* Lung Biology Center, Center for Occupational and Environmental Health, Cardiovascular Research Institute and the
Department of Medicine, University of California, San Francisco, California 94143; and Elan Pharmaceuticals, South San
Francisco, California 94080
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The integrin 9
1 has been shown to be
widely expressed on smooth muscle and epithelial cells,
and to mediate adhesion to the extracellular matrix
proteins osteopontin and tenascin-C. We have found
that the peptide sequence this integrin recognizes in
tenascin-C is highly homologous to the sequence recognized by the closely related integrin
4
1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine
whether
9
1 also recognizes VCAM-1, and whether any such interaction would be biologically significant.
In this report, we demonstrate that
9
1 mediates stable cell adhesion to recombinant VCAM-1 and to
VCAM-1 induced on human umbilical vein endothelial
cells by tumor necrosis factor-
. Furthermore, we show
that
9
1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1
and tenascin-C. Finally,
9
1 and
4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible
role for
9
1/VCAM-1 interactions in extravasation of
neutrophils at sites of acute inflammation.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
INTEGRINS are heterodimeric receptors for extracellular
matrix and cell surface counter-receptors which play
important roles in embryonic development, inflammation, wound healing, and tumorigenesis (Hynes, 1987,
1992
; Ruoslahti and Pierschbacher, 1987
). Integrin ligand-binding specificity is determined by structural features of
each subunit, but there is considerable ligand-binding
overlap among integrin heterodimers. One clue to ligand-binding overlap has been the degree of sequence homology among integrin
subunits. For example, the integrin
subunits
5,
v,
II
, and
8 are all closely related, and integrin heterodimers containing these
subunits recognize
ligands containing the peptide sequence arginine-glycine-aspartic acid (Hynes, 1992
; Schnapp et al., 1995
). Similarly,
the
m,
L, and
x subunits are highly homologous to one
another and recognize closely related immunoglobulin
family members as ligands (Hynes, 1992
). We previously
cloned and sequenced the integrin
9 subunit, and have
shown that it forms a single integrin heterodimer,
9
1 (Palmer et al., 1993
). The
9 subunit cDNA sequence is
41% identical to the integrin
4 subunit sequence, but
27% identical to any other integrin subunit, identifying
9 and
4 as sole members of a subfamily of integrin
subunits.
In an effort to understand the structural basis of 9
1
ligand-binding in more detail, we recently mapped the
9
1 ligand-binding site in the extracellular matrix protein
tenascin-C (Yokosaki et al., 1994
).
9
1 binds to a single
exposed loop in the third fibronectin type III repeat of tenascin-C (B-C loop) to a minimal sequence EIDGIEL
(Schneider et al., 1998
; Yokosaki et al., 1998
). We noticed
that a critical portion of this sequence (IDG) is homologous to the tripeptide sequence IDS present in the previously mapped ligand-binding site for the
4
1 ligand, vascular cell adhesion molecule-1 (VCAM-11; Clements et
al., 1994
; Yokosaki et al., 1998
). Therefore, we undertook
the current study to determine whether
9
1 recognizes VCAM-1 as a ligand and whether or not any such interaction is biologically significant.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents
BSA, formyl-methionylleucylphenylalanine (FMLP), and dextran were
purchased from Sigma Chemical Co. Recombinant human tumor necrosis
factor (TNF)-, recombinant human interferon (IFN)-
(specific activity
of 107 U/mg), and recombinant interleukin 8 (IL-8) were obtained from R&D Systems, Inc. Fluorescent reagent, 2',7'-bis-(carboxyethyl)-5,6-carboxy-fluorescein acetoxymethyl ester (BCECF-AM) was purchased from
Molecular Probes, Inc. A recombinant form of the third fibronectin type
III repeat of chicken tenascin-C (Prieto et al., 1993
) containing alanine substitution mutations within the RGD site (TNfn3RAA), was obtained from Anita Prieto and Kathryn Crossin (Scripps Research Institute, La
Jolla, CA) and prepared in Escherichia coli. A recombinant VCAM-1/IgG
chimera (Yednock et al., 1995
) was produced in baculovirus as previously
described. Recombinant intercellular adhesion molecule-1 (ICAM-1)-C
fusion protein was a gift from B. Imhof (Centre Medicale Universitaire,
Geneva, Switzerland) to D. Erle (University of California, San Francisco,
CA). Ficoll-hypaque plus for isolation of neutrophils from venous blood
was purchased from Pharmacia Biotech, Inc. and used according to the
manufacturer's specifications.
