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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 602-609
IMMUNOBIOLOGY
Soluble VCAM-1 binding to  4 integrins is cell-type specific and
activation dependent and is disrupted during apoptosis in T cells
David M. Rose,
Pina M. Cardarelli,
Ronald R. Cobb, and
Mark H. Ginsberg
From the Department of Vascular Biology, The Scripps Research
Institute, La Jolla, and Tanabe Research Laboratories, San Diego, CA.
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Abstract |
Soluble vascular cell adhesion molecule-1 (sVCAM-1) is generated
during inflammation and can alter lymphocyte functions. The authors
report that the binding of sVCAM-1 to  4 integrin-bearing cells is a
dynamically regulated, active cellular process. Binding of recombinant
sVCAM-1 to 4 integrins on peripheral blood mononuclear cells was
cell-type specific. Circulating CD16+ NK cells constitutively bound sVCAM-1 with high affinity, whereas a subpopulation of
T-lymphocytes, primarily CD45RO+ (memory), bound sVCAM-1 only after
phorbol ester stimulation. sVCAM-1 binding to homogenous stable cell
lines was also cell-type specific, and required active cellular
processes because it was blocked by the inhibition of ATP synthesis and by Fas-induced apoptosis. Indeed, the loss of high-affinity VCAM-1 binding was an early event in apoptosis. Furthermore, an
H-Ras/Raf-initiated signaling pathway also suppressed sVCAM-1 binding
to 4 1 integrins. Collectively, these results showed that the
capacity of 4 integrins to bind VCAM-1 is actively regulated and
that this regulation may control 4 integrin-dependent cellular functions.
(Blood. 2000;95:602-609)
© 2000 by The American Society of Hematology.
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Introduction |
The  4 integrins are adhesion receptors widely
expressed on mononuclear leukocytes and eosinophils.1 A
major ligand for 4 integrins is vascular cell adhesion molecule-1
(VCAM-1).2 VCAM-1 is a member of the immunoglobulin
superfamily and is expressed on vascular endothelium at sites of
inflammation and on selected other sites, such as thymic cortical
epithelium and bone marrow stroma.2,3 Cells expressing 4
integrins can interact with VCAM-1 to mediate events such as the
recruitment of leukocytes to sites of inflammation and the development
and activation of lymphocytes.4,5
In addition to its cell surface expression, VCAM-1 has been found in a
soluble form in the culture supernatant of cytokine-treated endothelial
cells, in human serum, and in the synovial fluid of patients with
rheumatoid arthritis.6,7 The origin of this soluble VCAM-1
(sVCAM-1) is not entirely known, but release from the cell surface by
proteolytic cleavage is possible.8 sVCAM-1 may act to
suppress further leukocyte migration to tissues by removing the ligand
for 4 integrins. The soluble form of VCAM-1 could also act as a
competitive inhibitor of ligand binding. In addition, the interaction
of the sVCAM-1 with 4 integrins could provide a signal to alter cell
function. Indeed, in T cells, sVCAM-1 acts as a
chemotractant,9 and sVCAM-1 binding inhibits T-cell activation.10
Integrin function can be dynamically regulated without changes in
integrin expression, a process termed "inside-out
signaling."11,12 Two general mechanisms account for such
regulation. One involves a change in the affinity of ligand binding,
operationally defined as "activation."11
Alternatively, affinity-independent mechanisms, such as changes in cell
shape, spreading, and lateral mobility/clustering of integrins, also
modulate cell adhesion.13-15 Many integrins, such as
IIb3, 51, and M2, manifest active cellular regulation of the affinity of ligand binding.16,17 In sharp
contrast, active cellular regulation of 41 affinity for
soluble ligand is controversial, and a recent review emphasized that
unlike other integrins, the affinity of 41 may not be
physiologically regulated.13 It is clear that soluble
ligand binding to 41 can be increased by exogenous agents such as
Mn++ or activating antibodies.18 A report
has demonstrated increased soluble fibronectin (FN) binding to 41
on T cells stimulated by L-selectin engagement, but its significance is
unclear.19 Furthermore, several studies have implicated
41 activation indirectly with reported antibodies such as
monoclonal antibody (mAb) 15/7, but whether these antibodies truly
recognize high-affinity integrin conformations has been
questioned.9,13,20,21
In the current study, we investigated the regulation of sVCAM-1
binding to 41 integrin using a sVCAM-1-immunoglobulin fusion protein as ligand. We report that sVCAM-1 binding to 4 integrins was
cell-type-specific and energy dependent and that it was disrupted during apoptosis. Specific intracellular signaling pathways also regulated sVCAM-1 binding to 41. Thus, the function of the
41 integrin can be physiologically regulated by changes in
soluble ligand binding.
