| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
4ß1 Integrin1
*
Department of Cell Biology, Institut Cochin de Génétique Moléculaire, Paris, France; and
Weill Medical College, Cornell University, New York, NY 10021
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
4 integrin, and a direct interaction between
ANX1 and the 4 integrin could be documented by
immunoprecipitation experiments. Moreover, ANX1 competed with the
endothelial integrin counterreceptor, VCAM-1, for binding to
4 integrin. These results indicate that ANX1 plays an
important physiological role in modulating monocyte firm adhesion to
the endothelium. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
4ß1 integrin (also
known as VLA-4 or CD49d/CD29), the
Mß2 integrin (Mac-1 or
CD11b/CD18), and the
Lß2 integrin (LFA-1 or
CD11a) (1). The interactions of leukocyte integrins with
ICAM-1 and VCAM-1, two Ig-like adhesion molecules expressed by
endothelial cells, determine the firm adhesion of activated leukocytes
to the EC, leukocyte emigration, and their recruitment into the
inflammatory site.
Annexin 1 (ANX1, lipocortin 1) belongs to a family of multifunctional
proteins that all bind acidic phospholipids in the presence of calcium
ions. The observation that cell-associated ANX1 was up-regulated by
glucocorticoids (GCs) suggested that ANX1 itself might participate in
the anti-inflammatory effects of these compounds (2).
A single administration of dexamethasone in laboratory animals
increased ANX1 levels in circulating leukocytes (3).
Conversely, lowering endogenous steroids by adrenalectomy or by a
sustained treatment with the steroid antagonist mifepristone (RU486)
reduced the amount of cell-associated ANX1 protein and mRNA by 
50%
(4). In inflammatory models, exogenous ANX1 inhibited
recruitment of polymorphonuclear (PMN) leukocytes in vivo (5, 6). Moreover, treatments with Ab to ANX1 blocked the
anti-inflammatory effect of dexamethasone in animal models of acute
inflammation (7). In these models, the inhibition of cell
migration to inflammatory sites appeared to be one of the mechanisms by
which GCs exert their anti-inflammatory effect.
Among the different classes of peripheral blood leukocytes, both PMN
and monocytes have been shown to contain large amounts of ANX1
(8, 9, 10, 11). The monocytic cell line U937 expresses increased
amounts of endogenous ANX1 upon cell differentiation into macrophages
(12), and GC treatment of differentiated U937 cells
produced translocation of the ANX1 to external leaflet of the plasma
membrane (13). Adhesion of monocytes to the vascular
endothelium during inflammation is a critical step preceding their
recruitment into extravascular tissue (14). Therefore, in
the present study, we have investigated whether ANX1 modulates monocyte
adhesion to human microvascular endothelial cells and have
characterized the molecular mechanisms involved. We show that both
exogenous rANX1 and endogenous ANX1 inhibit the firm adhesion of U937
cells to EC, most likely through direct interaction with the
4ß1 integrin. These
data indicate that the ANX1 can mediate inhibition of monocyte
migration through the endothelium, as part of the anti-inflammatory
actions of ANX1.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Wild-type U937 cells, stable clones of U937-expressing antisense ANX1 mRNA (15), and control U937 transfected with the empty expression vector were maintained in culture in RPMI medium (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% (v/v, 7.7 mg/ml) glutamine. G-418 (200 µg/ml) was used to maintain stable transfected clones. A human microvascular endothelial cell line (HBMEC) from adult human bone marrow (16) was used to study U937 cell adhesion. HBMEC cells display a normal pattern of regulation of adhesion molecules (17); they were maintained in DMEM supplemented with 5% FCS, 10 mM HEPES, and antibiotics at the same concentrations as above. In some experiments, U937 cells were differentiated into macrophages by incubation for 24 h with 6 ng/ml of PMA, as described (12), before use in adhesion studies.