Antibodies, Cells, and Cell Culture
Mouse mAbs, Y9A2 against human 9
1 (Wang et al., 1996
) and
AN100226M (100226) against
4 (Kent et al., 1995
), were prepared as
previously described. Mouse mAbs, W6/32 against human MHC and IB4
against the integrin
2 subunit, were prepared from hybridomas obtained
from American Type Tissue Collection. Mouse monoclonal antihuman
VCAM-1 (CD106) was purchased from R&D Systems. FITC-labeled mouse monoclonal anti-CD16 antibody was purchased from Caltag. Human umbilical vein endothelial (HUVE) cells were purchased from Clonetics and grown in endothelial cell growth media (EGM) containing 2% FBS, 10 ng/ml human recombinant EGF, 50 µg/ml gentamycin, 50 ng/
ml amphotericin B, 12 µg/ml bovine brain extract, and 1 µg/ml hydrocortisone and were used between passage 3 and 10.
9- and mock-transfected
SW480 and CHO cells were generated by transfection with the previously
described full-length
9 expression plasmid pcDNAIneo
9 (Yokosaki et
al., 1994
) or the empty vector pcDNAIneo (Invitrogen Corp.) by calcium
phosphate precipitation. Transfected cells were maintained in Dulbecco's
minimal essential medium(DMEM) supplemented with 10% FCS and the
neomycin analogue G-418 (1 mg/ml; Life Technologies, Inc.). Both cell
lines continuously expressed high surface levels of
9
1 as determined by
flow cytometry with Y9A2 (Yokosaki, 1996, 1998).
Flow Cytometry
Cultured cells were harvested by trypsinization and rinsed with PBS. Nonspecific binding was blocked with normal goat serum at 4°C for 10 min. Cells were then incubated with primary antibodies (unconjugated or conjugated with FITC) for 20 min at 4°C, followed by secondary antibodies conjugated with phycoerythrin (Chemicon International, Inc.). Between incubations, cells were washed twice with PBS. The stained cells were resuspended in 100 µl of PBS and fluorescence was quantified on 5,000 cells with a FACScan® (Becton Dickinson and Co.).
Immunoprecipitation and Western Blotting
Cells were lysed in immunoprecipitation buffer (100 mM Tris-HCl, pH
7.5, 150 mM NaCl, 1 mM CaCl2, 1% Triton X-100, 0.1% SDS, and 0.1%
NP-40) supplemented with 10 µg/ml pepstatin (Sigma Chemical Co.), 10 µg/ml leupeptin, 5 µg/ml aprotinin (Calbiochem-Novabiochem Corp.),
and 1 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co.). Human
neutrophils (107) were incubated with 1 mM diisopropyl flurophosphate
(Sigma Chemical Co.) for 15 min before cell lysis. After preclearing with
protein G-Sepharose, the supernatant was incubated with primary antibody for 2 h at 4°C and immune complexes were captured by protein
G-Sepharose for 45 min at 4°C. The beads were washed five times, and
boiled in 2.5× nonreducing Laemmli sample buffer, and samples were
separated by SDS-PAGE on 7.5% gels under reducing conditions and
transferred to Immobilon membranes. Membranes were blocked with 4%
casein, incubated with affinity-purified anti-9 cytoplasmic domain antiserum 1057 (Palmer et al., 1993
), and developed with luminol.
Cell Adhesion Assays
Wells of nontissue culture treated polystyrene 96-well flat bottomed microtiter plates (Nunc Inc.) were coated by incubation with 100 µl VCAM-1/Ig or TNfn3RAA for 1 h at 37°C. After incubation, wells were washed with PBS, then blocked with 1% BSA in DMEM at 37°C for 30 min. Control wells were filled with 1% BSA in DMEM. SW480 or CHO cells were detached using trypsin/EDTA and resuspended in serum-free DMEM. For blocking experiments, cells were incubated with 10 µg/ml Y9A2 and/ or 100226, for 15 min at 4°C before plating. The plates were centrifuged (top side up) at 10 g for 5 min before incubation for 1 h at 37°C in humidified 5% CO2. Nonadherent cells were removed by centrifugation (top side down) at 48 g for 5 min. Attached cells were fixed with 1% formaldehyde and stained with 0.5% crystal violet, and the wells were washed with PBS. The relative number of cells in each well was evaluated after solubilization in 40 µl of 2% Triton X-100 by measuring the absorbance at 595 nm in a microplate reader (Bio-Rad Laboratories). All determinations were carried out in triplicate.
