(Received for publication, November 19, 1996, and in revised form, May 12, 1997)
From the Monocytes in the blood circulation migrate across
endothelial cell monolayers lining the blood vessels and infiltrate
into the underlying tissues in inflammation. However, little is known about the mechanisms by which leukocytes migrate across the endothelial barrier after binding and what molecules participate in the process. Addition of the human monocytic cell line THP-1 to interleukin-1 In the early stages of inflammation, monocytes and other
leukocytes in the blood circulation migrate across endothelial cell monolayers lining the blood vessels and enter the perivascular tissues.
The migration of leukocytes involves multiple steps, and various types
of adhesion molecules participate in these processes, including
selectins mediating initial tethering and rolling of leukocytes over
the endothelial cells, and integrins on leukocytes interacting with
adhesion molecules belonging to the immunoglobulin superfamily
expressed on the endothelial cells (1, 2). In acute inflammation, the
expression and activation of adhesion molecules are regulated by
mediators such as thrombin, inflammatory cytokines, and chemokines
(2).
Although many observations have focused on the molecules participating
in the events from tethering to adhesion of leukocytes to endothelial
cells, little is known about the mechanisms whereby leukocytes migrate
across the endothelial barrier after binding and which molecules
participate in the process.
Platelet/endothelial cell adhesion molecule-1
(PECAM-1)1 is one of the
adhesion molecules that is concentrated at intercellular junctions
between endothelial cells (3). Anti-PECAM-1 monoclonal antibody (mAb)
or soluble PECAM-1 inhibits the transmigration of leukocytes through
endothelial cell monolayers in vitro without interfering
with the leukocyte's potential to adhere tightly to the apical surface
of endothelial cells (4). For neutrophils, integrin-associated protein
(CD47) present on both neutrophils and endothelial cells is supposed to
be essential for invasion (5). Activation of intercellular adhesion
molecule-1 (ICAM-1) by binding of T cells has been reported to
transduce a signal into endothelial cells, which induces tyrosine
phosphorylation of the actin-binding protein cortactin, indicating
alterations in the cytoskeleton (6). These findings suggesting the
possibility that binding itself induces changes in endothelial cells
leading to relaxation of interendothelial cell junctions are
significant.
To delineate the mechanism whereby monocytes can transmigrate through
the endothelium during inflammation, we first investigated the changes
in protein phosphorylation patterns of interleukin-1 HUVEC were purchased from Kurabo (Osaka, Japan)
and were cultured on gelatin-coated culture flasks (Iwaki glass, Tokyo,
Japan) with EGM-UV medium (Kurabo). Human monocytic THP-1 cells
(Japanese Cancer Research Resources Bank, Tokyo, Japan), monoblastic
U937 cells (American Type Culture Collection, Rockville, MD),
promyelocytic HL-60 cells (Fujisaki Cell Center, Hayashibara
Biochemical Labs., Inc., Okayama, Japan), and T leukemic MOLT-16 cells
(Fujisaki Cell Center) were maintained in RPMI 1640 (Nissui
Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum
(Life Technologies, Inc.), 10 mM HEPES, 100 units/ml
penicillin, and 50 µg/ml streptomycin.
Human IL-1 Six-cm culture dishes (Falcon 3002, Becton
Dickinson) were coated with 4 ml of a 100 µg/ml solution of gelatin
(Iwaki Glass) in phosphate-buffered saline (PBS) for 2 h, and
HUVEC were grown to confluency on the coated dishes. HUVEC were
stimulated with IL-1 After washing the mixed cultures of HUVEC and leukemic cells with PBS,
the cells were lysed with 500 µl of an extraction buffer (1% Triton
X-100, 1% Nonidet P-40, 150 mM NaCl, 2 mM
Na3VO4, 10 mM NaF, 2 mM
phenylmethylsulfonyl fluoride, 250 µg/ml leupeptin, 2 mM
EDTA, 50 mM Tris, pH 7.5) with the aid of a cell scraper. The lysates were stood on ice for 30 min with occasional mixing. One
hundred µl of the lysates were then transferred to new tubes as the
whole cell lysate. The residual lysates were centrifuged at 13,000 × g for 30 min, and the supernatants were collected and
used as the cell extract. Four hundred µl of extraction buffer solutions containing 1% SDS were added to each remaining pellet and
were dissolved by vigorous pipetting. These fractions were defined as
the insoluble fraction.