Abs, recombinant proteins, and peptides
The following Abs were used: A polyclonal anti-ANX1 Ab
(1:1000) (18) was used for immunofluorescence analysis as
well as immunoprecipitation (IP) studies. A mAb, 1-B (5, 19), was used to block biological activities of ANX1. Ab against
the 4 integrin (FITC-conjugated CD49d mAb,
diluted 1/20) was from Immunotech (Luminy, France). Ab directed against
LFA-1 (CD11a mAb diluted 1/40) and Ab directed against VCAM-1 (1/40)
were from R&D Systems (Minneapolis, MN). The Ab against
4 integrin used for IP (1/100) was from Santa
Cruz Biotechnology (Santa Cruz, CA), as was anti-actin Ab (1/100).
A goat anti-rabbit Cyanin 3 secondary Ab (diluted 1/300) and a goat
anti-mouse FITC (1/300) were both from Boehringer Mannheim
(Mannheim, Germany). The Ab against CXCR4 was from PharMingen (San
Diego, CA) (monoclonal 12GA5-N, at 0.26 mg/ml).
The ANX1 N-terminal acetylated, 2–25 synthetic peptide was kindly provided by Dr. M. Perretti (The William Harvey Institute, London, U.K.).
Recombinant ANX1, annexin 5 (ANX5), and a chimeric protein ANX1–5, were expressed as GST-fusion proteins in pGEX vectors, as described (6). Fusion proteins were purified from bacterial lysates by affinity chromatography using prepacked glutathione-Sepharose 4B, and the GST moiety was removed by cleavage with thrombin (20). All three recombinant proteins were further purified by fast protein liquid chromatography (Pharmacia). Proteins were >95% pure, as determined by SDS-PAGE, fast protein liquid chromatography profiles, and Western blot analysis. Endotoxin contamination was less than 20 pg/ml, as measured by the Limulus amebocyte chromogenic assay (Calbiochem, La Jolla, CA).
Adhesion of U937 cells to endothelial cells
Adhesion of U937 cells to HBMEC was measured, as previously
described (21). Briefly, 1 x
106 U937 cells were radiolabeled by incubation in
100 µl of RPMI containing 10% FCS and 20 µCi of
51Cr (as sodium chromate) for 1 h at 37°C.
After washing three times with HBSS labeled U937 cells were resuspended
in RPMI 1640 medium containing 10% FCS, and were then incubated at
37°C for 30 min with or without human rANX1 (6). Next,
200 µl (2 x 105) labeled U937
cells, treated or not with ANX1, were added to each well of
96-well plates containing confluent HBMEC monolayers. The HBMEC had
either been pretreated or not pretreated with 5 ng/ml human rTNF-
for 18 h (Amersham, Arlington Heights, IL).
After incubation of U937 on HBMEC monolayers for 1 h at 37°C, nonadherent U937 cells were removed by four consecutive washes. U937 cells remaining adherent to HBMEC monolayers after washing were then lysed with 1% Triton X-100 and gamma emissions quantified with a LKB (1282-Compugamma-CS) gamma counter. The percentage of adherent U937 was calculated from 6 to 12 independent wells per condition, as the ratio between radioactivity recovered in the cell lysate and the total amount of radioactivity in the initial aliquot of labeled U937 cells.
For analysis of the effects of externalized endogenous ANX1 on U937 adhesion to HBMEC, U937 cells that had been first differentiated with PMA and then treated with dexamethasone to externalize the up-regulated endogenous ANX1 were incubated with unstimulated HBMEC monolayers and further processed to quantitate adhesion, as described above.
Radioiodination of rANX1 and 125I-ANX1-binding assay
rANX1 was radioiodinated using the chloramine T method
(22). Small quantities of rANX1 (500 ng in 50 µl of PBS,
pH. 7.2) were added to a mixture of 15 µl of chloramine T, according
to Lowenthal et al. (23). The radiolabeled protein was
applied to a prepacked Sephadex G-50 column prepacked in PBS (with 1%
BSA) and purified. Specific radioactivity was further calculated and
was 20 µCi/µg of protein.