For adhesion assays on HUVE cells, confluent monolayers of HUVE
cells were prepared in 96-well plates in 250 µl of EGM with 2% FBS.
Plates were washed twice with serum-free DMEM, then stimulated for
24 h at 37°C with TNF- (3 ng/ml) or IFN-
(3 ng/ml) in serum-free DMEM. SW480 cells were detached using trypsin/EDTA and labeled with 2 µM BCECF-AM at room temperature for 30 min. Then cells were
washed three times with serum-free DMEM and incubated with blocking
antibody, Y9A2 (10 µg/ml), 100226 (10 µg/ml), or combinations of these
antibodies for 15 min on ice. In some experiments, HUVE cells were incubated with CD106 (5 µg/ml) for 15 min at 37°C. 50,000 cells in 200 µl of serum-free DMEM were added to each well, and plates were centrifuged at
20 g for 5 min, and covered with aluminum foil to prevent photobleaching.
Plates were then incubated for 60 min at 37°C in 5% CO2. After incubation, nonadherent cells were removed by washing twice with serum-free
DMEM. Finally, 200 µl of the same medium was added to each well, and
fluorescence was quantified with a fluorometer (Fluoroskan II; Labsystems) at excitation wavelength 485 nm and emission wavelength 538 nm. The adherent ratio (%) was calculated as follows: (fluorescence from experimental sample
fluorescence from negative control sample)
total
fluorescence added to chamber. All determinations were carried out in triplicate.
Neutrophil Migration Assays
Neutrophils were purified from human peripheral venous blood containing 20 U/ml of heparin. Neutrophils were isolated by ficoll-hypaque density gradient centrifugation, followed by 3% dextran sedimentation (Gresham et al., 1986). Erythrocytes were subjected to hypotonic lysis, remaining neutrophils were washed and resuspended in PBS. The isolated
neutrophils were >95% pure and >95% viable as assessed by Wright-Giemsa staining and trypan blue exclusion, respectively. Cell migration
was analyzed essentially as described by Marks et al. (1991)
. In brief, glass
coverslips were placed in 35-mm culture dishes and incubated with 100 µl
serum-free media containing 10 µg/ml VCAM-1/Ig, 10 µg/ml TNfn3RAA,
and 5 µg/ml of ICAM-1 or 1% BSA for 60 min at 37°C, washed, and then
incubated with 1% BSA for 30 min. Neutrophils were incubated with no
antibody, Y9A2 (10 µg/ml), 100226 (10 µg/ml), IB4 (20 µg/ml), or combinations of antibodies for 15 min at 4°C, and were then incubated for 10 min at 37°C with or without 10 nM FMLP. 104 cells were plated onto the
coverslip area of each well and allowed to attach at 37°C for 5 min. Dishes
were then placed on a videomicroscope stage and individual fields (200×)
were recorded for 3 min. Three different fields were examined in each
chamber. To count the number of migrating cells in a given field, outlines
were made of each cell. Cells were considered to have migrated when
both the leading edge and tail of the cell moved
7 µm from their initial
position. At least 40 neutrophils were analyzed per field and the ratio of
migrating to total cells was calculated.
Neutrophil Transmigration Assays
Transendothelial neutrophil migration was assessed as described by Cooper et al. (1995). HUVE cells were plated onto polycarbonate inserts
(Transwell, 6.5-mm diameter, 8-µm pore for 24-well plate; Costar Corp.)