The cell extracts
were incubated with 1 µg of anti-Tyr(P) 4G10 for 2 h or with 4 µg of anti-p125FAK 2A7 for 16-18 h at 4 °C with
continuous mixing. Protein G-Sepharose (Pharmacia, Uppsala, Sweden) was
washed twice with Tris-buffered saline (150 mM NaCl, 10 mM Tris, pH 7.4) and once with a washing buffer (1% Triton
X-100, 1% Nonidet P-40, 150 mM NaCl, 50 mM
Tris, pH 7.5), and the resin pellet was resuspended in washing buffer. The resins from 100-µl volumes of 50% suspensions were mixed with the HUVEC lysates and were incubated for 2 h at 4 °C with
continuous mixing. The resins were washed three times with washing
buffer and then resuspended in 40 µl of a 2 × SDS-sample buffer
(100 mM Tris, 5% SDS, 30% glycerol, 5%
2-mercaptoethanol, pH 6.8), and boiled for 5 min. After centrifugation,
20 µl of the supernatants were subjected to electrophoresis on a
7.5% polyacrylamide gel in the presence of SDS and transferred to
nitrocellulose filters. In the case of direct immunoblotting, samples
were treated with half their volume of 3 × SDS-sample buffer (150 mM Tris, 7.5% SDS, 45% glycerol, 7.5% 2-mercaptoethanol,
pH 6.8), and 20 µl of the treated samples were subjected to
electrophoresis.
After blocking nonspecific binding with Block Ace (Yukijirushi,
Sapporo, Japan), the filters were probed with the antibody of interest
for 2-3 h at room temperature (rt), followed by either horseradish
peroxidase-labeled rabbit anti-mouse Igs (Dako Japan, Kyoto, Japan),
horseradish peroxidase-labeled swine anti-rabbit Igs (Dako, Japan), or
a Vectastain ABC-PO kit for goat IgG (Vector Laboratories, Burlingame,
CA) for 2-3 h at rt. Washing of the membranes was performed with
Tris-buffered saline containing 0.05% Tween 20. The bands were
visualized with the enhanced chemiluminescence detection system
(Amersham, Buckingamshire, United Kingdom) as directed by the
manufacturer. In the case of reprobing the same membranes with a
different first antibody, the horseradish peroxidase of the already
bound second antibody was inactivated by treating the filters with
Block Ace supplemented with 0.1% NaN3 for 16-18 h at rt.
Quantification of the density of the detected blots was performed by
scanning densitometry using ImageMaster DTS (Pharmacia).
Polystyrene chamber slides (Nippon
InterMed, Tokyo, Japan) were coated with gelatin for 2 h. HUVEC
were plated on the slides and cultured to confluency. HUVEC were then
stimulated with 0.5 ng/ml IL-1 Ten thousand HUVEC were seeded in each
well of gelatin-coated 96-well culture plates (Iwaki Glass) and
cultured for 48 h. Confluent cultures of HUVEC were stimulated
with IL-1 First we
investigated the changes in the tyrosine phosphorylation levels of
molecules in HUVEC after adding human leukemic THP-1, U937, HL-60, or
MOLT-16 cells. To simulate the activated state of blood vessels in
inflammation, HUVEC were pretreated with IL-1
The residual immunoprecipitates obtained by immunoprecipitation with
anti-p125FAK 2A7 were probed with anti-p125FAK C-20. As
shown in Fig. 1C, the pattern of immunoblots detected with
polyclonal anti-p125FAK C-20 was almost identical to the
pattern that was obtained with anti-Tyr(P) PY20, indicating that the
decrease in p125FAK band resulted from a decrease in the amount
of p125FAK protein itself and not from tyrosine
de-phosphorylation of the protein. To further clarify the reason for
the decrease in p125FAK, changes in the amount of
p125FAK in whole cell lysates, cell extracts, and in insoluble
fractions were investigated by direct immunoblotting with
anti-p125FAK C-20. As shown in Fig. 1D, decreased
p125FAK levels induced by monocytic cell treatment was observed
in whole cell lysates. In addition, the pattern of p125FAK
levels in cell extracts was identical to that in whole cell lysates, and no p125FAK was detected in the insoluble fractions under
the same detection conditions (data not shown). From these results, we
assumed that the decrease in the amount of p125FAK induced by
monocytic cell seeding resulted from a decrease in p125FAK
protein and not from a decrease in solubility of the protein. However,
obvious candidates for the degradation products of p125FAK
could not be observed. The decrease in p125FAK was observed not
only in THP-1-treated HUVEC but also in U937-treated HUVEC (Fig.