Binding studies of 125I-ANX1 to cells were conducted according to Dower et al. (24). Briefly, aliquots of 2 x 105 cells were incubated in RPMI medium in the presence of varying concentrations of 125I-ANX1 at 4°C for 4 h. To avoid any potential internalization of the labeled protein, experiments were conducted at 4°C, a temperature known to inhibit endocytosis. Bound 125I-ANX1 was separated from free by centrifugation through a 30% sucrose cushion. Nonspecific binding was measured in the presence of a 50–100 molar excess of unlabeled ANX1.
Immunofluorescence and confocal imaging
U937 cells (treated or not with PMA and/or dexamethasone) were
harvested, and aliquots of 1 x 106 cells
were incubated at 37°C in the presence of 100 nM rANX1. Cells were
then fixed with 2% paraformaldehyde for 10 min at room temperature
(RT) and washed three times in a buffer consisting of 25 mM HEPES, 1 mM
CaCl2, 1 mM MgCl2, and 1%
BSA. Cells were incubated for 2 h at RT with a polyclonal
anti-ANX1 Ab (1:1000), or with mAbs directed against the
4 integrin (FITC-conjugated CD49d mAb, diluted
1/20) or against LFA-1 (CD11 mAb, diluted 1/40). Anti-actin Ab was
used as the control of cellular membrane integrity. PBS/BSA was used as
negative control. A goat anti-rabbit Cyanin 3 secondary Ab (diluted
1/300) and a goat anti-mouse FITC secondary Ab (diluted 1/300) were
used, as was appropriate, for 30 min at RT. After four washes, samples
were mounted in moviol (Sigma, St. Louis, MO). Images were obtained
with a laser-scanning confocal microscope (MRC-1000; Bio-Rad, Hercules,
CA) mounted on a Nikon Optiphot microscope. Dual detection was
performed with separate photomultiplier tubes, and resultant images
were merged.
Polarization of U937 cells
U937 cells (107) were added to the upper chamber of Transwell-Clear inserts (3 µm diameter pore; Costar, Cambridge, MA). SDF-1 (R&D Systems) was added at a concentration of 10-7 M to the lower chamber of the transwell. After 18 h at 37°C, U937 cells that had migrated into the lower chamber were harvested, washed, and incubated with human rANX1 (100 nM). After fixation in paraformaldehyde, cells were processed for immunofluorescence studies, as described above. A mAb against CXCR4 (12GA5-N terminal region 0.26 mg/ml; PharMingen) was used as marker of cell polarization.
Immunoprecipitation
Cells (107) were collected after 30-min
incubation with human rANX1 (100 nM) or human rVCAM-1 (10 nM; R&D
Systems) or both, and lysed in 500 µl of a lysis buffer (10 mM Tris,
pH 7.2, 150 mM NaCl, 1 mM CaCl2, 1 mM
MgCl2 and 1% Nonidet P-40) for 10 min on ice in
presence of protease inhibitors (100 mM PMSF, 1 µg/ml leupeptin, 1
µg/ml aprotinin). Cell lysates were preincubated with protein
A-Sepharose and preimmune sera and centrifuged, and the recovered
supernatants were incubated at 4°C overnight with protein A-Sepharose
and appropriate Abs: either polyclonal anti-ANX1, anti-VCAM-1,
or anti-4 integrin. After two washes in
low salt (150 mM NaCl) and three washes in high salt (500 mM NaCl)
buffers, Laemmli sample buffer (25) was added to pellets
and samples were boiled for 5 min. Samples were then analyzed by
SDS-PAGE, and separated proteins transferred onto nitrocellulose
membranes. The membranes were saturated in 5% BSA and then incubated
with a polyclonal Ab directed against the 4
integrin (dilution 1/100) or ANX1 (1/100) or VCAM-1 (1 µg/ml). An
anti-rabbit or anti-mouse Ig HRP secondary Ab (1/10,000;
Amersham) was employed, and peroxidase reaction was developed using
enhanced chemiluminescence (ECL; Amersham).