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Materials and methods |
Antibodies and reagents
The antihuman 1 mAb, 8A2, was the generous gift of Drs N. Kovach
and J. Harlan (University of Washington, Seattle, WA). Antihuman 4
(9F10), antihuman 1 (9EG7), and fluorescein isothiocyanate (FITC)-conjugated antihuman CD3 (HIT3a) mAbs were purchased from PharMingen (San Diego, CA). Function-blocking antihuman 4 (HP2/1) and the anti-Fas (CH11) IgM were purchased from Immunotech (Westbrook, ME). The antihuman CD3 (OKT3) hybridoma was obtained from American Type
Culture Collection (ATCC; Rockville, MD) and IgG purified as previously
described.17 Phorbol 12-myristate 13-acetate (PMA) and
sodium azide were purchased from Sigma Chemical (St. Louis, MO), and
2-deoxyglucose was purchased from Calbiochem (La Jolla, CA). Anti-Tac
antibody, 7G7B6, was obtained from ATCC, and biotinylated with
biotin-N-hydroxy-succinimide was obtained from Sigma Chemical. Phycoerythrin (PE)-conjugated annexin V was purchased from R&D Systems
(Minneapolis, MN). PE-conjugated streptavidin was purchased from
Molecular Probes (Eugene, OR). A VCAM-Ig fusion protein containing the
2 N-terminal domains of human VCAM-1 fused to the human IgG1 heavy
chain was a generous gift of Dr Roy Lobb (Biogen, Cambridge, MA) and
was prepared as previously described.18 A VCAM-C fusion protein containing the 7 Ig domains of human VCAM-1 fused to the murine
C segment was a gift from Dr. Richard M. Wright (Novartis Pharmaceuticals, Summit, NJ). Purified human plasma FN was purchased from Life Technologies (Grand Island, NY) and fluoresced as follows: FN
was resuspended in phosphate-buffered saline at 5 mg/mL and was added
to a reaction buffer containing 0.1 mol/L NaHCO3, pH 9, and
2 mg/mL FITC-celite (Sigma Chemical). The solution was incubated for 60 minutes at room temperature in the dark. The FITC-FN was purified on a
PD-10 Sephadex column from AmershamPharmacia Biotech (Wikstroms,
Sweden) and analyzed by spectrophotometry.
Cells
The T-leukemic cell lines Jurkat and HUT-78, the monocytic
cell lines THP-1 and U937, and the rat basophilic leukemia (RBL) cell
line were purchased from ATCC and cultured in RPMI-1640 medium (Biowhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (Biowhittaker), 1% glutamine, 1% penicillin, and 1%
streptomycin (Sigma Chemical). Human lymphocytes were purified from
peripheral blood from normal donors by centrifugation through a
Ficoll-Paque gradient (Pharmacia Biotech, Uppsala, Sweden), panning for
monocytes on tissue culture plastic, and passaging over a nylon wool
column.17 Chinese hamster ovary (CHO) cells expressing both
human 4 and 1 integrin subunits were a generous gift from Dr
Yoshikazu Takada (The Scripps Research Institute, La Jolla, CA) and
have been described elsewhere.22
cDNA constructs and transfections
The chimeric Tac-5 and Tac-1 constructs in CDM8 vector
have been previously described.23 The pDCR-H-Ras (G12V) was
a gift from Dr M. H. Wigler (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The vector encoding RafBxB has been
previously described.24 CHO-41 cells were transiently
transfected using lipofectamine (Gibco BRL, Grand Island, NY) according
to the manufacturer's instructions, as previously
described.25
Construction and expression of VCAM-1-immunoglobulin fusion protein
Total RNA was isolated from HUVECs stimulated with IL-1 using
Ultraspec (BioTecX, Houston, TX) following the instructions of the
manufacturer. First-strand cDNA was generated using the GeneAmp kit of
Perkin/Elmer (Norwalk, CT) and oligo-dT as a primer following the
manufacturer's instructions. The entire coding region of the VCAM-1
cDNA was cloned to generate pRmHaVCAM. The entire coding sequence of
the extracellular domain (7 Ig-like domains) of VCAM-1 was polymerase
chain reaction-amplified from pRmHaVCAM using the following primers:
5'-GCTAGCGTCGACATGCCTGGGAAGATGGTC-3'; 5'-AGCGTCAGCCTCAGGAGAAAAATAGTC-3'. The resultant
fragment was then subcloned into the NheI site of pBB4Ig, which
contains the human Fc coding sequences at the carboxy terminus of the
recombinant protein. Recombinant protein was expressed in Sf9 cells
grown in SF900 II SFM (Life Technologies, Gaithersburg, MD).
Recombinant protein was purified using Protein A filters (Nygene,
Goldens Bridge, NY). Analysis of recombinant protein by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a major molecular species of ~240 kd under nonreducing conditions and ~115 kd under reducing conditions. These are the predicted weights of the dimer and
the monomer, respectively.
