated that phorbol myristate
acetate (PMA) increased the adhesion of K562 cells to pFn without an
increase in mAb 9EG7-dependent epitope expression. The same study
showed that mAb 9EG7 stimulated the adhesion to Fn via
5
1-integrins in the presence of
Mn2+, a divalent cation that increases mAb 9EG7-dependent
epitope expression (2, 3, 25). To verify the existence of
a functional mAb 9EG7-dependent epitope in bovine endothelial cells,
biotinylated CPAE cells were incubated with mAb 9EG7 in the presence or
absence of 5 mM Mn2+. The results of immunoprecipitation
with this Ab showed two major bands after staining with streptavidin
(Fig. 9A, lanes 2 and 3) that aligned with bands of
5- and
1-subunits immunoprecipitated with mAb HA5 (lane
1). These two bands were the only major bands present above 67.5 kDa on the blot, suggesting that
5
1-integrin was a major complex
immunoprecipitated by mAb 9EG7. The increased band intensity with
Mn2+ treatment (lane 3) confirmed that the mAb
9EG7-dependent epitope exists on
5
1-integrins expressed on the surface of
CPAE cells. Reprobing with a polyclonal Ab against the
5-subunit confirmed the upper band (Fig. 9B).
Accordingly, Mn2+ induced the surface expression of the mAb
9EG7-dependent
1-epitope in bovine endothelial cells,
thus verifying the existence of a functional mAb 9EG7 epitope in our
bovine cells.
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Effect of 1-activating antibodies on cell adhesion.
Addition of mAb 9EG7 can activate the
5
1-integrin without Mn2+,
presumably by shifting the
1-subunit from an inactive to
an active state (33). If true, then the TNF-
treatment
of CPAE cells may have caused inactivation of
5
1-integrins, resulting in reduced
adhesion to pFn, which should be recoverable by
1-activating Abs. To test this concept, suspended
control and TNF-
-treated CPAE cells were preincubated with 10 µg/ml of the
1-activating antibodies mAb 12G10 and mAb
9EG7 before being plated on pFn-coated wells. Neither activating Ab
significantly (P < 0.05) increased adhesion of control
cells to immobilized pFn, yet both Abs significantly (P < 0.05) increased adhesion of TNF-
-treated cells by 30-40%, thus reversing the adhesion deficit (Fig.
10). The restoration of TNF-treated
CPAE cells by the
1-activating antibodies was, in turn,
prevented by the
1-blocking mAb AIIB2 (data not shown). These
1-activating antibodies rescue adhesion of
TNF-
-treated but not control cells, supporting the concept that
TNF-
treatment causes a significant population of
1-integrins to become inactive.
|
Soluble 125I-pFn binding and ECM incorporation to CPAE
monolayers after TNF- treatment.
Because the
5
1-integrin has been shown to
mediate soluble Fn binding to human fibroblasts (18), we
then explored the possibility that the binding of soluble pFn to the
cell layers may also be reduced in TNF-
-treated monolayers in which
5
1-integrin activity was reduced.
125I-pFn binding at 4°C was measured after 5, 10, 20, 30, or 60 min of incubation with TNF-
. Contrary to our expectations, the
binding of soluble Fn was significantly (P < 0.05)
increased five- to sixfold after TNF-
treatment at 60 min (Fig.
11A).
|
|
Effect of RGD peptides and mAb 13 on fluid-phase pFn binding to
TNF--treated CPAE monolayers.
It has been suggested that
5
1-integrins
can facilitate pFn incorporation in the ECM (1, 18, 51).
However, despite the reduction of
1-integrin function,
we observed an increase in soluble pFn binding and ECM incorporation
after TNF-
treatment. Because it is possible that
1-integrins, although losing adhesive activity for
immobilized Fn, may still play a role in soluble ligand binding, we
repeated the binding studies using control and TNF-
-treated CPAE
monolayers exposed to RGD peptides and mAb 13 to investigate a role for
1-integrins in soluble pFn binding. TNF-
-treated
monolayers again showed increased binding of 125I-pFn
compared with controls, but only the control cells were inhibited
(P < 0.05) when incubated with the RGD peptide
compared with RGE peptide (Fig.
13A).