Statistics
Data, reported as mean ± SEM of n separate observations, were analyzed by a two-tailed Student’s t test with p value threshold of 0.05, which was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ANX1 is externalized by PMN after contact with endothelium (9) and by U937 cells after treatment with dexamethasone (13). To study whether extracellular ANX1 modulates the adhesion of U937 monocytes to endothelial cells, we first evaluated its paracrine effects of rANX1 in a monocyte adhesion assay (21).
HBMEC monolayers were treated with TNF- to induce expression of
E-selectin and VCAM-1, as previously reported (17).
Adhesion of U937 cells to TNF--activated HBMEC was examined starting
12 h after exposure of the latter to TNF-.
51Cr-labeled U937 cells were seeded on monolayers
of control or stimulated HBMEC for varying time periods, the monolayers
were then washed, and the radioactivity associated with cells remaining
adherent was measured (Fig. 1
A). Pretreatment of U937
cells with human rANX1 resulted in a rapid inhibition of U937 cell
adhesion to TNF--stimulated HBMEC (Fig. 1A, closed
squares). ANX1 alone produced 40% inhibition of U937 cell adhesion,
which represented inhibition of adhesion comparable with that induced
by Abs that blocked the 4 integrin, LFA-1, or
VCAM. Maximal inhibition (43%) was observed 2 h after ANX1
treatment of the U937. To confirm that inhibition of monocyte adhesion
was due to ANX1, U937 cells were seeded on HBMEC monolayers either in
the presence or absence of the anti-ANX1 mAb 1-B or in the presence
of unrelated control Ab (Fig. 1B). The specific
anti-ANX1 mAb 1-B reversed most of the ANX1-inhibitory effect on
U937 adhesion, whereas control Ab did not reverse the inhibition of
adhesion.
|
). Correspondingly,
the calculated for ANX1 was 89 nM, for the ANX1–5 chimera 33 nM, while
the EC50 for ANX5 was 690 nM, almost 10-fold
higher. Indeed, the most potent inhibitor of U937 adhesion was the
ANX1–5 chimeric protein. In comparison, the maximal inhibition of
adhesion observed with the N-terminal peptide of ANX1 (Ac 2–26)
reached 75% of that of ANX1. This maximum inhibition with peptide
alone required a molar concentration 20 times higher than that of
intact ANX1 (EC50 1.6 µM). These data
indicate that the inhibitory effect of ANX1 on U937 cell adhesion
depends mainly on its N-terminal domain. The bell-shaped pattern of the
dose-response curves may relate either to toxic effects at highest
concentrations or to additional specific characteristics of ANX1
binding sites, such as dimerization.
|
ANX1 presumably binds to a surface site on the U937 plasma
membrane that blocks adhesion of U937 cells to HBMEC. Binding assays
with 125I-ANX1 were performed at 4°C (to avoid
endocytosis of bound ligand) to characterize this putative binding
site. The binding of 125I-ANX1 to U937 cells was
biphasic. Nonlinear regression analysis of binding data showed a
saturable binding site characterized by a
Kd of 14.3 + 6.1 nM, and a maximal
binding of 94,000 + 24,000 sites per cell (Fig. 3). This high affinity
125I-ANX1 binding site was not significantly
modified by calcium-chelating compounds: in the presence of EDTA/EGTA,
the Kd was 14.8 + 5.9 nM, and maximal
binding was 63,000 + 12,600 sites per cell. In contrast, a second, low
affinity, binding component, which could not be characterized because
saturation was not achieved, was blunted when U937 cells were incubated
with 125I-ANX1 in the presence of 1 mM EDTA and
EGTA. These findings are consistent with the hypothesis that the
N-terminal domain (rather than the Ca2+-binding
domain) is principally involved in the inhibitory effects of ANX1 on
U937 cell binding to endothelium.
|
To investigate whether endogenous ANX1 could have an autocrine
effect on U937 function, U937 cells were first differentiated in vitro
into macrophages by incubating with PMA (13) and then were
treated for 12 h with 1 µM dexamethasone to promote the
externalization of ANX1 to the outer side of the plasma membrane
(13). PMA-differentiated U937 macrophages display more
adhesion than do undifferentiated U937 cells, no matter whether the
differentiated U937 cells have been treated or have not been treated
with dexamethasone. Compared with adhesion of PMA-differentiated U937,
adhesion of dexamethasone-treated, PMA-differentiated U937 was
decreased by 40% (Fig. 4A).