in 200 µl of serum-containing EGM, and allowed to grow to confluence
over 72 h. 500 µl serum-free DMEM was added to the lower chamber of
each well. 24 h before addition of neutrophils, upper chambers were
washed twice with serum-free media and new medium with or without 3 ng/ml of TNF-
. Immediately before the addition of neutrophils, the upper chambers were washed twice with serum-free DMEM and medium in
the lower chamber was replaced with 500 µl serum-free DMEM or serum-free DMEM with 10 nM FMLP or 50 ng/ml IL-8. In some experiments
HUVE cells were incubated with CD106 (5 µg/ml) at 37°C for 15 min. Purified neutrophils were incubated with no antibody, Y9A2 (10 µg/ml),
100226 (10 µg/ml), IB4 (20 µg/ml), W6/32 (10 µg/ml), or combinations of
antibodies for 15 min at 4°C, and 2 × 105 cells in 200 µl of media were
added to each upper chamber. After 3 h at 37°C in 5% CO2, nonadherent
cells in the upper chamber were removed. Medium, including migrated
neutrophils from the lower chamber, was collected, the lower chamber
was rinsed several times to collect all the neutrophils that had transmigrated, and the absence of additional adherent neutrophils was confirmed
microscopically. The medium and all washes were pooled and resuspended, and cells were counted with a hemocytometer. All determinations were carried out in duplicate and repeated at least twice.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
9
1 Mediates Static Adhesion of Resting
9-transfected SW480 Cells and CHO Cells
to VCAM-1
To determine whether VCAM-1 could function as a ligand
for 9
1, we performed cell adhesion assays with two different cell lines, SW480 and CHO, that had been stably
transfected with either an
9-expression plasmid or empty
vector. Both cell lines stably expressed
9
1 on the cell
surface as demonstrated by flow cytometry with the anti-
9
1 antibody Y9A2 (Fig. 1, A and B). Adhesion assays were performed on plates coated with either the known
9
1 ligand, recombinant TNfn3RAA (Fig. 1, C and D),
or recombinant VCAM-1/Ig (Fig. 1, E and F). For both
cell lines,
9-transfectants adhered to both TNfn3 and to
VCAM-1 in a concentration-dependent manner, whereas
mock-transfectants did not adhere to either substrate. Adhesion of each
9-transfected cell line was completely inhibited by the anti-
9
1 antibody, Y9A2, demonstrating
that this effect was mediated by
9
1.
|
9
1 Mediates Adhesion to TNF-
-activated, but not
to IFN-
-activated HUVE Cells, Via Interaction with
Induced VCAM-1
To determine whether 9
1-mediated adhesion to
VCAM-1 was biologically significant, we next examined
the role of this integrin in adhesion of cells to resting
HUVE cells, and to HUVE cells that had been activated
by incubation with TNF-
(3 ng/ml), a well characterized
inducer of VCAM-1 expression, or IFN-
(3 ng/ml), a cytokine that does not induce VCAM-1 expression. The effects of each cytokine on VCAM-1 expression under the
conditions used in these experiments were examined by
flow cytometry with anti-VCAM-1 antibody CD106 (Fig.
2, B-D). As expected, resting HUVE cells (Fig. 2 B) and
HUVE cells stimulated with IFN-
(Fig. 2 D) did not express detectable levels of VCAM-1, but VCAM-1 was dramatically induced by TNF-
(Fig. 2 C). All cell lines examined demonstrated baseline adhesion to resting HUVE
cells, and demonstrated a similar level of adhesion to
HUVE activated by IFN-
, and this baseline adhesion was
unaffected by anti-
9
1 antibody (Fig. 2 A). However, only
9-transfected cells demonstrated enhanced adhesion
to TNF-
-treated HUVE. This enhanced adhesion was returned completely to basal levels by antibody to either
9
1(Y9A2) or to VCAM-1 (CD106), demonstrating that
it was due to an interaction between
9
1 and VCAM-1.
|
9
1 Is Expressed on Neutrophils
We have previously demonstrated that 9
1 is widely expressed on epithelial and smooth muscle cells (Palmer et al.,
1993
), but expression on leukocytes has not been reported.
To determine whether
9
1 is expressed on cells likely to
encounter activated endothelial cells, we performed flow
cytometry on whole blood leukocytes with the
9
1 antibody Y9A2. We evaluated expression on neutrophils,
monocytes, and lymphocytes by gating on each population separately, based on differential light scattering. From a
separate atopic donor we evaluated expression on eosinophils, which were separated from other leukocytes based
on light scattering and the absence of surface expression of
CD16. In parallel, we examined expression of the structurally related integrin subunit,
4.
9
1 was not detected on
lymphocytes or eosinophils and was expressed at low levels on monocytes (Fig. 3 A). In contrast,
9
1 was highly and uniformly expressed on human neutrophils. As expected,
4 was highly expressed on lymphocytes, monocytes, and eosinophils, but was also detected on neutrophils, albeit at considerably lower levels.
|
Expression of 9 on neutrophils was further confirmed
by immunoprecipitation with Y9A2 followed by Western
blotting with an affinity-purified antiserum raised against
a unique portion of the
9 cytoplasmic domain. A band of
160 kD (appropriate molecular mass for
9) was detected
in lysate of human neutrophils after immunoprecipitation
with Y9A2, but not after immunoprecipitation with the
control antibody R6G9 (Fig. 3 B).