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DISCUSSION |
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We have previously shown that the addition of either recombinant
TNF-, soluble RGD-containing peptides, or polyclonal antibodies against
5
1-integrins to the culture
medium of previously confluent CPAE monolayers increased their protein
permeability in association with disruption of the fine fibrillar
Fn-rich matrix (10, 48, 49). The addition of soluble human
pFn can prevent and reverse the increase in monolayer protein
permeability caused by TNF-
, and specificity was apparent, since
other RGD-containing proteins such as fibrinogen and vitronectin will
not work (10, 48, 49). In the current study, we observed
reduced colocalization of
5
1-integrins
with fine Fn fibers in the ECM after TNF-
exposure, which correlated
with reduced adhesion of the CPAE cells to immobilized Fn. Curtis et
al. (11) demonstrated that the protein permeability increase and Fn matrix rearrangement is not the result of proteolysis of Fn within the subendothelial ECM after TNF-
treatment, since no
Fn degradation could be detected in the medium or cell layer/matrix. In
addition, protease inhibitors did not prevent the increase in protein
permeability after TNF-
(11). Thus we hypothesized that
this reduced ability of CPAE cells to adhere to matrix Fn may be
because of changes in either the surface expression of
5
1-integrins or the activation state of
the
1-subunit. Cytokines such as TNF-
,
interleukin-1
, and IFN-
have been shown to modulate the surface
expression of integrins on a variety of cells (12, 14, 17,
31). Although exposure of HUVECs (40) to 100 ng/ml TNF-
can decrease the expression of the
5
1-integrin, we were unable to detect any
significant reduction in the expression of
5
1-integrins after exposure of CPAE
monolayers to ~9 ng/ml TNF-
, as used in our experimental protocol.
Although mAb 13, a blocking Ab against the 1-subunit,
significantly inhibited adhesion of control cells to pFn, only partial adhesion blocking occurred. Other integrins on the cell surface may
also be involved in the adhesion of CPAE cells to immobilized Fn such
as
v
3,
v
5,
and
IIB
3. We observed that blocking the
v
3-integrin with mAb LM609 also inhibited
adhesion to immobilized Fn. Defilippi et al. (13, 14)
showed that cotreatment of HUVECs with TNF-
and IFN-
decreased
the surface expression of
v
3-integrin, whereas B. Gao, K. Powell, and T. M. Saba (unpublished results) demonstrated that surface expression of
v
3-integrins on CPAE cells doubled after
exposure to TNF-
for 18 h, as used in the current
study. There was also no additive blocking effect against adhesion when cells were coincubated with mAb 13 and mAb LM609 together, raising the possibility that the
5
1- and
v
3-integrins work in concert in mediating
the adhesion of CPAE cells to immobilized Fn. In support of this
concept is the finding that M21 melanoma cells require surface
expression of both
5
1- and
v
3-integrins to attach and spread on
Fn-coated surfaces (7). Although
5
1- and
v
3-integrins may both play a role in
maintaining pulmonary endothelial monolayer barrier function, the
increased protein permeability caused by TNF-
appears not to be the
result of decreased surface expression of these receptors. Furthermore,
unlike
5
1-integrins,
v
3-integrins were not colocalized with Fn
in the matrix, suggesting a specific role for
5
1-integrins in the adhesion of CPAE
monolayers to Fn-rich matrix. Other RGD-dependent integrins, such as
v
5 and
IIB
3, may also play a role in CPAE cell
adhesion to immobilized Fn, and their role may be addressed in future studies.
The ability of 1-integrin-activating Abs to restore the
adhesion of TNF-
-treated CPAE cells to Fn surfaces, without
affecting the adhesion activity of control cells, suggests that a loss
of
5
1-integrin activity may be
responsible for the observed increased protein permeability. The role
for
5
1 is further supported by the
ability of mAb 9EG7 to immunoprecipitate
5
1-integrin subunits. One must also
consider the possibility that TNF-
treatment may have caused a shift
of a population of
5
1-integrins from an active to an inactive state, incapable of binding immobilized pFn.
Mould et al. (33) have shown that an inactive pool of
5
1-integrins incapable of binding ligand
can indeed exist and that mAb 9EG7 can rescue this inactive pool with
respect to ligand binding. An association between decreased surface
expression of the mAb 9EG7-dependent epitope and decreased cell
adhesion to Fn has also been previously suggested (26). We
did not observe decreased expression of this epitope after TNF-
in
the current study. Despite a lack of changes in surface expression in
our study, this epitope appeared to be functionally responsive to
Mn2+, a well-known characteristic of this epitope (2,
3). However, a direct relationship between mAb 9EG7-dependent
epitope expression and cell adhesion to substrate Fn does not always
hold true. For example, PMA treatment of K562 cells increased the
ability to adhere to Fn without increased surface expression of the mAb
9EG7-dependent epitope (3).
The possibility that TNF--induced loss of adhesion may be mediated
by the inactivation of integrins is also supported by several other
studies. Ruegg et al. (42) cotreated human endothelial cells with 200 ng/ml TNF-
and 330 ng/ml IFN-
and observed a loss
of adhesion to immobilized vitronectin, denatured collagen, and
fibrinogen, but not to Fn. This loss of adhesion was the result of
inactivation of
v
3-integrins, even in the
presence of their increased surface expression. The mechanism by which
1 deactivation can be caused by TNF-
, as suggested by
our current data, can only be speculated. Blystone et al.