This decrease in adhesion was reversed when the dexamethasone-treated,
PMA-differentiated U937 macrophages were coincubated with a mAb
directed against ANX1 (mAb 1-B at 20 µg/ml), indicating that ANX1
mediates the inhibition of adhesion that is associated with
dexamethasone treatment. A control Ab not directed against ANX1 did not
modify the inhibition of U937 adhesion induced by dexamethasone
treatment.
|
B). Taken together, our
results indicate that both exogenous ANX1 acting in a paracrine manner
and endogenous ANX1 acting in an autocrine manner serve as potent
inhibitors of monocyte adhesion to endothelial cells.
ANX1 colocalizes with 4ß1, but not
with Lß2 integrins on U937 cells
Because the results described above suggest that ANX1 inhibits
monocytic cell adhesion to EC by interacting with or masking an
adhesion molecule at the surface of U937 cells, we next characterized
the expression of cell surface adhesion molecules by flow cytometry of
U937 cells before and after treatment with PMA, or treatment with PMA
and then dexamethasone. These cells expressed both
4ß1 and
Lß2 integrins at their
surface (Fig. 5, upper and
lower panels, respectively). Dexamethasone treatment did not
decrease and even increased integrin expression compared with untreated
cells (not shown). Redistribution of
4ß1 and
Lß2 integrins was
analyzed by confocal microscopy using cells incubated with ANX1 for 30
min at 37°C (Fig. 6). Exogenous ANX1
(Fig. 6B, red) was mostly concentrated to one pole. ANX1 did
not colocalize with the
Lß2 integrin (Fig. 6B, upper series, green), whereas it colocalized with the
4ß1 integrin (Fig. 6B, lower series, green), as indicated by merging confocal
images (Fig. 6B, lower series I + II, yellow). The plot of
fluorescent signals within the cells confirmed the colocalization of
the two fluorochromes in cells doubly labeled with anti-ANX1 and
anti-4 integrin Abs, but not in cells
doubly labeled with anti-ANX1 and
anti-L integrin Abs (Fig. 6C).
Similar results were also obtained in cells treated with PMA and
dexamethasone, in which endogenous externalized ANX1 colocalized with
the 4 integrin, but not with LFA-1
(L) (Fig. 6, D–F). In an effort to
characterize more precisely the subcellular colocalization of ANX1 with
the 4 integrin, we took advantage of the
expression of the chemokine receptor CXCR4 at the surface of U937
cells. Preliminary unpublished experiments have shown that U937 cells
are chemoattracted by the CXCR4 ligand SDF-1. Chemotaxis is associated
with particular morphological changes and with redistribution of CXCR4
to the leading edge of the cell (not shown). U937 cells were plated in
the upper chamber of transwell plates, and 100 nM SDF-1 was added to
the lower chamber. After 18-h incubation, U937 cells that had migrated
into the lower chamber were harvested and compared with control resting
U937 cells for their distribution of CXCR4 and of ANX1 binding sites
(Fig. 7). As expected, CXCR4 displayed a
homogeneous distribution at the plasma membrane of resting cells,
whereas it concentrated in one pole of SDF-1-activated polarized U937
cells, presumably at the leading edge. Exogenous ANX1 was mostly
concentrated in one pole of resting cells and only partially
colocalized with CXCR4. In SDF-1-activated cells, ANX1 seemed to be
more diffusely distributed than in resting cells but, interestingly, it
was almost completely excluded from the area of the plasma membrane in
which CXCR4 molecules were concentrated.