1C, lanes 4 and 5), although no change was
detected in HUVEC treated with HL-60 and MOLT-16 (Fig. 1C, lanes
6-9).
We investigated whether levels of proteins in
IL-1
VCAM-1 and To further characterize the decrease in the
amount of p125FAK, the kinetics of the changes in
p125FAK after addition of THP-1 cells were investigated. As
shown in Fig. 3, the decrease in
p125FAK was detected from 5 min after the addition of THP-1
cells (lane 5) and reached a maximum 15-30 min later
(lanes 6 and 7) in IL-1
To investigate whether THP-1 treatment induces changes in
the cytoskeletal structure of HUVEC, the HUVEC were stimulated with or
without IL-1
The decrease in the amount of
p125FAK induced by THP-1 cells was observed in IL-1
It is well known that IL-1
Focal contacts are regions of the cell that come in direct contact
with the extracellular matrix, providing anchorage sites for actin
stress fibers and forming a link between the extracellular matrix and
the actin cytoskeleton (19). The p125FAK molecule is a tyrosine
kinase co-localized in focal contacts with several other molecules,
such as talin and tensin (20-22), and plays a central role in
integrin-mediated signal transduction from the extracellular matrix
(20-24). In this study, we showed that binding of THP-1 cells to
IL-1 A decrease in the density of actin fibers induced by THP-1 was also
observed in HUVEC in parallel with the decrease in p125FAK. It
has been well documented that the formation of actin stress fibers
parallels the formation of focal adhesion and is accompanied by
increased tyrosine phosphorylation of p125FAK (27-29).
Integrity of the actin cytoskeleton has also been reported to be
required for the increased phosphorylation of p125FAK in
response to a variety of extracellular stimuli (23, 30). Therefore, it
can be postulated that the decrease in actin stress fibers is closely
associated with the decrease in p125FAK.
It is unclear why the p125FAK protein level drops so rapidly.
Recently, it was reported that p125FAK is cleaved by calpain, a
calcium-dependent cysteine protease, in platelets (31).
Therefore, we investigated whether the decrease in p125FAK
could be prevented by calpeptin, a membrane-permeable inhibitor of
calpain, or a cysteine protease inhibitor E-64. However, pretreatment of HUVEC by these inhibitors at a concentration of up to 50 µM could not affect the decrease in p125FAK (data
not shown). In addition, little change was observed in the amount of
talin which interacts with p125FAK (32) and is cleaved by
calpain preferentially (33). From these results, it is unlikely that
calpain is responsible for the decrease in p125FAK. The
molecular mechanisms of the decrease in p125FAK are still
inconclusive, even though we have also tried to inhibit the decrease in
p125FAK by other protease inhibitors.
The decrease in p125FAK in IL-1 The molecules participating in the interactions between THP-1 cells and
HUVEC remain to be clarified. The candidate molecule that triggers the
decrease in p125FAK is considered to be an adhesion molecule
present on THP-1 cells rather than a newly secreted soluble factor
induced by interaction of THP-1 cells with activated HUVEC because the
cell-free culture supernatant obtained after co-culture of THP-1 cells
with IL-1 We thank Drs. Mark Micallef and Tsunetaka
Ohta for helpful discussions and for review of the manuscript. We thank
Shigeto Yamamoto for preparing the figures.