(4) demonstrated that
v
3-integrins can negatively influence
5
1-integrin activity with respect to Fn-mediated phagocytosis by suppressing calcium/calmodulin-dependent protein kinase II (CaMKII) activity. Furthermore, Bouvard et al. (5) have shown that CaMKII mediates CHO cell adhesion to
Fn by regulating the affinity state of the
5
1-integrin. Based on these findings, one
could speculate that the twofold increase in surface expression of
v
3-integrin on CPAE cells after TNF-
exposure may have caused a reduction of CaMKII activity, thereby reducing
5
1-integrin activity and
contributing to the decrease in adhesion of CPAE cells to the Fn matrix.
In addition to their role in cell adhesion to ECM,
5
1-integrins may facilitate the binding
of soluble Fn to the cell layer and its matrix assembly (1, 18,
23, 27, 51). Our findings of decreased
1-integrin-mediated binding of soluble pFn after TNF-
are consistent with the belief that a population of
5
1-integrins expressed on the surface of
TNF-
-treated CPAE cells are in a low-affinity or inactive state.
However, the reduction we observed in
1-mediated pFn
binding was greatly offset by a dramatic increase in pFn binding to the
cell layer, which could not be blocked by mAb 13 or RGD peptide. The
5
1-integrins have the ability to assist
in the assembly of soluble Fn in ECM when transfected in CHO cells
(50), but mAb LM609, which blocks such matrix assembly of
Fn, did not inhibit binding of soluble pFn to the cell layer in our
current study. Thus the increased binding of soluble pFn does not
appear to be integrin mediated; however, it may be mediated by tissue
transglutaminase, since its extracellular activity is increased in CPAE
cells after TNF-
, causing nonreducible Fn multimer formation in the
matrix (8).
In response to binding an immobilized ligand, such as Fn, major integrins will cluster within focal adhesion complexes, resulting in interactions with actin-associated cytoskeletal proteins (6). The structural link between the ECM, integrins, and the actin cytoskeleton stabilizes cell adhesion and contributes to the mechanical basis by which integrins can influence endothelial cell shape. Changes in the ability of the cells to bind to the ECM results in cytoskeletal reorganization and global changes in cell shape (30, 44). Sims et al. (44) showed that changes in cell shape can result from cytoskeletal tension, generated by an actomyosin filament sliding mechanism, and that this tension can be physically resisted by cell surface integrins bound to immobilized adhesion sites within the ECM, including the RGD or cell attachment site in Fn. Thus the ability of integrins to bind to matrix proteins may exert a unique control on cell shape. Loss of this binding could result in a loss of monolayer confluence and the development of intercellular gaps, with an increase in endothelial protein permeability like we observed.
In summary, our findings demonstrate that TNF-, which increases
protein permeability and reorganizes the Fn matrix, also reduces the
ability for CPAE cells to adhere to immobilized Fn. This loss of
adhesion to an Fn substrate appears to be related to inactivation or a
reduced function of
5
1-integrins.
However, in parallel, TNF-
treatment caused both an increase in
1-integrin-independent binding of soluble pFn to the
CPAE cell layer and its ECM incorporation. This suggests that TNF-
released in the plasma during postoperative bacterial sepsis or lung
inflammatory injury may have a dual role. It may initially increase the
permeability of the lung vascular barrier to facilitate the
transvascular flux of plasma proteins essential to pulmonary host
defense but, thereafter, lead to enhanced incorporation of pFn in the
subendothelial matrix to rapidly stabilize the endothelial barrier.
![]() |
ACKNOWLEDGEMENTS |
---|
The technical assistance of Edward Lewis, Van Fronhofer, Eshin Cho, and Kara L. Powell and the secretarial assistance of Debbie Moran is acknowledged. We thank Caroline Damsky at the University of California-San Francisco for the generous gift of the mAb clone AIIB2 originally provided to the laboratory in support of the thesis research of T. M. Curtis.
![]() |
FOOTNOTES |
---|
This study was supported primarily by National Institutes of Health Grants GM-21447 (T. M. Saba) and in part by GM-51540 (S. E. LaFlamme). Trainee support was provided by T32-HL-07194 and F32-HL-010201 (T. M. Curtis), T32 HL-07529 (R. F. Rotundo), and T32-GM-07033 (A. Mastrangelo).
Address for reprint requests and other correspondence: T. M. Saba, Center for Cell Biology and Cancer Research, MC-165, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: sabat{at}mail.amc.edu).
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.
10.1152/ajplung.00145.2000
Received 5 May 2000; accepted in final form 2 October 2001.
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