9
1 Mediates Migration of FMLP-activated
Neutrophils on TNfn3 or VCAM-1
To determine whether 9
1 expression on neutrophils
was biologically significant, we initially sought to examine
static adhesion of neutrophils to dishes coated with either
TNfn3RAA or VCAM-1. However, in the absence of antibodies against
2 integrins, neutrophils avidly adhered to
all surfaces examined, and in the presence of
2 integrin
blocking antibodies, neutrophils could not be induced to
adhere to either VCAM-1 or TNfn3RAA by incubation
with MnCl2, FMLP, phorbol esters, or the
1 activating
antibody TS2/16 (data not shown). Therefore, we examined the possible role of
9
1 in another important neutrophil function, cell migration. Migration was examined
by counting the numbers of individual neutrophils that migrated on chambers coated with either TNfn3RAA or
VCAM-1 in the presence or absence of the activating agonist FMLP (10 nM). In the absence of FMLP, very few
neutrophils migrated on either substrate (Fig. 4 A), and
antibodies against
9
1,
4, or
2 integrins had no effect.
In the presence of FMLP, neutrophil migration was significantly enhanced on TNfn3RAA, an effect that was abolished by antibody against
9
1. FMLP also enhanced neutrophil migration on VCAM-1, and this effect was partially
inhibited by antibodies against
9
1 or
4, and completely
inhibited by the combination of both antibodies. These
data demonstrate a significant role for
9
1 in mediating
neutrophil migration on both substrates. Antibody against
2 integrins had no effect on neutrophil migration on
FMLP-induced neutrophil migration on TNfn3RAA or
VCAM-1. However, as expected, antibody against
2 inhibited FMLP-induced migration on the
2 integrin ligand
ICAM-1, whereas antibodies against
9
1 or
4 had no effect (Fig. 4 B).
|
9
1 Mediates Migration of Neutrophils through
Activated HUVE Cell Monolayers
We next sought to determine whether the effect of 9
1
and
4 integrin(s) described above was relevant to an in
vitro model of neutrophil extravasation-migration across
endothelial monolayers. HUVE cells were grown to confluence on the top side of permeable filter supports and incubated in the presence or absence of TNF-
(3 ng/ml).
Purified neutrophils were added to the apical compartment in the presence or absence of FMLP added to the
basal compartment. These studies were performed in the
absence of blocking antibodies, or in the presence of antibodies against
9
1,
4,
2, VCAM-1, control antibody
against MHC, or combinations of these antibodies. As expected, in the absence of blocking antibodies, FMLP
greatly increased neutrophil migration into the bottom
compartment, and this effect was augmented by pretreatment of HUVE cells with TNF-
(Fig. 5 A). No antibody
affected basal migration across unstimulated HUVE cells
or FMLP-induced migration across unstimulated HUVE cells (Fig. 5 B). However, antibody against either
9
1 or
4 inhibited the augmented migration induced by TNF-
.
Antibody against VCAM-1 was equally effective in inhibiting migration across TNF-
-treated HUVE cells, suggesting that TNF-
augmented transmigration was mediated by an interaction between
9
1 and
4 integrins and
VCAM-1. As previously reported, antibody against
2 integrins also partially inhibited transmigration in response
to FMLP, but this effect was surprisingly small. Essentially
identical results were obtained when IL-8 was used as a
chemoattractant in place of FMLP (data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of the current study demonstrate that the inducible endothelial cell immunoglobulin family member,
VCAM-1, is an effective ligand for the integrin 9
1. This
receptor-ligand interaction is sufficient to support adhesion of
9-transfected cell lines to VCAM-1 and to TNF-
-activated HUVE cells, an effect that is mediated by the
binding of
9
1 to VCAM-1. Furthermore,
9
1 is uniformly and specifically expressed on normal resting human neutrophils, and mediates both neutrophil migration
on a fragment of tenascin-C or VCAM-1 and transmigration of neutrophils across TNF-
-activated endothelial
monolayers. Together, these data suggest a previously unsuspected role for
9
1 and VCAM-1 in extravasation of
neutrophils at sites of acute inflammation.
In addition to 9
1, we found detectable, albeit low, levels of the structurally related integrin
4 subunit on resting
human neutrophils. This finding is consistent with several
previous reports of
4 expression on neutrophils from a
variety of species (Issekutz et al., 1996
; Gao and Issekutz,
1997
; Davenpeck et al., 1998
). Although the level of expression of
4 we detected on human neutrophils was one
to two orders of magnitude lower than expression on eosinophils, monocytes, and lymphocytes, this low level
expression appeared to be biologically significant, since
antibody against
4 partially inhibited migration of neutrophils on VCAM-1 and migration across TNF-activated
endothelial monolayers. Recently,
4
1 has been shown
to mediate both neutrophil adhesion to VCAM-1 (Davenpeck et al., 1998
) and neutrophil transmigration across fibroblast monolayers (Gao and Issekutz, 1997
). As expected,
4 integrins did not contribute to migration on
TNfn3RAA, since this fragment of tenascin is not a ligand
for either
4 integrin.