Fujisaki Institute,
Department of Medical
Engineering,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(IL-1
)-stimulated human umbilical vein endothelial cells (HUVEC) induced a decrease in the amount of focal adhesion kinase
(p125FAK) protein, a tyrosine kinase localized at focal
contacts and essential for cell attachment to the extracellular matrix,
whereas little change was observed in the amount of other molecules
associated with cell adhesion such as vascular cell adhesion
molecule-1,
-catenin, and talin. A maximum decrease in the
amount of p125FAK was observed 15-30 min after addition of
THP-1 cells to HUVEC, after which the level of p125FAK
gradually recovered. A reduction in the density of actin stress fibers
in IL-1
-activated HUVEC was observed in parallel with the decrease
in p125FAK. The p125FAK decrease was partially
inhibited by preventing THP-1 binding to HUVEC using a mixture of
antibodies to adhesion molecules. We suggest that the decrease in
p125FAK triggered by binding of monocytes in inflammation
facilitates the transendothelial migration of the monocytes by altering
the adhesiveness of endothelial cells to the extracellular matrix.
(IL-1
)-stimulated human umbilical vein endothelial cells (HUVEC) overlayered with human monocytic THP-1 cells and found that the addition of THP-1 cells induces a decrease in the amount of a phosphorylated 120-130-kDa protein(s) in HUVEC. In this study, we show
that the decreased protein is focal adhesion kinase (p125FAK),
a tyrosine kinase present at focal contact sites, and we discuss the
possible involvement of this alteration in the process of leukocyte
migration at sites of inflammation.
Cell Lines
was purchased from Genzyme
(Cambridge, MA). Human natural tumor necrosis factor-
(TNF-
)
(specific activity of 2 × 106 Japan reference
unit/mg) was prepared in our laboratory (7). Mouse monoclonal
anti-phosphotyrosine (anti-Tyr(P)) PY20, anti-Tyr(P) 4G10,
anti-p125FAK 2A7, and anti-
4 integrin SG/73 were purchased
from Seikagaku Corp. (Tokyo, Japan). Rabbit polyclonal
anti-p125FAK C-20 antibody and goat polyclonal anti-vascular
cell adhesion molecule-1 (VCAM-1) C-19 antibody were from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-
2 integrin mAb MEM 48 was
purchased from R&D Systems (Abingdon, UK), and anti-sialyl
Lex CSLEX1 (IgM) was purchased from Becton Dickinson
(Bedford, MA). Anti-talin mAb TA205 was purchased from Genosys
(Cambridge, UK). Rabbit polyclonal anti-
-catenin was purchased from
Sigma.
or TNF-
for 5 h and subsequently
overlayered with various human leukemic cells for the indicated times
at different cell densities.
for 5 h and were subsequently
layered with 1.5 × 104 THP-1 cells at different
periods. After removing the supernatant and washing, the cells were
fixed with a mixture of acetone and methanol (1:1 v/v) for 20 min at
20 °C, and after washing with PBS, the cells were incubated with
2.5 units/ml of rhodamine phalloidin (Wako Pure Chemical Industries,
Osaka, Japan) for 1 h at rt. After washing, the slides were
mounted using 50% glycerol in PBS and observed under a fluorescence
microscope (model BHF, Olympus, Tokyo, Japan).