Soluble ligand-binding assay
Cells (5 × 105) were resuspended in Dulbecco's
minimum essential medium (DMEM; Biowhittaker) containing
0.1% bovine serum albumin (BSA; Sigma Chemical) or in a modified
Tyrode's buffer (150 mmol/L NaCl, 2.5 mmol/L KCl, 12 mmol/L NaHCO3, 1 mg/mL glucose, and 1 mg/mL BSA) with
1 mmol/L MnCl2. A modified Tyrode's buffer was used with
MnCl2 because this divalent cation precipitates in
phosphate-containing solutions. The VCAM-Ig fusion protein, or FITC-FN,
was added to the mixture and incubated for 30 minutes at room
temperature (preliminary studies indicated no significant differences
in ligand binding at room temperature and 37°C). Afterward, cells
were washed twice with 0.5 mL of their respective buffers. If cells
were also being analyzed for surface antigen expression, they were
incubated with primary antibody for 30 minutes on ice and again washed
twice. Cells were then resuspended in the same buffer containing either PE- or FITC-conjugated donkey antihuman IgG (Jackson Immunoresearch, West Grove, PA) at a 1:100 dilution (if they were dual labeled, FITC-conjugated antimouse IgG was also added) and incubated for 30 minutes. Cells were washed twice and fluorescence analyzed on a
FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) using
CellQuest software.
Analysis of apoptosis
The assessment of apoptosis was measured by the binding of
PE-conjugated annexin V to externalized phosphatidylserine by flow cytometry according to the manufacturer's instructions (R&D Systems).
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Results |
sVCAM-1 binding to isolated human peripheral blood cells
Because the interaction of 4 integrins with VCAM-1 modulates the
functions of mononuclear leukocytes, we examined the regulation of
sVCAM-1 binding to human peripheral blood lymphoid cells. These cells
were analyzed for both CD3 expression and sVCAM-1 (7 Ig-domain form)
binding by 2-color flow cytometry. A population of CD3-negative cells
bound sVCAM-1, whereas few (typically less than 2%) of the CD3-positive T-lymphocytes manifested such constitutive binding (Figure
1) The 4 integrins were functional on these CD3+
T-lymphocytes because 60% of these cells bound sVCAM-1 in the presence
of an exogenous integrin activator, MnCl2 (data not shown).
Stimulation with PMA (50 ng/mL) for 30 minutes resulted in
increased sVCAM-1 binding in a subgroup representing ~15% of the
CD3-positive T-lymphocytes (Figure 1). Binding in each case was 4
integrin specific because it was completely blocked by an anti-4
antibody, HP2/1. Furthermore, binding was not simply ascribable to the
dimeric nature of the VCAM-Ig construct. A monomeric VCAM-1 construct
containing the 7 extracellular Ig domains fused to murine light
chain showed similar binding characteristics (data not shown). These
results indicated that a subgroup of peripheral T-lymphocytes responds to PMA stimulation with increased sVCAM-1 binding to 4 integrins.
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| Fig 1.
sVCAM-1 binding to isolated peripheral blood lymphocytes.
Human peripheral blood lymphoid cells were isolated as described in
"Materials and Methods." Cells were resuspended in Tyrode's
buffer containing 1 mmol/L CaCl2, 1 mmol/L
MgCl2, and 0.1% BSA and incubated for 30 minutes at room
temperature in the absence of stimulation (upper panels) or in the
presence of PMA (50 ng/mL) (lower panels). In the presence
or absence of the function blocking anti-4 mAb HP2/1, 7 Ig-domain
VCAM-Ig (20 µg/mL) was added to the cells, and the mixture was
incubated for 30 minutes at room temperature. After washing, the cells
were stained with FITC-anti-CD3 mAb (HIT3a). At the same time, they
were stained with PE-conjugated donkey antihuman IgG to detect the
VCAM-Ig fusion protein as described in "Materials and Methods."
The percentage of cells in quadrants is indicated. Results are
representative of 3 separate experiments.
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We next used surface markers to characterize the subpopulation of
CD3-positive T-lymphocytes that bound sVCAM-1 in response to PMA
stimulation. Resting CD45RO+ (memory) and CD45RA+ (naive) T-lymphocytes
showed little difference in sVCAM-1 binding (Figure 2). However, after PMA
stimulation, CD45RO+ cells bound much more sVCAM-1 than CD45RA+ cells
(Figure 2). This CD45RO+ cell response was not simply a response to the
differences in 4-integrin expression because sVCAM-1 binding could
be induced on both CD45RO+ and CD45RA+ cells after exogenous activation
with MnCl2 (data not shown). These results showed that
CD45RO+ memory T-lymphocytes responded to phorbol ester stimulation by
increasing soluble VCAM-1 binding.
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| Fig 2.
PMA stimulates sVCAM-1 binding to CD45RO-positive
peripheral T-lymphocytes.