Adhesion of activated neutrophils to endothelial cells at
sites of inflammation is well known to require the participation of integrins sharing the 2 subunit (Arfors et al.,
1987
) which bind to two other members of the immunoglobulin family expressed on endothelial cells, ICAM-1
(Marlin and Springer, 1987
; Diamond et al., 1990
) and
ICAM-2 (Staunton et al., 1989
). ICAM-1 is constitutively expressed on many epithelia, but expression is dramatically induced by a variety of inflammatory stimuli, including TNF-
. Our data do not address the role of
9
1 or
4
integrins in stable adhesion of neutrophils, since we were
not able to maintain adhesion of these cells to any substrate in the presence of
2 integrin blocking antibodies.
This effect could be due to a critical role of these integrins
in adhesion or to an inhibitory signaling pathway through which antibody-mediated ligation of
2 integrins inhibits
the function of other integrins, such as
9
1. However, the
mechanisms underlying the subsequent steps in neutrophil
extravasation, including detachment from sites of initial
adhesion and subsequent migration across the endothelial
cell surface and components of the underlying extracellular matrix, are not as well understood. The data in this
manuscript suggest, at least in the model system used,
that
9
1 and
4 integrins are likely to play important
roles. Both integrins could contribute to migration across
VCAM-1 expressing endothelial cells and shared ligands
such as osteopontin (Smith et al., 1996
; Bayless et al.,
1998
), and
9
1 could be critical for migration across tenascin-C that is present outside the vasculature at sites of
inflammation (Erickson, 1993
).
A role for 2 integrin-independent processes in neutrophil extravasation in vivo has been suggested by several
sets of observations, including studies of neutrophil extravasation into the liver in response to endotoxin (Essani
et al., 1997
) and neutrophil migration into the alveolar
spaces of the lung in response to intratracheal instillation
of live bacteria (Doerschuk et al., 1990
). Recent studies
demonstrating neutrophil extravasation into the lungs and
peritoneal cavity in
2 integrin knockout mice also demonstrate the importance of mechanisms independent of
2
integrins (Mizgerd et al., 1997
). The extent to which these
events are mediated by
9
1 and/or
4 integrins needs to
be determined from in vivo studies. We have recently succeeded in generating mice expressing a null mutation in
the
9 subunit gene, but these mice die within 10 d of birth
(unpublished observation). However, the development of
bone marrow chimeras from this line should allow us to directly examine these questions.
In addition to the expression on neutrophils described in
this report, 9
1 is widely expressed on muscle cells, surface epithelial cells, and hepatocytes (Palmer et al., 1993
).
It is unclear what role, if any, interactions with VCAM-1
might have at these sites. VCAM-1 has also been reported
to be expressed on muscle cells under various conditions
(Rosen et al., 1992
; Sheppard et al., 1994
), so it is conceivable that
9
1/VCAM-1 interactions may be biologically
significant in muscle as well. Such an effect could explain
the apparent contradiction between reports, based on antibody inhibition, that
4
1/VCAM-1 binding plays a critical role in myotube formation (Rosen et al., 1992
) and the
normal muscle development of
4 knockout cells in chimeric mice (Yang et al., 1996
), if the
4 knockout led to a
developmentally regulated increase in
9
1 expression.
In summary, we have identified VCAM-1 as a novel and
biologically significant ligand for the integrin 9
1, have
demonstrated that this integrin is expressed on neutrophils
and mediates neutrophil migration on two relevant ligands
and neutrophil transmigration across activated endothelial
monolayers. These findings support a role for
9
1/
VCAM-1 interactions in extravasation of neutrophils at
sites of inflammation.
![]() |
Footnotes |
---|
Address correspondence to Dean Sheppard, Lung Biology Center, UCSF Box 0854, San Francisco, CA 94143. Tel.: (415) 206-5901. Fax: (415) 206-4123. E-mail: deans{at}itsa.ucsf.edu
Received for publication 23 November 1998 and in revised form 24 February 1999.
We thank Amha Atakilit and David Erle for technical and intellectual assistance with flow cytometric analysis.