or TNF-
at 37 °C for 5 h and then washed once
with assay medium (RPMI 1640 supplemented with 0.1% bovine serum
albumin (Armour Pharmaceutical, Kankakee, IL), 10 mM HEPES,
100 units/ml penicillin, and 50 µg/ml streptomycin) before addition
of isotope-labeled THP-1 cells. THP-1 cells were labeled with
51CrO4 (Amersham) at 37 °C for 1 h. After washing three times with the culture medium, 5 × 104 labeled THP-1 cells suspended in the assay medium were
added to each well in 100-µl volumes. In inhibition experiments using adhesion-blocking antibodies, 51Cr-labeled THP-1 cells
incubated with 50 µg/ml mAbs to adhesion molecules for 60 min at rt
and washed twice were used. After mild centrifugation at 40 × g for 1 min, the plates were incubated at 37 °C for 30 min. The nonadherent cells were removed by washing twice with the assay
medium, and the adherent THP-1 cells were lysed with 1 N
NaOH. Radioactivity from samples of supernatants from each well and the
original THP-1 cell suspension was determined by gamma counter, and the
percentage of THP-1 cells adhering to HUVEC in each well was
calculated.
The p125FAK Level in IL-1-stimulated HUVEC Is
Decreased by Co-culture with Monocytic Cell Lines
for 5 h. After
treating HUVEC with leukemic cells, the cells were lysed with the
extraction buffer, and cell extracts were prepared. Tyrosine
phosphorylation patterns were assessed by immunoprecipitation with
anti-Tyr(P) 4G10 and subsequent immunoblotting with anti-Tyr(P) PY20.
As shown in Fig. 1A, several
tyrosine-phosphorylated proteins were observed in IL-1
-stimulated
HUVEC in the absence of the leukemic cell lines (lane 1). In
the case of co-culture with THP-1 cells, a decrease in
tyrosine-phosphorylated 120-130-kDa proteins in IL-1
-stimulated
HUVEC was very obvious (lanes 2 and 3).
Considering the molecular size and the levels of expression of the
phosphorylated molecule(s) observed in our experiments, p125FAK
was selected as a probable candidate for the tyrosine-phosphorylated 120-130-kDa protein observed in HUVEC. To confirm the identity of the
protein(s) banding at 120-130 kDa, cell extracts from HUVEC co-cultured with leukemic cell lines were immunoprecipitated with anti-p125FAK 2A7 and probed with anti-Tyr(P) PY20. As shown in
Fig. 1B, anti-p125FAK 2A7 immunoprecipitated a
120-130 kDa protein, and the amount of immunoprecipitated molecule(s)
was reduced by THP-1 co-culture in a manner depending on the number of
seeded THP-1 cells (Fig. 1B, lanes 2 and 3). This
indicates that a component of the tyrosine-phosphorylated 120-130-kDa
band is identical to p125FAK.
Fig. 1.
Decreased p125FAK in
IL-1-stimulated HUVEC by co-culture with monocytic cell lines.
Confluent HUVEC cultures on gelatin-coated culture dishes were treated
with 0.5 ng/ml IL-1
for 5 h and were subsequently overlayered
with 6 × 105 cells/2 ml or 2 × 106
cells/2 ml of the human leukemic cells for 30 min. A, the
cells were lysed with extraction buffer, and the cell extracts were subjected to immunoprecipitation with anti-Tyr(P) 4G10. The
immunoprecipitates were then immunoblotted with anti-Tyr(P) PY-20.
Molecular markers (kDa) are indicated on the left, and the
tyrosine-phosphorylated 120-130-kDa bands (arrow) are
indicated on the right. B, the cells were lysed
with extraction buffer, and the cell extracts were subjected to
immunoprecipitation with anti-p125FAK 2A7. One-half of the
immunoprecipitates were immunoblotted with anti-Tyr(P) PY-20. The
position of the p125FAK band is indicated on the
right. C, the other half of the
immunoprecipitates were immunoblotted with anti-p125FAK C-20.
The position of the p125FAK band is indicated on the right.
D, the cells were lysed with the extraction buffer, and the
whole cell lysates were immunoblotted directly with
anti-p125FAK C-20. The position of the p125FAK band is
indicated on the right. IP, immunoprecipitation;
IB, immunoblotting.