|
|
|
The adhesion molecule VCAM-1 expressed by endothelial cells is
known to interact with the
4ß1 integrin. In our
biological assay, ANX1-mediated inhibition of U937 adhesion to EC was
blunted by incubation with the extracellular domain of VCAM-1 (Fig. 1B). In the same assay, the extracellular domain of VCAM-1
could not block cell adhesion directly. These findings, together with
the observations from confocal microscopy experiments, that ANX1 and
4ß1 integrin
colocalize, suggest that ANX1 and VCAM-1 may compete for binding to the
4ß1 integrin. To test
this hypothesis, U937 cells were incubated with rANX1 and/or the
extracellular domain of VCAM-1 for 30 min at 37°C, then were lysed
and the lysates incubated with specific Abs directed against either
ANX1 or VCAM-1. Immunoprecipitated proteins were blotted on membranes
and probed with an anti-4
integrin-specific Ab. Both ANX1 and VCAM-1 coprecipitated with the
4 integrin (Fig. 8A, lanes 1 and
3). When U937 cells were incubated with both ANX1 and VCAM-1
(Fig. 8A, lane 2) no 4
integrin could be coprecipitated with ANX1, indicating that ANX1 and
VCAM-1 compete for binding to the
4ß1 integrin on intact
cells. Control IP with anti-4 Abs, probed
with anti-VCAM-1 or anti-ANX1 Abs, confirmed that both ANX1 and
VCAM-1 may be found in molecular complexes with
4 integrin (Fig. 8B). A similar
observation was made in U937 treated with PMA and dexamethasone to
externalize endogenous ANX1 (Fig. 8C).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The inhibitory effect is mediated by the unique N terminus of the ANX1 molecule. Thus, both rANX1 and a chimeric fusion protein between the N-terminal domain of ANX1 and the core domain of ANX5, ANX1–5 chimera, which share the same N-terminal segment, inhibited monocyte adhesion to a similar extent when they were incubated with U937 cells, whereas rANX5, which has a different N terminus, displayed only one-hundredth as much inhibition on a molar basis. In a previous report, an N-terminal peptide present in ANX1 that spans aa 2–26 was observed to block PMN adhesion to endothelial cells in vitro (26). We showed in this study that this peptide also blocks U937 cell adhesion; the efficacy of the peptide as an inhibitor is 20-fold less than that of ANX1 itself. This finding, together with the 3-fold greater potency of the ANX1–5 chimera compared with ANX-1, suggests that the annexin core domain does participate in the correct folding of the N-terminal domain. The predominant role of the N-terminal domain in this inhibitory effect of ANX1 is further supported by the binding studies that show the lack effect of Ca2+ on inhibition of adhesion: namely, that the high affinity binding of ANX1 to U937 cells is not affected by Ca2+ depletion.
Monocytes contain abundant intracellular stores of ANX1. Under physiological conditions of cell migration, monocytes and PMN display marked increases in ANX1 associated with the outer surface of the plasma membrane in vivo (13) (11). We investigated in this study whether or not ANX1 externalization had functional significance for cell adhesion, using U937 cells in which ANX1 production had first been stimulated by PMA, and then ANX1 externalization then effectuated by dexamethasone treatment (13). PMA + dexamethasone-treated U937 cells adhered significantly less to activated HBMEC than did control monocytes. We confirmed that the action of dexamethasone to decrease PMA-induced monocyte adhesion to EC was mediated by externalization of endogenous ANX1, by showing that exposure of the PMA + dexamethasone-treated U937 to an anti-ANX1 Ab reversed the inhibitory effect that dexamethasone exerted on cell adhesion. Furthermore, stable U937 clones expressing antisense ANX1 mRNA contained 20–30% less ANX1 than did the parental U937 cells (15), and adhered better to HBMEC than did stable U937 clones transfected with an empty expression vector. These data confirm that autocrine ANX1 is directly involved in the inhibition of cell adhesion in this model.