This work was supported by National Institutes of Health grants HLAI33259, HL47412, HL53949, and HL56385 to D. Sheppard.
![]() |
Abbreviations used in this paper |
---|
EGM, endothelial cell growth media; FMLP, formyl-methionylleucylphenylalanine; HUVE, human umbilical vein endothelial; ICAM, intercellular adhesion molecule; IFN, interferon; TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion molecule-1.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Arfors, K., C. Lundberg, L. Lindblom, K. Lundberg, P.G. Beatty, and J.M. Harlan. 1987. A monoclonal antibody to the membrane glycoprotein complex CD18 inhibits polymorphonuclear leukocyte accumulation and plasma leakage in vivo. Blood. 69: 338-340 [Abstract]. |
2. |
Bayless, K.J.,
G.A. Meininger,
J.M. Scholtz, and
G.E. Davis.
1998.
Osteopontin
is a ligand for the alpha4beta1 integrin.
J. Cell Sci.
111:
1165-1174
|
3. |
Clements, J.M.,
P. Newham,
M. Shepherd,
R. Gilbert,
T.J. Dudgeon,
L.A. Needham,
R.M. Edwards,
L. Berry,
A. Brass, and
M.J. Humphries.
1994.
Identification of a key integrin-binding sequence in VCAM-1 homologous
to the LDV active site in fibronectin.
J. Cell Sci.
107:
2127-2135
|
4. |
Cooper, D.,
F.P. Lindberg,
J.R. Gamble,
E.J. Brown, and
M.A. Vadas.
1995.
Transendothelial migration of neutrophils involves integrin-associated protein (CD47).
Proc. Natl. Acad. Sci. USA.
92:
3978-3982
|
5. |
Davenpeck, K.L.,
S.A. Sterbinsky, and
B.S. Bochner.
1998.
Rat neutrophils
express alpha4 and beta1 integrins and bind to vascular cell adhesion
molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1
(MAdCAM-1).
Blood.
91:
2341-2346
|
6. | Diamond, M.S., D.E. Staunton, A.R. de Fougerolles, S.A. Stacker, A.J. Garcia, M.L. Hibbs, and T.A. Springer. 1990. ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18). J. Cell Biol. 111: 3129-3139 [Abstract]. |
7. | Doerschuk, C.M., R.K. Winn, H.O. Coxson, and J.M. Harlan. 1990. CD18-dependent and -independent mechanisms of neutrophil adherence in the pulmonary and systemic microvasculature of rabbits. J. Immunol. 114: 2327-2333 . |
8. | Erickson, H.P.. 1993. Tenascin-C, tenascin-R, tenascin-X: a family of talented proteins in search of functions. Curr. Opin. Cell Biol. 5: 869-876 |
9. | Essani, N.A., M.L. Bajt, A. Farhood, S.L. Vonderfecht, and H. Jaeschke. 1997. Transcriptional activation of vascular cell adhesion molecule-1 gene in vivo and its role in the pathophysiology of neutrophil-induced liver injury in murine endotoxin shock. J. Immunol. 158: 5941-5948 [Abstract]. |
10. | Gao, J.X., and A.C. Issekutz. 1997. The beta 1 integrin, very late activation antigen-4 on human neutrophils can contribute to neutrophil migration through connective tissue fibroblast barriers. Immunology. 90: 448-454 |
11. |
Gresham, H.D.,
L.T. Clement,
J.E. Lehmeyer, and
F.M. Griffin Jr..
1986.
Simulation of human neutrophil Fc receptor-mediated phagocytosis by a low molecular weight cytokine.
J. Immunol.
137:
868-875
|
12. | Hynes, R.O.. 1987. Integrins: a family of cell surface receptors. Cell. 48: 549-554 |
13. | Hynes, R.O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 69: 11-25 |
14. | Issekutz, T.B., M. Miyasaka, and A.C. Issekutz. 1996. Rat neutrophils express very late activation antigen 4 and it mediates migration to arthritic joint and dermal inflammation. J. Exp. Med. 183: 2175-2184 [Abstract]. |
15. | Kent, S.J., S.J. Karlik, C. Cannon, D.K. Hines, T.A. Yednock, L.C. Fritz, and H.C. Horner. 1995. A monoclonal antibody to alpha 4 integrin suppresses and reverses active experimental allergic encephalomyelitis. J. Neuroimmunol. 58: 1-10 |
16. | Marks, P.W., B. Hendey, and F.R. Maxfield. 1991. Attachment to fibronectin or vitronectin makes human neutrophil migration sensitive to alterations in cytosolic free calcium concentration. J. Cell Biol. 112: 149-158 [Abstract]. |
17. | Marlin, S.D., and T.A. Springer. 1987. Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1). Cell. 51: 813-819 |
18. |
Mizgerd, J.P.,
H. Kubo,
G.J. Kutkoski,
S.D. Bhagwan,
K. Scharffetter-Kochanek,
A.L. Beaudet, and
C.M. Doerschuk.