[View Larger Version of this Image (31K GIF file)]
-activated HUVEC other than p125FAK were reduced by
THP-1 seeding or not. Talin present at focal contacts (8) such as is
p125FAK, VCAM-1 expressed on the cell surface of
cytokine-activated endothelial cells (9), and
-catenin co-localized
at the sites of intercellular junctions with cadherin and
-catenin
(10), were probed with their respective antibodies on the same
transferred membrane. As shown in Fig. 2,
no changes in the amount of VCAM-1 and
-catenin were observed in
whole cell lysates of HUVEC co-cultured with THP-1 cells. In the case
of talin, a slight decrease was observed, and a possible degradation
fragment of approximately 200 kDa was identified. However, the extent
of the decrease in talin was far less than that observed in
p125FAK. In U937-treated HUVEC, patterns for the probed
proteins were almost the same as those observed in HUVEC treated with
THP-1 cells (Fig. 2).
Fig. 2.
Decreased levels of proteins in HUVEC induced
by co-culture with monocytic cell lines. Confluent HUVEC cultures
on gelatin-coated culture dishes were treated with 0.5 ng/ml IL-1 for 5 h and were subsequently overlayered with 6 × 105 cells/2 ml or 2 × 106 cells/2 ml
THP-1 or U937 cells for 30 min. The cells were lysed with extraction
buffer, and the whole cell lysates were immunoblotted directly with
anti-p125FAK C-20, anti-talin, anti-VCAM-1, or anti-
-catenin
antibodies on the same transferred membrane. Molecular markers (kDa)
are indicated on the left, and the positions of the proteins
of interest are indicated on the right.
[View Larger Version of this Image (39K GIF file)]
-catenin were not detected in whole cell lysates obtained
from 2 × 106 THP-1 cells alone, although talin was
faintly detectable (lane 4). In subsequent experiments we
included
-catenin as a control to show that equal amounts of HUVEC
protein were included in each sample of our assays.
-treated HUVEC
-stimulated HUVEC.
Although the amount of p125FAK did not return to initial
levels, a tendency for recovery of p125FAK was observed
(lane 10) 4 h from THP-1 cell seeding. Interestingly, the p125FAK degradation was not observed in unstimulated HUVEC
(lanes 1-3).
Fig. 3.
Kinetics of p125FAK
decrease induced by THP-1 binding to IL-1-treated HUVEC.
Confluent cultures of HUVEC on gelatin-coated culture dishes were
untreated (lanes 1-3) or treated with 0.5 ng/ml IL-1
(lanes 4-10) for 5 h and were subsequently overlayered with 1 × 106 THP-1 cells for the indicated times. The
cells were lysed with extraction buffer, and the whole cell lysates
were immunoblotted directly with anti-p125FAK C-20.
Subsequently,
-catenin on the same membrane was also probed. The
positions of the p125FAK and
-catenin bands are indicated on
the right.
[View Larger Version of this Image (39K GIF file)]
for 5 h and were subsequently overlayered with THP-1 cells. The cells were fixed, stained with rhodamine phalloidin, and observed by fluorescence microscopy. Well organized actin stress
fibers were observed in both unstimulated (Fig.
4A) and IL-1
-stimulated
HUVEC (Fig. 4E) before seeding of THP-1 cells. The well
developed stress fibers were also observed in unstimulated HUVEC which
were overlayered with THP-1 cells (Fig. 4, B-D). In contrast, THP-1 seeding markedly reduced the number, thickness, and
length of actin stress fibers in HUVEC preactivated with IL-1
(Fig.
4, F-H). The changes were observed from 30 min after
seeding THP-1 cells and continued for at least 2 h.
Fig. 4.
Changes in cytoskeletal structure induced by
THP-1 binding. Confluent cultures of HUVEC on gelatin-coated
plastic chamber slides were incubated in the absence (A-D)
or presence (E-H) of 0.5 ng/ml IL-1 for 5 h.
The HUVEC were untreated (A and E) or covered with 1.5 × 104 THP-1 cells for 0.5 h
(B and F), 1 h (C and
G), and 2 h (D and H). The cells
were then fixed and stained with rhodamine phalloidin. In panel
F, T represents adherent THP-1 cells.