Leukocyte adhesion and extravasation, key phenomena in inflammation,
depend on a complex array of interactions between adhesion molecules
present on leukocytes and those on endothelial cells. For example, the
4ß1 integrin, present
on monocytes and lymphocytes, interacts with its counter structure,
VCAM-1, present on activated endothelium (27, 28). The
4ß1 integrin mediates
several functions such as tethering, rolling, and firm arrest on VCAM-1
(29, 30, 31). Monocytes express multiple integrins in vitro,
including the 4ß1,
Mß2, and
Lß2 integrins. In
vivo, each of these integrins has a distinct function in cell
migration, because only simultaneous blockade of all three integrins at
the same time completely abolishes monocyte accumulation in tissues
(32). Previous observations that blocking adhesion
molecules hampers inflammatory reactions (33) prompted us
to investigate whether ANX1 interacts with one or more adhesion
molecules expressed by U937 cells. We first established by flow
cytometry that these U937 cells express both the
4ß1 and the
Lß2 integrins. The
bone marrow endothelial cells used in our study express ICAM-1 and
VCAM-1 adhesion molecules upon stimulation with TNF-
(17). Using confocal microscopy, we showed that exogenous
rANX1 colocalizes on the U937 cell membrane with
4 integrin, but not with the
Lß2 integrin on the
U937 cell membrane. Similar results were also obtained with U937 cells
in which endogenous ANX1 had been externalized by sequential treatment
with PMA and then dexamethasone. IP studies confirmed and extended
these findings. Since ANX1 not only coprecipitated with the
4 integrin, but also competed with VCAM-1 for
binding to the 4 integrin, our results provide
a plausible mechanism by which externalized autocrine or paracrine ANX1
may inhibit firm adhesion.
As a result of chemoattractant activation, leukocytes become polarized and migrate in the direction of the chemotactic gradient. During this process, chemoattractant receptors and integrins are redistributed in different areas of the plasma membrane. Whereas in most cases chemoattractant receptors are concentrated at the leading edge of the cells (34), integrins are redistributed at the trailing edge or uropod (35, 36). In SDF-1-activated polarized U937 cells, CXCR4 receptors become concentrated at one pole of the cell, which is almost completely devoid of ANX1. ANX1, in turn, appears to be redistributed at the trailing edge of the cell. The association between ANX1 and integrins thus seems to be maintained in activated polarized cells.
ANX 1 has long been known as a mediator of the anti-inflammatory properties of GCs. Whereas early studies focused on such intracellular effects of cytoplasmic ANX1 as the inhibition of phospholipase A2 (15, 37, 38), more recent reports indicated that ANX1 may have additional important physiological actions after it becomes externalized at the outer side of the plasma membrane (6, 9, 11). Our data showing that ANX1 inhibits monocyte adhesion to endothelium through an autocrine/paracrine interaction with surface integrins strongly support this hypothesis. Accordingly, the fate of activated leukocytes, which either migrate into tissues or detach from the endothelium, may depend upon a fine balance between leukocyte integrins and other species of regulatory molecules, such as ANX1, that are expressed on the cell surface. To obtain a clearer picture of ANX1 functions affecting leukocyte physiology is an important issue that remains to be solved to understand which are the precise molecular signals and what are the cellular mechanisms that promote ANX1 externalization.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Egle Solito at the current address: Department of Neuroendrocrinology, Division of Neuroscience, Imperial College School of Medicine, Charing Cross Hospital, Fulham Palace Road, London W6 8RF U.K.
3 Current address: Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, U.K.
4 Abbreviations used in this paper: EC, endothelial cell; ANX1/ANX5, annexin 1/5; ANX1–5, chimeric annexin 1-annexin 5; CXCR, CXC chemokine receptor; GC, glucocorticoid; HBMEC, human bone marrow endothelial cell; 125I-ANX1, 125I-labeled ANX1; IP, immunoprecipitation; PMN, polymorphonuclear; RT, room temperature; SDF, stromal cell-derived factor.