Human peripheral blood T-lymphocytes were isolated as described in
"Materials and Methods". Cells were resuspended in Tyrode's
buffer containing 1 mmol/L CaCl2, 1 mmol/L
MgCl2, and 0.1% BSA (solid line) or 5 mmol/L EDTA (shaded
line). They were incubated for 30 minutes at room temperature in the
absence of stimulation (upper panels) or in the presence of PMA (50 ng/ml) (lower panels). 7 Ig-domain VCAM-Ig (20 µg/mL)
was added, and binding was allowed to proceed for 30 minutes at room
temperature. After they were washed, cells were incubated with
anti-CD45RA mAb (HI100) or anti-CD45RO mAb (UCHL1). Binding of VCAM-Ig
and anti-CD45 mAbs was detected with PE-conjugated donkey antihuman IgG
and FITC-conjugated donkey antimouse IgG, respectively, as described in
"Materials and Methods." Histograms depict sVCAM-1 binding to
cells gated for CD45 expression. To obtain a quantitative estimate of
sVCAM-1 binding, we calculated a numeric activation index (AI) defined
as 100 × (Fo Fr)/FmaxFr), where Fo is
the mean fluorescence intensity of sVCAM-1 binding, Fr is background
fluorescence in the presence of 5 mmol/L EDTA, and Fmax is
the fluorescence intensity in the presence of 1 mmol/L
MnCl2 (not shown). Results are representative of 3 separate
experiments.
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We also characterized the CD3-negative cells that manifested
constitutive VCAM-1 binding. This population of cells had the size and
granularity of lymphoid cells in flow cytometry. They were not,
however, depleted by passage through a nylon wool column, suggesting
that they were not B-lymphocytes. Using CD16 as a surface marker of
natural killer (NK) cells, we found that more than 50% of circulating
CD16+ NK cells constitutively bound sVCAM-1 and that this
binding was inhibited by the function-blocking anti-4 antibody, HP2/1 (Figure 3). Thus, most
circulating CD16+ NK cells express 4 integrins that constitutively
bind soluble VCAM-1. Analysis of sVCAM-1 binding to circulating B
cells was not possible because of the interaction of our secondary
detection antibody with surface-bound IgG or Fc-receptors (data not
shown).
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| Fig 3.
CD16-positive human peripheral blood natural killer cells
constitutively bind sVCAM-1.
Human peripheral blood lymphoid cells were isolated as described in
"Materials and Methods". Cells were resuspended in Tyrode's
buffer containing 1 mmol/L, CaCl2, 1 mmol/L
MgCl2, and 0.1% BSA. In the presence or absence of the
function blocking anti-4 mAb HP2/1 (10 µg/mL), 7 Ig-domainVCAM-Ig
(20 µg/mL) was added, and cells were incubated for 30 minutes at room
temperature. After they were washed, cells were incubated with
FITC-anti-CD16 mAb.3G8 Binding of VCAM-Ig was
detected with PE-conjugated donkey antihuman IgG as described in
"Materials and Methods." The percentage of cells in quadrants is
indicated. Results are representative of 3 separate experiments.
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Cell-type-specific binding of sVCAM-1 to 41 integrin
To assess sVCAM-1 binding to homogenous cell populations, we
examined binding to stable cell lines. In the Jurkat T-leukemia cell
line, sVCAM-1 (7 Ig domain) bound tightly (EC50 ~50
nmol/L), and binding was completely blocked by the anti-4 antibody
HP2/1 (Figure 4a) and by the anti-VCAM-1
antibody P8B1(data not shown). The addition of 1 mmol/L
MnCl2, to activate 41 integrins exogenously resulted
in ~2-fold greater maximal sVCAM-1 binding at saturation. Thus,
~50% of the functionally expressed 41 integrins constitutively bound sVCAM-1. Similar results were obtained with 41-transfected CHO cells (CHO-41) (Figure 5). In
contrast to sVCAM-1 binding, soluble FN showed no detectable binding to
Jurkat cells unless integrins were exogenously activated with
MnCl2 (Figure 4B). More than 80% of this binding was
inhibited by HP2/1, reflecting the 5-fold greater abundance of 41
than other FN binding integrins on these cells (data not shown).
Furthermore, the Mn++-induced soluble FN binding was
inhibited by antibodies hfn7.1 and FN1-8 (data not shown), which
recognize the ninth and tenth type 2I homology segments of
FN.26,27 These results suggest that this binding occurs
through the central cell-binding region of FN, as has been
reported.28 In addition, minimal sVCAM-1 binding to
41 was seen with a VCAM-Ig construct containing only the 2 N-terminal Ig-like domains unless cells were exogenously activated with
1 mmol/L MnCl2 (Figure 4c).
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| Fig 4.
High affinity binding of a sVCAM-Ig fusion protein and
soluble FN to 41 integrins on Jurkat cells.