[View Larger Version of this Image (111K GIF file)]
and TNF-
Treatment of HUVEC on the Decrease in
p125FAK in HUVEC
-treated
HUVEC but not in unstimulated HUVEC. Therefore, we investigated further
whether THP-1 cells could induce the decrease in p125FAK in
HUVEC stimulated with inflammatory stimuli other than IL-1
. HUVEC
were stimulated with TNF-
for 5 h and were then co-cultured with THP-1 for 30 min. The cells were lysed and subjected to
immunoblotting with anti-p125FAK C-20. As shown in Fig.
5A, decrease in the amount of
p125FAK after addition of THP-1 was observed not only in
IL-1
-activated HUVEC (lanes 2-5) but also in
TNF-
-activated HUVEC (lanes 7-10) in a manner dependent
on the concentration of the cytokines added. We simultaneously
investigated the binding of THP-1 cells to HUVEC activated with these
inflammatory stimuli. 51Cr-labeled THP-1 cells were added
to activated HUVEC and incubated for 30 min. As shown in Fig.
5B, it was observed that IL-1
and TNF-
treatment of
HUVEC augmented THP-1 binding to HUVEC in a dose-dependent
manner, suggesting a correlation between decrease in p125FAK
and binding of THP-1 cells to cytokine-activated HUVEC.
Fig. 5.
Effect of IL-1 and TNF-
treatment of
HUVEC on the decrease in p125FAK induced by
THP-1 and the adherence of THP-1 cells. A, confluent HUVEC
cultures on gelatin-coated culture dishes were treated with IL-1
(lanes 2-5) or TNF-
(lanes 7-10) for 5 h. Subsequently, 1 × 106 THP-1 cells were added and
incubated for 30 min. The cells were lysed with extraction buffer, and
the whole cell lysates were subjected to immunoblotting with
anti-p125FAK C-20. Subsequently,
-catenin on the same
membrane was also probed. The position of the p125FAK and
-catenin bands are indicated on the right. B,
HUVEC grown on a gelatin-coated 96-well microplate were treated with
IL-1
or TNF-
for 5 h. 51Cr-labeled THP-1 cells
were added to the activated HUVEC and incubated for 30 min. Nonadherent
cells were removed, and the adherence of THP-1 cells was determined.
Values shown represent the mean ± S.D. of triplicate wells.
[View Larger Version of this Image (44K GIF file)]
and TNF-
induce the expression of adhesion molecules such as ICAM-1, VCAM-1, and
E-selectin on the surface of endothelial cells (9, 11, 12). Therefore, we investigated whether pretreating THP-1 cells with a blocking antibody to the counter-receptors for ICAM-1, VCAM-1, or E-selectin could inhibit the decrease in p125FAK or not. THP-1 cells have
been reported to express
2 integrin, a
subunit of the
2
integrin family,
4 integrin, an
subunit of very late antigen-4,
and sialyl Lex (13).
2 integrins, very late antigen-4,
and sialyl Lex are known to interact with ICAM-1, VCAM-1,
and E-selectin, respectively (14-18). THP-1 cells were incubated with
50 µg/ml anti-
4, anti-
2, anti-sialyl Lex, or a
mixture of these three mAbs at rt for 1 h. After washing three
times with medium, the antibody-pretreated THP-1 cells were seeded over
IL-1
-activated HUVEC. As shown in Fig.
6A, pretreatment of THP-1
cells with a mixture of anti-
4, anti-
2, and anti-sialyl Lex inhibited the decrease in p125FAK in
IL-1
-activated HUVEC (lane 6), whereas treatment with
either of these mAbs alone could not inhibit the decrease in
p125FAK (lanes 3-5). Fig. 6B shows the
result of quantification of the density of the detected blots in Fig.
6A. In the case of cell adhesion assays, only treatment with
a mixture of the three mAbs similarly inhibited the adherence of THP-1
cells to activated HUVEC (Fig. 6C, lane 6).
Fig. 6.