Received for publication November 11, 1999. Accepted for publication May 22, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-activated vascular endothelium under flow in vitro. J. Immunol. 157:326.[Abstract]
4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80:413.[Medline]
triggers a chemotactic response and induces cell polarization in human B lymphocytes. Eur. J. Immunol. 28:2197.[Medline]
This article has been cited by other articles:
|
|
 |
|
  R. D. Ye, F. Boulay, J. M. Wang, C. Dahlgren, C. Gerard, M. Parmentier, C. N. Serhan, and P. M. Murphy International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the Formyl Peptide Receptor (FPR) Family Pharmacol. Rev., June 1, 2009; 61(2): 119 - 161. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Prevost, J. V. Mitsios, H. Kato, J. E. Burke, E. A. Dennis, T. Shimizu, and S. J. Shattil Group IVA cytosolic phospholipase A2 (cPLA2{alpha}) and integrin {alpha}IIb{beta}3 reinforce each other's functions during {alpha}IIb{beta}3 signaling in platelets Blood, January 8, 2009; 113(2): 447 - 457. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Yohannes, J. Chang, G. J. Christ, K. P. Davies, and M. R. Chance Proteomics Analysis Identifies Molecular Targets Related to Diabetes Mellitus-associated Bladder Dysfunction Mol. Cell. Proteomics, July 1, 2008; 7(7): 1270 - 1285. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. P. G. Hayhoe, A. M. Kamal, E. Solito, R. J. Flower, D. Cooper, and M. Perretti Annexin 1 and its bioactive peptide inhibit neutrophil-endothelium interactions under flow: indication of distinct receptor involvement Blood, March 1, 2006; 107(5): 2123 - 2130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Spessotto, M. Cervi, M. T. Mucignat, G. Mungiguerra, I. Sartoretto, R. Doliana, and A. Colombatti beta 1 Integrin-dependent Cell Adhesion to EMILIN-1 Is Mediated by the gC1q Domain J. Biol. Chem., February 14, 2003; 278(8): 6160 - 6167. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Perretti, F. Ingegnoli, S. K. Wheller, M. C. Blades, E. Solito, and C. Pitzalis Annexin 1 Modulates Monocyte-Endothelial Cell Interaction In Vitro and Cell Migration In Vivo in the Human SCID Mouse Transplantation Model J. Immunol., August 15, 2002; 169(4): 2085 - 2092. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Gerke and S. E. Moss Annexins: From Structure to Function Physiol Rev, April 1, 2002; 82(2): 331 - 371. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. LA, M. D'AMICO, S. BANDIERA, C. DI FILIPPO, S. M. OLIANI, F. N. E. GAVINS, R. J. FLOWER, and M. PERRETTI Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action FASEB J, October 1, 2001; 15(12): 2247 - 2256. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. de Coupade, M. N. Ajuebor, F. Russo-Marie, M. Perretti, and E. Solito Cytokine Modulation of Liver Annexin 1 Expression during Experimental Endotoxemia Am. J. Pathol., October 1, 2001; 159(4): 1435 - 1443. [Abstract] [Full Text]
|
|
|
|
|
|
M. Perretti, S. J. Getting, E. Solito, P. M. Murphy, and J.-L. Gao Involvement of the Receptor for Formylated Peptides in the in Vivo Anti-Migratory Actions of Annexin 1 and its Mimetics Am. J. Pathol., June 1, 2001; 158(6): 1969 - 1973. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. J. Strausbaugh and S. D. Rosen A Potential Role for Annexin 1 as a Physiologic Mediator of Glucocorticoid-Induced L-Selectin Shedding from Myeloid Cells J. Immunol., May 15, 2001; 166(10): 6294 - 6300. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Srikrishna, K. Panneerselvam, V. Westphal, V. Abraham, A. Varki, and H. H. Freeze Two Proteins Modulating Transendothelial Migration of Leukocytes Recognize Novel Carboxylated Glycans on Endothelial Cells J. Immunol., April 1, 2001; 166(7): 4678 - 4688. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or not (
) with 5 ng/ml TNF-
) showed a
reduced firm adhesion to HBMEC. U937 cell adhesion was measured, as
described in Materials and Methods. Bars indicate mean
± SEM of triplicates measurements; data are representative of six
independent experiments giving similar results. *, Significant
difference between ANX1-treated and control cells, p <
0.001; **, significant difference between TNF-