1997.
Neutrophil emigration
in the skin, lungs and peritoneum: differential requirements for CD11/CD18
revealed by CD18-deficient mice.
J. Exp. Med.
186:
1357-1364
|
19. | Palmer, E.L., C. Ruegg, R. Ferrando, R. Pytela, and D. Sheppard. 1993. Sequence and tissue distribution of the integrin alpha 9 subunit, a novel partner of beta 1 that is widely distributed in epithelia and muscle. J. Cell Biol. 123: 1289-1297 [Abstract]. |
20. | Prieto, A.L., G.M. Edelman, and K.L. Crossin. 1993. Multiple integrins mediate cell attachment to cytotactin/tenascin. Proc. Natl. Acad. Sci. USA. 90: 10154-10158 [Abstract]. |
21. | Rosen, G.D., J.R. Sanes, R. La Chance, J.M. Cunningham, J. Roman, and D.C. Dean. 1992. Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis. Cell. 69: 1107-1119 |
22. | Ruoslahti, E., and M.D. Pierschbacher. 1987. New perspectives in cell adhesion: RGD and integrins. Science. 238: 491-497 |
23. |
Schnapp, L.M.,
N. Hatch,
D. Ramos,
I.V. Kliminskaya,
D. Sheppard, and
R. Pytela.
1995.
The human integrin ![]() ![]() |
24. | Schneider, H., R.P. Harbottle, Y. Yokosaki, J. Kunde, D. Sheppard, and C. Coutelle. 1998. A novel peptide, PLAEIDGIELTY, for the targeting of alpha9/beta1-integrins. FEBS Lett. 429: 269-273 |
25. | Sheppard, A.M., M.D. Onken, G.D. Rosen, P.G. Noakes, and D.C. Dean. 1994. Expanding roles for alpha 4 integrin and its ligands in development. Cell Adhes. Commun 2: 27-43 |
26. |
Smith, L.L.,
H.-K. Cheung,
L.E. Ling,
J. Chen,
D. Sheppard,
R. Pytela, and
C.M. Giachelli.
1996.
Osteopontin N-terminal domain contains a cryptic adhesive sequence recognized by ![]() ![]() |
27. | Staunton, D.E., M.L. Dustin, and T.A. Springer. 1989. Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1. Nature. 339: 61-64 |
28. | Wang, A., Y. Yokosaki, R. Ferrand, J. Balmas, and D. Sheppard. 1996. Differential regulation of airway epithelial integrins by growth factors. Am. J. Respir. Cell Mol. Biol. 15: 664-672 [Abstract]. |
29. | Yang, J.T., T.A. Rando, W.A. Mohler, H. Rayburn, H.M. Blau, and R.O. Hynes. 1996. Genetic analysis of alpha 4 integrin functions in the development of mouse skeletal muscle. J. Cell Biol. 135: 829-835 [Abstract]. |
30. |
Yednock, T.A.,
C. Cannon,
C. Vandevert,
E.G. Goldbach,
G. Shaw,
D.K. Ellis,
C. Liaw,
L.C. Fritz, and
L.I. Tanner.
1995.
Alpha 4 beta 1 integrin-dependent cell adhesion is regulated by a low affinity receptor pool that is conformationally responsive to ligand.
J. Biol. Chem.
270:
28740-28750
|
31. |
Yokosaki, Y.,
E.L. Palmer,
A.L. Prieto,
K.L. Crossin,
M.A. Bourdon,
R. Pytela, and
D. Sheppard.
1994.
The integrin ![]() ![]() |
32. |
Yokosaki, Y.,
H. Monis,
J. Chen, and
D. Sheppard.
1996.
Differential effects of
the integrins ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
33. |
Yokosaki, Y.,
N. Matsuura,
S. Higashiyama,
I. Murakami,
M. Obara,
M. Yamakido,
N. Shigeto,
J. Chen, and
D. Sheppard.
1998.
Identification of the
ligand binding site for the integrin alpha9/beta1 in the third fibronectin type
III repeat of tenascin-C.
J. Biol. Chem.
273:
11423-11428
|