Cells were resuspended in DMEM containing 0.1% BSA or in a Tyrode's
buffer containing 1 mmol/L MnCl2 and 0.1% BSA
(Mn++). The indicated concentrations of VCAM-Ig (A) or
FITC-FN (B) were added, and the mixtures were incubated for 30 minutes
at room temperature.  , Mn++ added;  , no
addition. Where indicated by  and  ,
function-blocking anti-4 mAb HP2/1 was added before the soluble
ligand. VCAM-Ig binding was evaluated with FITC-conjugated donkey
antihuman IgG using flow cytometry as described in "Materials and
Methods" and expressed as geometric mean fluorescence intensity
(MFI). (C) Jurkat cells were resuspended in either DMEM containing
0.1% BSA (solid line), DMEM with the addition of 5 mmol/L EDTA (shaded
line), or Tyrode's buffer containing 1 mmol/L MnCl2
(dotted line). VCAM-Ig containing only the first 2 N-terminal or all 7 Ig domains (20 µg/mL) was added to the cells, and the mixtures were
incubated at room temperature for 30 minutes. Results are
representative of 3 separate experiments.
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| Fig 5.
Cell-type-specific binding of sVCAM-1 to 41
integrin.
Jurkat, CHO-41, RBL, THP-1, U937, or HUT-78 was resuspended in
either DMEM containing 0.1% BSA (solid line), DMEM with the addition
of 5 mmol/L EDTA (shaded line), or Tyrode's buffer containing 1 mmol/L
MnCl2 (dotted line). Seven Ig-domain VCAM-Ig (20 µg/mL)
was added, and the mixture was incubated at room temperature for 30 minutes. VCAM-Ig binding was detected with FITC-conjugated donkey
antihuman IgG as described in "Materials and Methods." Results
are representative of 3 separate experiments.
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To assess further the cell-type specificity of sVCAM-1 binding (the 7 Ig domain form), we assayed other 4 expressing cell lines. In
contrast to Jurkat cells, the human T-leukemic line HUT-78 bound
sVCAM-1 minimally (Figure 5). However, the 41 on these cells was
functional because soluble VCAM-Ig binding could be induced either by
treatment with MnCl2 or by the 1 integrin activating
antibodies 8A2 and 9EG7 (data not shown). Differences in sVCAM-1
binding were not attributable to differences in 41 expression
because they express similar integrin levels, reflected by the similar
sVCAM-1 binding in the presence of MnCl2. The human monocytic lines THP-1 and U937 also bound little sVCAM-1
without exogenous activation, whereas RBL bound sVCAM-1 in a manner
similar to Jurkat and CHO-41 cells (Figure 5). These results
indicated that the capacity of 41 integrin to bind sVCAM-1 is
dependent on the cellular context.
sVCAM-1 binding to 41 integrin is dependent on active cellular
processes
We next questioned whether binding of sVCAM-1 to 41 is a
passive or an active cellular event. Treating Jurkat and CHO-41 cells with the metabolic inhibitors sodium azide and 2-deoxyglucose resulted in a time-dependent suppression of sVCAM-1 binding (Figure 6). This decrease in sVCAM-1 binding was
not caused by a loss of 41 integrin expression because the
binding could be recovered by exogenous activation with Mn++. Thus,
metabolic energy is required to maintain sVCAM-Ig binding to 41
integrin.
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| Fig 6.
Cellular-energy depletion suppresses sVCAM-Ig binding.
Jurkat (A) or CHO-41 (B) cells were placed in glucose-free
Tyrode's buffer containing 2-deoxyglucose (2 mg/mL) and sodium azide
(0.1% wt/vol) and were incubated at 37°C for 0, 30, 60, or 120 minutes. Subsequently, cells were resuspended in DMEM or Tyrode's
buffer containing 1 mmol/L MnCl2 (Mn++), and 7 Ig-domain VCAM-Ig binding was assessed as described in "Materials
and Methods". VCAM-Ig binding was expressed as a percentage of
initial binding in cells incubated in the absence of 2-deoxyglucose and
sodium azide. Results are the mean ± SEM of 3 independent
experiments.  , Mn++ added; , no
addition.
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sVCAM-1 binding is disrupted during apoptosis
We next determined whether a physiologic relevant stimulus could
modulate the activation of 41. An early feature of apoptotic cell
death is loss of cell adhesion. Thus, we examined the effect of
Fas-induced apoptotic cell death on sVCAM-1 binding to Jurkat cells.
Treatment of Jurkat cells with anti-Fas antibody resulted in apoptosis,
as measured by annexin-V binding to surface-expressed phosphatidylserine (Figure 7). The
annexin-V positive cells (apoptotic) manifested markedly reduced
sVCAM-1 binding compared with annexin-V-negative cells (Figure 7B).
However, the surface expression of 4 on apoptotic cells was not
markedly different from that on nonapoptotic cells (Figure 7D).