Inhibition of the decrease in
p125FAK and adherence of THP-1 cells by
pretreatment with mAbs to adhesion molecules. A, THP-1 cells
were incubated with 50 µg/ml mAbs to adhesion molecules for 60 min at
rt. After washing, the pretreated THP-1 cells were seeded on
IL-1-stimulated HUVEC, and the plates were incubated for 30 min. The
cells were lysed and subjected to immunoblotting with
anti-p125FAK C-20. Subsequently,
-catenin on the same
membrane was also probed. The positions of the p125FAK and
-catenin bands are indicated on the right. B,
intensities of immunoblotted p125FAK were quantified by
densitometer. C, HUVEC grown on a gelatin-coated 96-well
microplate were treated with IL-1
for 5 h.
51Cr-Labeled and mAb-treated THP-1 cells were added to the
activated HUVEC and incubated for 30 min. Nonadherent cells were
removed, and the adherence of THP-1 cells was determined. Values shown represent the mean ± S.D. of quadruplicate wells. Control mAbs, mixture of class-matched irrelevant mAbs.
[View Larger Version of this Image (23K GIF file)]
-stimulated HUVEC induces a decrease in the amount of the
p125FAK molecule in HUVEC. It has been reported that inhibition
of the function of p125FAK by p41/43FRNK
(pp125FAK-related non-kinase) blocked the formation of focal
contacts, indicating a functional relation between p125FAK and
the formation of focal contacts (25). Moreover, the loss of
p125FAK has been reported to be a prerequisite for cell
detachment (26). Taken together, it was considered that the decrease in
p125FAK in HUVEC indicates a decrease in the function of focal
contacts, resulting in decreased strength of attachment of the
endothelial cell to the extracellular matrix. The decrease in the
adhesiveness of endothelial cells would enable monocytes to migrate
beneath the endothelial cells more easily.
-stimulated HUVEC was induced
not only by monocytic THP-1 cells but also by monoblastic U937 cells.
Furthermore, the decrease induced by THP-1 was also observed in HUVEC
grown on collagen type I or fibronectin (data not shown), indicating
that the decrease in p125FAK was independent of the
extracellular matrix on which the HUVEC were grown. These results
indicate that the decrease in the amount of tyrosine-phosphorylated
p125FAK might be a commonly observed event in
cytokine-activated HUVEC.
-activated HUVEC did not induce a decrease in
p125FAK levels (data not shown). With regard to the
counter-receptor(s) on the surface of HUVEC responsible for the
transduction of the p125FAK-modifying signal, although the
decrease in the amount of p125FAK was partially blocked by a
mixture of neutralizing mAbs against ICAM-1, VCAM-1, and E-selectin
pathways, a direct role for these three adhesion molecules in the
transmission of a regulatory signal has yet to be established. It is
possible that adhesion molecules such as ICAM-1, VCAM-1, and
E-selectin, the expression of which is augmented by inflammatory
cytokines, enable THP-1 cells to bind tightly to HUVEC, resulting in
the effective transduction of the p125FAK reducing signal
induced by other molecule(s) into HUVEC.
*
This study was supported by Special Coordination Funds for
Promoting Science and Technology (Joint Research Utilizing Scientific and Technological Potential in Region) of the Science and Technology Agency of the Japanese Government.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.
§
To whom correspondence should be addressed: Fujisaki Institute,
Hayashibara Biochemical Laboratories, Inc., 675-1 Fujisaki, Okayama
702, Japan. Tel.: 81-86-276-3141; Fax: 81-86-276-6885.
1
The abbreviations used are: PECAM-1,
platelet/endothelial cell adhesion molecule-1; ICAM-1,
intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion
molecule-1; HUVEC, human umbilical vein endothelial cells;
p125FAK, focal adhesion kinase; IL-1, interleukin-1
;
TNF-
, tumor necrosis factor-
; anti-Tyr(P), anti-phosphotyrosine;
mAb, monoclonal antibody; rt, room temperature; PBS, phosphate-buffered
saline.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.