Furthermore, binding to apoptotic cells could be restored by adding the
activating antibody 8A2 (Figure 7C). In addition, 8A2-induced sVCAM-1
binding increased annexin-V binding to the previously negative cells
(Figure 7C). This suggests that sVCAM-1 binding promotes Fas-induced
apoptosis in these cells. Through kinetic analysis, we found that the
reduction in sVCAM-1 binding occurred rapidly after Fas cross-linking.
A large proportion of cells lost sVCAM-1 binding while they remained
annexin-V negative during the first 30 minutes after Fas ligation
(Figure 7E). These results indicated that the disruption of sVCAM-1
binding is an early event in apoptosis.
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| Fig 7.
Fas-induced apoptosis suppresses sVCAM-Ig binding to
Jurkat cells.
Jurkat cells were untreated (A) or were treated with anti-Fas antibody
CH11 (300 ng/mL) for 4 hours (B-D). 7 Ig-domain VCAM-Ig binding was
assayed as described in "Materials and Methods" (A-C). (D) Cells
were stained with the anti-4-integrin mAb 9F10. Outer membrane
phosphatidylserine exposure was assayed by PE-conjugated annexin-V
binding, as described in "Materials and Methods". The percentage
of cells in each quadrant is indicated. Note the marked reduction in
VCAM-Ig binding to annexin-V(+) cells (B) without a major reduction in
4 expression (D). VCAM-Ig binding to annexin-V(+) cells is restored
by the activating antibody 8A2 (C). 8A2 also markedly increased
annexin-V binding to previously negative cells (C). (E) Kinetics of
sVCAM-1 and annexin-V binding to Jurkat cells after anti-Fas antibody
treatment was analyzed. Cells that bound VCAM-Ig (V+) or failed to bind
VCAM-Ig (V) are indicated. Similarly, annexin-V binding (A+) and
nonbinding (A) cells are also indicated. Note that the
A()/V() cells initially increased coordinately with the
decrease in A()/V(+) cells, before any increase in A(+) cells,
and note the nearly complete absence of V(+)/A(+) cells, indicating
that in apoptotic cells 4-integrin activation is blocked. Results
are representative of 3 separate experiments.
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Suppression of 41-mediated sVCAM-1 binding by a
Ras/Raf-initiated signaling pathway
A Ras/Raf-initiated signaling pathway suppressed the activation of
chimeric integrins.25 Thus, we questioned whether such a
signaling pathway could regulate 41-mediated soluble VCAM-1 binding. CHO-41 cells were transiently cotransfected with cDNA encoding constitutively active H-Ras (H-RasG12V) or Raf-1
(RafBxB) and Tac-5 (used as a surface marker of
transfection). Forty-eight hours after transfection, cells were
analyzed for Tac expression and VCAM-Ig binding by flow cytometry.
Transfection of Tac-5 alone, or in combination with vector control
DNA, resulted in little change in sVCAM-1 binding (Figure
8). Transfection with activated forms of
H-Ras or Raf resulted in an approximately 50% reduction in sVCAM-1
binding (Figure 8), similar to the suppression seen by the
over-expression of free 1 cytoplasmic domain (Tac-1) (Figure 8),
which has also been described for chimeric integrins.29 This suppression was cell-autonomous; only transfected
cells showed reduced sVCAM-1 binding. In addition, this reduced binding
was not caused by a loss of 41 integrins given that activating
the integrins with mAb 9EG7 (data not shown) could restore the binding. sVCAM-1 binding was also suppressed by the overexpression of wild-type H-Ras, but dominant negative H-Ras N17 and constitutively active CDC42
and Rac-1 had no effect on soluble ligand binding (data not shown).
Thus, as with chimeric integrins, the binding of soluble ligands to
41 integrin is regulated by a Ras/Raf-mediated suppression pathway.
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| Fig 8.
Activated H-Ras and Raf-1 suppress 41
integrin-mediated VCAM-1 ligand binding.
CHO-41 cells were transiently transfected with cDNA encoding
Tac-5 or Tac-1 alone or with a combination of Tac-5 plus
vector control DNA, H-Ras G12V, or RafBxB. After 48 hours,
cells were analyzed for Tac expression and soluble VCAM-Ig binding by
2-color flow cytometry. 7 Ig-domain sVCAM-Ig binding was evaluated in
low Tac-expressing cells, designated Tac() (), and high Tac
expressing cells, designated Tac(+) (). Percentage
inhibition of VCAM-Ig binding was calculated relative to cells
transfected with Tac-5 cDNA alone. Results are the means ± SEM
of 3 separate experiments.
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Discussion |
Soluble VCAM-1 is generated at sites of inflammation and regulates
lymphocyte function.9,10 We now report that active cellular
processes regulate the binding of sVCAM-1 to the integrin 41.
Binding of sVCAM-1 to 41 is cell-type specific in primary cells
and in cell lines. In addition, binding is energy dependent and is
disrupted during apoptosis. sVCAM-1 binding is also suppressed by
constitutively active variants of Ras and Raf-1. CD45RO+ peripheral blood T cells are stimulated to bind sVCAM-1 by treatment with phorbol
ester. Thus, the affinity of 41 for its ligand, VCAM-1, is
regulated by active cellular events. Inside-out signaling through 4
integrins may regulate events such as leukocyte trafficking, migration,
survival, and hematopoiesis.
Activation-dependent binding of soluble ligands to 41 integrin is
differentially regulated. We found that a soluble recombinant fusion
protein containing the complete extracellular 7 Ig domains of VCAM-1
bound spontaneously to 41 on Jurkat cells. In contrast, soluble
FN failed to bind to these cells unless the integrins were exogenously
activated, as has been described.19 Furthermore, a soluble
VCAM-Ig protein consisting of the first 2 N-terminal Ig domains was a
poor soluble ligand for 41 compared with the 7 Ig domain form. Ig
domains 1 and 4 of VCAM-1 can mediate 41-dependent cell adhesion
independently.30 Thus, it is unclear whether domain 4 is
solely responsible for the active binding of sVCAM-1 or whether there
is cooperation between domains 1 and 4. Mutational analysis of each of
these domains is ongoing and will provide insight into this question.
The binding observed in this study did not result simply from the
dimeric nature of the fusion protein; a monomeric 7 domain VCAM-1
showed similar binding characteristics. However, cooperative
interaction between domains 1 and 4 on a single VCAM-1 molecule with 1 or more 41 integrins could play a role in soluble ligand binding.
Interestingly, a splice variant of VCAM-1 lacking domain 4 has been
described.31 It will be of interest to determine whether
differential expression of the 2 VCAM-1 variants and their interaction
with 41 integrin may regulate the migration or development of
4 integrin-expressing cells.
The cellular context in which the 41 is expressed controls
sVCAM-1 binding. Cell-type differences in adhesion to immobilized VCAM-1 correlated with sVCAM-1 binding. In general, cell types previously reported to adhere avidly to VCAM-1 (ie, Jurkat and CHO-41) also showed the greatest sVCAM-1 binding. In contrast, cell types that adhere poorly to VCAM-1 (ie, THP-1 and HUT-78) bound
less soluble ligand.20,32 In addition, the binding of sVCAM-1 to 41 integrin is an energy-dependent process because metabolic inhibitors suppressed soluble binding. Other investigators have failed to observe activation-dependent binding of a 7 Ig domain sVCAM-Ig fusion protein to 4 integrin-expressing
cells.33,34 However, some of these studies were performed
on a single cell type such as the erythroleukemic line
K562,33 a cell line previously reported to express 1
integrins in a low-affinity state.35 Others failed to
observe active sVCAM-1 binding to 41 at 4 °C.34 Because we found that VCAM-1 binding is energy dependent, the process
may have been blocked in experiments conducted at 4°C. Thus,
sVCAM-1 binding is active and cell type-specific.
The differences in sVCAM-1 binding properties of 4 integrins on
subgroups of blood mononuclear cells suggest important variations in
4 integrin-dependent functions. Many circulating CD16+ NK cells
express 4 integrins in a high-affinity state based on constitutive sVCAM-1 binding. NK cells act as a first line of defense against many
pathogens and accumulate rapidly in tissues early in the course of
inflammation.36,37 The high-activation state of 4 integrins on NK cells may facilitate the attachment to endothelium early in inflammation, when VCAM-1 may be expressed at relatively low
levels. Furthermore, high integrin affinity promotes cell migration at
low-substrate densities.38 Consequently, NK cells may
preferentially migrate at sites of low VCAM-1 expression. In contrast
to NK cells, most circulating CD3-positive T-lymphocytes express 4
integrins that fail to bind sVCAM-1. Cellular activation may be
required for the expression of high-affinity 4 integrins on these
cells. PMA stimulation activated the 4 integrins, primarily on
CD45RO+ memory T-lymphocytes rather than on CD45RA+ naïve T
cells. Future studies will determine whether more physiologically relevant stimuli, such as chemokines and
T-cell-antigen-receptor engagement, will also activate
4 integrins on this T-cell subset. The activation of 4 integrins
on these cells may facilitate their interaction with VCAM-1 and their
recruitment to sites of inflammation. This is consistent with a
paradigm of lymphocyte recirculation in which memory, but not
naïve, T-lymphocytes preferentially recirculate through
peripheral blood vessels at inflammatory sites at which VCAM-1 is
expressed.39,40 sVCAM-1 exhibits chemotactic activity for
CD45RO+ memory T cells.9 This, too, may facilitate the
influx of memory T cells to sites of inflammation. Interestingly, Giblin et al19 reported preferential activation of 41
and FN binding on naive T cells stimulated by L-selectins. Thus, the differential activation of 41 and ligand preference on naive and
memory T cells may facilitate the recruitment of each of these T-cell
subsets to selective sites.
A soluble form of VCAM-1 is present in the blood and synovial fluid of
patients with inflammatory diseases at concentrations of 10 to 20 nmol/L,9 and higher levels could occur at localized regions
of inflammation. The ED50 of ~50 nmol/L for the binding of sVCAM-1 to Jurkat cells indicates that the pathologic levels of
sVCAM-1 could occupy a significant fraction of activated 41. Perhaps sVCAM-1 acts as a competitive ligand inhibitor blocking adhesion to inflamed vascular endothelium. Furthermore, Kitani et
al10 have shown that sVCAM-1 inhibits T-cell proliferation possibly because of the suppression of IL-2 production. Recent studies
suggest that sVCAM-1 binding to T cells may induce apoptotic cell
death (Ishii JK, et al, manuscript submitted for publication). Hence, though the expression of VCAM-1 on vascular endothelium is
involved in the recruitment of 4-expressing cells to sites of
inflammation, sVCAM-1 may dampen or downregulate the inflammatory response by altering leukocyte trafficking, activation, and survival.
Specific intracellular signaling pathways regulate the binding of
sVCAM-1 to 4 1 integrins. We observed that activated variants
of H-Ras or Raf-1 inhibited 41 ligand binding. Thus, the
activation state of 41, like chimeric IIb3,25
is subject to downregulation by an integrin-suppression pathway.
Suppression of VCAM-1 binding to 41 could play a role in the
release of mononuclear cells from sites such as bone marrow and thymus.
For example, double-positive thymocytes express 41 in a
constitutively active state that facilitates adhesion to VCAM-1
expressed in the cortex and at the corticomedullary junction of
the thymus.3,41 A decrease in 41 affinity for VCAM-1
could be important in allowing the release of thymocytes from the
cortex, facilitating their migration to the medulla. We are now in a
position to study how extracellular stimuli, acting through Ras/Raf
signaling, modulate 41 affinity.
sVCAM-1 binding to 41 on Jurkat cells was disrupted early in
Fas-induced apoptosis. This disruption was not associated with a
significant loss of 4-integrin expression, suggesting that the
affinity of 41 for VCAM-1 was reduced. Cell detachment from the
substratum and neighboring cells is a hallmark feature of apoptosis.42,43 How this occurs is unknown, but
caspase-dependent proteolysis of proteins such as actin, fodrin,
-catenin, and FAK could play a role in this
process.44-46 Our results suggest that decreased
integrin affinity for ligands may play a role in the loss of
cell adhesion associated with apoptosis. Apoptosis could suppress
VCAM-1 binding by the induction of a suppressor-signaling pathway or by
the proteolytic cleavage of integrin activators or of
integrin cytoplasmic domains themselves.47,48 Our
results now make it possible to analyze this newly
observed early event in T-cell apoptosis.
Integrin-affinity modulation plays several roles in cellular processes,
such as adhesion, migration, and extracellular matrix assembly. The
prototypic integrin in which this form of regulation has been studied
is platelet IIb3. Using recombinant IIb3, a number of novel
integrin-regulatory molecules have been studied by genetic and
biochemical approaches.25,29,49 However, there are
cell-type and integrin-specific differences in integrin regulation. The
use of soluble VCAM-1 offers a useful means to study affinity modulation of 4 integrins expressed in their natural cellular environment. Thus, genetic strategies, such as those developed for
IIb3, can now be used to dissect the regulation of 41 function in mononuclear cells. sVCAM-1 itself appears to be a ligand
that regulates mononuclear cell function. Thus, the alteration of
VCAM-1 binding through 4-integrin activation is likely to contribute
to mononuclear cell-mediated inflammation and immune responses.
| |
Acknowledgments |
The authors thank Richard M. Wright (Novartis Pharmaceuticals, Summit,
NJ) for the VCAM-C fusion protein and Roy Lobb (Biogen, Cambridge,
MA) for the 2-domain VCAM-Ig.
| |
Footnotes |
Submitted May 13, 1999; accepted September 15, 1999.
Supported by grants from the National Institutes of Health and by grant
3FB-0164 from the California Breast Cancer Research Program of the
University of California.
Reprints: Mark H. Ginsberg, Department of Vascular Biology, The
Scripps Research Institute, 10550 N Torrey Pines Rd, VB-2, La Jolla, CA
92037; e-mail: ginsberg{at}scripps.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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October 26, 2001;
276(44):
40903 - 40909.
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L. C. Bridges, P. H. Tani, K. R. Hanson, C. M. Roberts, M. B. Judkins, and R. D. Bowditch
The Lymphocyte Metalloprotease MDC-L (ADAM 28) Is a Ligand for the Integrin alpha 4beta 1
J. Biol. Chem.,
January 25, 2002;
277(5):
3784 - 3792.
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Abstract
Introduction